Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus

Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus Siti Tasnim Makhtar, Sheau Wei Tan, Nur Amalina Nasruddin, Nor Azlina Abdul Aziz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-121500/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Mar, 2021 Read the published version in BMC Veterinary Research → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Feline morbillivirus (FeMV) is a member of genus Morbillivirus which has been associated with the chronic kidney disease in cats even though a definite relationship is still unclear. Morbilliviruses are associated with severe diseases such as Peste des petits ruminants, canine distemper and measles. FeMV has been detected in many countries including Malaysia. This study aims to develop a Taqman real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection. Results: A specific assay was developed since no amplification was observed in viral strains from the same Paramyxoviridae family, such as canine distemper virus (CDV), Newcastle disease virus (NDV) and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 x 10 4 copies/ L with Cq value of 34.32 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34% - 0.53% and 1.38% - 2.03%, respectively. Besides that, the clinical sample evaluation using this assay showed a higher detection rate, with 26 (37%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR. Conclusions: The Taqman-based real-time RT PCR assay targeting the N gene described here is more sensitive, specific, rapid and reproducible compared to the conventional RT-PCR assay targeting the N gene and it could be used to detect early infection in cats. Small Animal Medicine Feline Morbillivirus Taqman-based real-time RT-PCR N gene Figures Figure 1 Figure 2 Figure 3 Background Feline morbillivirus (FeMV) is a novel virus under the Paramyxoviridae family which has been associated with the occurrence of chronic kidney disease in cats. FeMV was first discovered in Hong Kong in 2012, followed by other countries, such as Japan, United States, Italy, Turkey, Thailand, Germany, and Brazil [1-7]. Similar to other morbilliviruses, the FeMV genome is composed of six genes (3’- N - P / V / C - M - F - H - L -5’) which encode for six structural and two non-structural proteins [7]. The matrix ( M ), fusion ( F ), and hemagglutinin ( H ) form the glycoprotein envelope surrounding the ribonucleocapsid [8]. The large ( L ) and phosphoprotein ( P ) form the RdRp complex responsible for all polymerase activities. The genomic or anti-genomic RNA is always encapsidated by the nucleoprotein ( N ), thus making the production of N as a requirement before productive replication can occur. In addition to that, a putative recombination involving fusion ( F ) and hemagglutinin ( H ) genes has been documented for a Japanese strain, but whether or not this new virus strain represents interjumping virus from the wildlife population to domestic cats is yet to be determined [9]. Since the discovery of this novel virus, a number of publications describes utilizing conventional PCR assays to screen FeMV [5-7, 10]. However, the screening of FeMV by conventional PCR assays can be hampered by the low quantity of RNA in the samples [10, 11]. Furthermore, screening large number of samples and performing quantitative analysis are either laborious or impossible to be performed with conventional PCR assays. Our previous study has detected 82 FeMV-positive cats out of 208 cats in Malaysia using conventional PCR [12]. In addition to that, a high range of 85% - 99% nucleotide similarities were obtained from the analysis of partial nucleocapsid ( N ) gene from these FeMV-positive cats. To our knowledge, there were only two published studies describing the development of quantitative-based assay to detect FeMV targeting the L and P / V / C gene [11, 13]. Given the need to further characterize this virus and its pathogenesis in cat populations, this research aims to develop Taqman real-time RT-PCR (qRT-PCR) assay based on N gene. N gene is an ideal target gene because it is a well-conserved gene and a major viral protein that folds and protects the viral RNA, playing a key role in virus replication [14]. The N gene sequence was used to design primers to develop Taqman-based real-time PCR assay. Both specificity and sensitivity of the assay were assessed and further evaluated with clinical samples alongside the conventional RT-PCR. Ultimately, developing this specific and sensitive assay for FeMV will galvanize the subsequent detailed examination on the significance of this virus in domestic cat populations. Results Optimization of the Taqman-based real-time RT-PCR (qRT-PCR) targeting N gene of FeMV The Taqman-based qRT-PCR was optimized by determining the optimal primers and probe concentration of which 0.8 L of 10 M primers and 0.2 L of 10 M probe with a final concentration of 400 nM and 100 nM, respectively were used. Therefore, the final reaction volume of 20 consisted of 10 L 2x SensiFAST Probe No-ROX Mix, 0.8 L of each forward and reverse primers, 0.2 L probe, 4 L converted cDNA template, and 4.2 L nuclease-free water. Sensitivity of Taqman-based real-time RT-PCR (qRT-PCR) targeting N gene of FeMV The concentration of the cRNA was 12 ng/L, which was equivalent to 1.74 x copies/L. A series of 10-fold dilution of the cRNA, ranging from 1.74 x to 1.74 x was reversed transcribed and then used to determine the sensitivity of the real-time PCR assay. The lower detection limit based on the cRNA copy number was achieved at 1.74 x copies/L, with a corresponding Cq value of 34.32 0.5 (Figure 1a). The standard curve of the qRT-PCR assay based on the cRNA copy number was plotted to be linear with the coefficient of determination (R 2 ) of 0.999, the slope of -3.402 and the efficiency of 96.8% (Figure 1b). Specificity of Taqman-based real-time RT-PCR (qRT-PCR) targeting N gene of FeMV Specificity of the qRT-PCR assay was assessed against the viral strains from the same Paramyxoviridae family, which were the Newcastle disease virus (NDV), canine distemper virus (CDV) and measles virus (MeV), and other feline viruses, such as feline leukemia virus (FeLV) and feline coronavirus (FCoV). None of the RNA viruses showed amplification signals, proving the high specificity of the assay (Figure 2). Besides that, the no-template control did not show any amplification signal. Reproducibility of Taqman-based real-time RT-PCR (qRT-PCR) targeting the N gene of FeMV The reproducibility of the qRT-PCR was determined by the intra- and inter-assay evaluation of the Cq value using RNAs from three different positive samples: UPM23, UPM52 and UPM202. The intra-assay was analyzed by using the RNAs from these three samples in triplicates per run whereas for the inter-assay assessment, the RNAs were amplified in triplicates in three different consecutive runs. The calculated SD and CV values for the intra-assay variations ranged from 0.10 to 0.17, and from 0.34% to 0.53%, respectively (Table 1). Meanwhile, for the inter-assay evaluation, the calculated SD and CV values ranged from 0.45 to 0.66 and 1.38% to 2.03%, respectively (Table 2). Table 1 Intra-assay variation Sample Cq value Mean Cq S.D. CV% Replicate 1 Replicate 2 Replicate 3 UPM23 28.00 28.01 27.83 27.95 0.1 0.36 UPM52 32.92 32.77 32.71 32.80 0.11 0.34 UPM202 32.17 32.26 32.49 32.31 0.17 0.53 Table 2 Inter-assay variation Sample Cq value Mean Cq S.D. CV% Assay 1 Assay 2 Assay 3 UPM23 27.42 27.66 28.42 27.83 0.52 1.87 UPM52 32.33 32.57 33.20 32.70 0.45 1.38 UPM202 31.90 32.44 33.21 32.51 0.66 2.03 Clinical samples evaluation using Taqman-based qRT-PCR and comparison with conventional RT-PCR Assay Urine samples (n = 55) and kidney samples (n = 16) were evaluated to determine the performance of the newly developed qRT-PCR assay (Table 3). Of these 71 samples, 22/55 (40.0%) urine and 3/16 (18.8%) kidney samples were positive for FeMV with log copy number ranging from 3.54 0.01 to 6.60 0.12. Besides that, of the 16 cats that had both kidney and urine samples collected, none of these cats had detectable FeMV RNA in both samples concurrently. For example, the three cats showing FeMV-positive kidney samples (UPM304, UPM312 and UPM321) had no detectable FeMV RNA in their urine samples. Conversely, one of the cats (UPM305) showing detectable FeMV RNA in its urine sample had no FeMV RNA in its kidney samples. In order to determine the sensitivity of Taqman qRT-PCR assay in detecting FeMV from the clinical samples, conventional RT-PCR using two different sets of published primers targeting N gene [9] was conducted on all 71 samples, of which only 11/55 (20%) urine samples were positive while no (0/16; 0%) kidney samples were detected positive. Interestingly, the cats deemed as healthy with their health status available had a higher positive rate of detection (13/20) compared to the cats diagnosed with a disease (3/10). The source of the cats, whether they came from shelters or were pet cats, might also be important in the FeMV-positive detection rate. A higher percentage of positive detection rate was observed among client-owned cats (46%; 18/39) compared to the shelter cats (43%; 7/16). Biological samples taken from the cats in shelters were also part of other study looking at the presence of the Platynosomum sp., a hepatic trematode found in the gall bladder and biliary ducts of cats. Incidentally, Platynosomum sp. was detected in 7 out of 16 (43.8%) shelter cats, of which 4 (57.1%) of them were also FeMV-positive. Table 3 Detection of FeMV using conventional RT-PCR and qRT-PCR quantification from clinical samples Sample Sample ID RT-PCR ( N gene) Log copy number (mean S.D. ) Source of cat Health status Urine UPM23 + 6.600.12 Client-owned Healthy Urine UPM52 + 4.940.02 Client-owned Healthy Urine UPM53 + 4.930.02 Client-owned Healthy Urine UPM202 + 4.770.02 Client-owned Healthy Urine UPM203 - BLD Client-owned Healthy Urine UPM204 - 4.860.06 Client-owned Healthy Urine UPM205 - 4.550.03 Client-owned Healthy Urine UPM207 - 4.670.02 Client-owned Healthy Urine UPM210 + 3.660.08 Client-owned Healthy Urine UPM219 - 3.800.14 Client-owned CKD stage IV Urine UPM220 + 4.110.05 Client-owned N/A Urine UPM221 - 3.660.02 Client-owned Healthy Urine UPM224 - BLD Client-owned Healthy Urine UPM225 - 3.540.01 Client-owned Healthy Urine UPM226 - 3.710.11 Client-owned Healthy Urine UPM227 - BLD Client-owned N/A Urine UPM228 - BLD Client-owned N/A Urine UPM229 - BLD Client-owned N/A Urine UPM231 + 4.290.23 Client-owned Healthy Urine UPM232 - BLD Client-owned CKD Stage II Urine UPM240 - BLD Client-owned Healthy Urine UPM241 - BLD Client-owned Healthy Urine UPM271 - BLD Client-owned Heart disease Urine UPM272 - BLD Client-owned CKD Stage II Urine UPM273 - BLD Client-owned N/A Urine UPM274 - BLD Client-owned Suspected CKD Urine UPM275 - BLD Client-owned FURD Urine UPM276 + 4.050.08 Client-owned Healthy Urine UPM277 - BLD Client-owned Healthy Urine UPM278 - BLD Client-owned N/A Urine UPM279 - BLD Client-owned N/A Urine UPM280 - BLD Client-owned Suspected CKD Urine UPM281 - BLD Client-owned N/A Urine UPM282 - 4.630.05 Client-owned FLUTD Urine UPM283 - BLD Client-owned Healthy Urine UPM284 - BLD Client-owned Healthy Urine UPM285 - 3.740.02 Client-owned N/A Urine UPM286 - 3.880.18 Client-owned Obstructive FLUTD Urine UPM287 - BLD Client-owned Suspected CKD Urine UPM304 - BLD Shelter N/A Urine UPM305 + 4.340.10 Shelter Platynosomum sp. Urine UPM306 - 3.760.26 Shelter N/A Urine UPM307 - BLD Shelter N/A Urine UPM308 - BLD Shelter Platynosomum sp. Urine UPM310 - BLD Shelter N/A Urine UPM311 - BLD Shelter Platynosomum sp Urine UPM312 - BLD Shelter Platynosomum sp Urine UPM313 - BLD Shelter N/A Urine UPM314 + 4.450.07 Shelter Platynosomum sp Urine UPM315 + 4.920.07 Shelter Platynosomum sp Urine UPM316 - BLD Shelter Platynosomum sp Urine UPM317 - BLD Shelter N/A Urine UPM318 - BLD Shelter N/A Urine UPM320 - BLD Shelter N/A Urine UPM321 - BLD Shelter N/A Kidney UPM304 - 3.800.03 Shelter N/A Kidney UPM305 - BLD Shelter Platynosomum sp. Kidney UPM306 - BLD Shelter N/A Kidney UPM307 - BLD Shelter N/A Kidney UPM308 - BLD Shelter Platynosomum sp. Kidney UPM310 - BLD Shelter N/A Kidney UPM311 - BLD Shelter Platynosomum sp. Kidney UPM312 - 3.700.10 Shelter Platynosomum sp. Kidney UPM313 - BLD Shelter N/A Kidney UPM314 - BLD Shelter Platynosomum sp. Kidney UPM315 - BLD Shelter Platynosomum sp. Kidney UPM316 - BLD Shelter Platynosomum sp. Kidney UPM317 - BLD Shelter N/A Kidney UPM318 - BLD Shelter N/A Kidney UPM320 - BLD Shelter N/A Kidney UPM321 - 3.890.08 Shelter N/A BLD denotes below limit of detection of qRT-PCR N/A denotes not available Discussion Feline morbillivirus (FeMV) has been speculated to cause chronic kidney disease (CKD) and it is commonly detected in urine and kidney tissue samples. Previously, conventional assays such as RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP) have been developed targeting the L gene of FeMV [4, 6, 7, 15, 16]. However, these conventional assays were qualitative and required additional step of gel electrophoresis to visualize the PCR product. In this study, a two-step Taqman-based qRT-PCR diagnostic assay for the detection of FeMV- N gene was developed. This quantitative assay was designed to detect the N gene, one of the most conserved regions in Morbilliviruses. Other quantitative-based assays specifically qRT-PCR has also been developed to detect FeMV, but all were designed to either target the L gene and P / V / C gene [11, 13]. Besides, these qRT-PCR assays had low positive rate of detection which could be due to limited availability of FeMV sequences at that time resulting in less specific primers and probe. Hence, it is crucial to design the primers and probe based on the latest sequences available. In addition, P gene has been shown to have the highest rates of nucleotide polymorphisms, ranging from 88.6% to 99.3% [9]. Besides that, a study conducted on Peste des petits ruminant virus (PPRV), another Morbillivirus member revealed that the L gene had the highest evolution rate at 9.75 x 10 -4 site per year while the N gene showed the lowest evolution rate at 1.1 x 10 -3 site per year [15]. Nucleoprotein ( N ) comprises of two regions: core domain ( N CORE ) and C-terminal domain (also known as N TAIL ) [16]. N CORE covers the first 400 amino acids of the N protein, and it is responsible for the RNA encapsidation prior to viral replication and translation. This core domain also consists of three previously identified conserved motifs. N protein also possesses nuclear export signal, nuclear localization signal (NLS) and RNA binding motifs. Both nuclear export signal and NLS are believed to transport the N protein to the nucleus of the host cell while the RNA binding site is considered to be involved in N - N self-interaction and interaction of N - N RNA monomers during the genomic RNA binding. N - TAIL region comprises of structurally variable region of approximately 120 to 150 amino acids which mediate the interaction with P gene [17]. Besides that, N gene is integral during the initial infection as the RNA synthesis by RNA-dependent RNA polymerase (RdRp) complex will only recognize and synthesize the viral RNA encapsidated by N protein as a template [8]. Given that the N gene is conserved compared to other genes of FeMV and detectable during the early phase of infection, a quantitative assay targeting the N gene of FeMV was developed in this study. In order to develop this assay, a partial sequence of 1.5kb N gene was obtained from seven local isolates. Upon obtaining the 1.5kb of FeMV- N gene sequences of these isolates, primers and probe for Taqman-based qRT-PCR assay were designed specifically to target 122bp of N gene. For the development of this assay, a standard curve was generated to accurately quantify FeMV. The sensitivity of the developed assay was done by quantifying 10-fold serial dilutions of standard complementary RNA (cRNA). The results showed the assay could detect up to 10,000 copy number of FeMV. Even though the resulting sensitivity of this newly developed qRT-PCR assay was low, this assay has done none of the other established assays had to date, which was utilizing newly designed primers and probe set targeting N gene. The specificity test of the assay was performed by running the assay against other viral strains from the same family, which were NDV, CDV and MeV, and two feline viruses, FeLV and FCoV. Absence of cross-reaction proved high specificity of the developed assay. In addition to that, the low sensitivity reported for this assay could be due to the highly specific designed primers based on stringent parameters as evident by the absence of amplification in other tested viruses. In a diagnostic test, a highly specific assay may compromise the sensitivity of the assay, resulting in an inverse relationship [18]. Furthermore, incorporating probe known for its specificity in this assay might also contribute to a highly specific qRT-PCR assay, compromising its sensitivity. In order to evaluate the reproducibility of the assay, intra- and inter-assay assessments were done by testing three different positive samples (UPM23, UPM52 and UPM202). The value of standard deviation calculated for both intra- and inter-assay indicated that the developed qRT-PCR assay was low in variability and had high reproducibility. Besides that, comparing the coefficient of variation (CV) of both intra- and inter-assay with previously reported study, the value of CV recorded in this study was relatively low, confirming the reproducibility of this assay [13]. In order to further evaluate the sensitivity of the qRT-PCR assay, clinical samples (n = 71) were tested to detect FeMV by targeting the N gene and compared with conventional RT-PCR assay. All samples which were detected positive by the conventional RT-PCR (11/71) were also detected by the qRT-PCR assay. However, there were 15 qRT-PCR FeMV-positive samples that were not detected by the conventional RT-PCR. Some of these samples had copy number of ~1000. Based on the standard curve, even though the resulted lowest limit of detection was ~10,000 copy number, the quantification on clinical samples proved that the newly designed qRT-PCR assay was able to amplify samples which had lower copy number. Evaluation of the clinical samples showed that healthy cats had a higher positive rate compared to cats diagnosed with a disease. Since this study was a cross-sectional study, it was difficult to interpret whether this finding was significant. Nevertheless, a prospective follow up of these cats may provide an insight on the clinical relationship of the cats with FeMV infection. Besides that, a higher rate of detection was observed among client-owned cats compared to shelter cats which was consistent with our previous study, but in contrast with other studies [1, 12]. This observation could be due to the differences in the shelter management among different countries. Shelter cats are usually placed in an enclosed area, limiting their interactions with free-roaming cats, and hence, reducing transmission from one cat to another. Furthermore, the transmission among client-owned cats may occur if the cats are living outdoors or if they are semi-roamer cats as they can interact with other free-roaming cats [19]. Besides that, a multi-cat household may have a higher chance to transmit the virus to other healthy cats already living within the household or newly introduced animals [2]. It was also noted that all cats were not detected positive for both urine and kidney samples as also observed by many studies [3, 20]. This observation may be caused by the early-stage infection, whereby the virus has yet to travel to the kidney. Consequently, this finding would suggest for a further study to observe the viral shedding and specifically, viral load over time accompanied with serum urea and creatinine level among FeMV-positive cats to determine the relationship of the severity of kidney disease and FeMV pathogenesis. The cats recruited in this study was also part of a study to detect the presence of Platynosomum sp., a fluke living in pancreatic ducts, bile ducts and gall bladder. This parasite requires two intermediate hosts, a snail and followed by a second intermediate host, such as skink, gecko, lizard or toad. A cat can be infected with Platynosomum sp. after ingesting an infected second intermediate host [21]. The clinical signs of Platynosomum sp. infection vary from asymptomatic to progressive disease, and it can also cause death due to hepatic failure and biliary tract obstruction. Biological samples of the liver, bile ducts and feces were obtained from the shelter cats in this study in which Platynosomum sp. was detected in 4 out of 7 FeMV-positive cats. Although this finding was incidental, co-infection of FeMV and Platynosomum sp. should be further explored, especially in shelter cat populations given that this cohort of cats could potentially harbor many pathogens. The developed qRT-PCR assay can be utilized for field samples given its high specificity and novelty of targeting the N gene of FeMV. Although there has yet a clear indication on whether or not this virus is involved in the pathogenesis of chronic kidney disease in cats, further studies are needed to determine the extent of infection among cats and its clinical significance. Conclusion In conclusion, a probe-based qRT-PCR assay targeting the N gene of FeMV has been successfully optimized and validated. This study is the first quantitative assay targeting the FeMV- N gene which is integral in RNA synthesis by RNA-dependent RNA polymerase (RdRp) complex. Even though the developed assay demonstrated a low sensitivity compared to previously designed qRT-PCR assay targeting L and P / V / C gene, compared to the conventional RT-PCR, it can detect samples that has low viral load. Hence, the developed qRT-PCR assay can be applied in diagnostic and quantification of the FeMV viral load, especially during the early stage of infection. Materials And Methods Total RNA extraction and cDNA synthesis The total RNA extraction for collected samples was performed by using Direct-zol ™ RNA MiniPrep Plus kit (Zymo Research, California, United States) following the manufacturer’s guidelines. Extracted RNA was first subjected to cDNA synthesis prior to conventional RT-PCR and qRT-PCR assays. cDNA synthesis was performed by using the SensiFAST ™ cDNA Synthesis Kit (Bioline, London, UK) with 20 L reaction consisting of 10 L purified RNA, 4 L of 5x TransAmp Buffer, 1 L reverse transcriptase and 5 L nuclease-free water. The reaction was carried out at 25 for 10 mins, 42 for 15 mins, 48 for 15 mins and 85 for 5 mins. The cDNA was stored in -20 until use. Taqman-based real-time RT-PCR (qRT-PCR) assay Primer set and probe were designed based on partial N gene sequences of FeMV-Malaysia isolates (UPM23, UPM52, UPM53, UPM210, UPM231, UPM305 and UPM315) using Integrated DNA Technologies (IDT) software (Table 4). The designed probe and primer set were then compared with the alignment of closely-related FeMV from other countries’ isolates: Japan (SS3, MiJP003, ChJP073), China (M252A) and Thailand (Thai-U16) (Table 4). Specific sequences for primers and probe used were as follows to yield 122bp PCR amplicons: forward primer, GGTCAAGAGATGGTGAGAAGAT; reverse primer, CCAGATTCACCTCCCGAATTA; and probe, TTTGCGCGAGAACTTGGGCTATCT. The FeMV Taqman probe was labeled with 6-FAM at 5’-end, and fluorescence quencher, ZEN and IBFQ at an internal site and 3’-end, respectively. Real-time RT-PCR was performed using CFX96 machine (Bio-Rad, California, USA) utilizing SensiFAST TM Probe NO-ROX Kit (Bioline, London, UK) following the manufacturer’s suggestion. Briefly, each reaction well was consisted of 4 L cDNA, 10 L of 2x SensiFAST Probe No-ROX Mix, a final concentration of 400 nM for each forward and reverse FeMV primers, 100 nM probe and nuclease-free water up to the final volume of 20 L. The assay was performed at 95 for 2 mins, followed by 35 cycles of 95 for 15 secs and 54 for 45 secs and each sample was run in triplicates. Table 4 GenBank accession number Isolate Accession Number SS3 LC036587 MiJP003 AB924121 ChJP073 AB924122 M252A JQ411016 Thai-U16 MF627832 UPM23 MN264638 UPM52 MN264639 UPM53 MN264640 UPM210 MN264641 UPM231 MN264642 UPM305 MN792827 UPM315 MN792828 Standard RNA preparation Generation of standard RNA was prepared by sub-cloning the partial sequence of 1.5kb FeMV- N gene into a pSC-A-amp/kan plasmid vector by using StrataClone PCR cloning kit (Agilent Technologies, California, United States) according to the manufacturer’s instruction. After linearization, RNA transcription was performed using the Riboprobe ® in vitro Transcription Systems (Promega, Wisconsin, United States) with the T7 RNA polymerase promoter site available in vector pSC-A-amp/kan following the manufacturer’s direction. After DNase treatment, the transcript was purified by phenol:chloroform purification and quantified by using spectrophotometer (Bio-Rad, California, USA). The copy number of cRNA calculated was 1.74 x 10 11 copies/L. cRNA was diluted in 10-fold serial dilution and converted to cDNA prior to standard curve generation. Specificity and sensitivity The specificity of the developed assay was assessed by testing on viral RNA from same genus, which were canine distemper virus (CDV) and measles virus (MeV), from same family, Newcastle disease virus (NDV), and from other feline viruses, which were feline leukemia virus (FeLV) and feline coronavirus (FCoV) (Table 5). The sensitivity of qRT-PCR assay was assessed by running 10-fold serial dilution of cRNA standard (1.74 x 10 11 to 1.74 x 10 2 copies/L) to detect the threshold limit of the assay. Ten different 10-fold serial dilution was performed by adding 1 L of RNA into 9 L of nuclease-free water. The mixture was then vortexed for at least 10 secs before the same step was repeated for the next dilution. Each of the diluted RNA along with the stock RNA control was converted into cDNA and subjected for qRT-PCR in triplicates. Table 5 List of viruses used in this study and its sources Virus Sources Canine distemper virus (CDV) Nobivac Puppy DP, Intervet Measles virus (MeV) Serum Institute of India LTD, Pune Newcastle disease virus (NDV) Department of Vet. Pathology and Microbiology, UPM Feline leukemia virus (FeLV) Department of Vet. Pathology and Microbiology, UPM Feline coronavirus (FCoV) Department of Vet. Pathology and Microbiology, UPM Reproducibility In order to assess reproducibility of the developed assay, three different positive samples were selected which were then subjected for intra- and inter-assay. Three different positive samples (UPM23, UPM52, UPM202) were assayed in the same run in triplicates to evaluate the intra-assay variations. In order to assess for inter-assay variations, the same three samples were subjected for three different consecutive runs in triplicates. Values for mean, standard deviation (SD) and coefficient of variations (CV) for both variation assays were calculated by using Microsoft Excel Software (version 2016, USA). Clinical samples collection Animal ethics application was approved by the Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia (UPM/IACUC/AUP-R037/2018). Convenient sampling was performed whereby urine (n = 55) and kidney samples (n = 16) were collected from veterinary hospital, private veterinary clinics around Klang Valley, Malaysia and animal shelters. The cats presented to the veterinary hospital or private veterinary clinics were either presented for annual health examination, neutering procedure or having health-related issues, such as kidney-related and heart diseases. Cats’ samples which their serum urea-creatinine data were available were further sub-grouped into cats with presence or absence of kidney-related disease based on the International Renal Interest Society (IRIS) Guidelines. The owner’s consent was requested prior to samples collection. When available, kidney samples (n = 16) from post-mortem and corresponding urine samples (n = 16) were collected from animal shelters. Sample processing A urine sample was collected into a sterile sample collection bottle either by cystocentesis or manual compression. The supernatant of urine was obtained after a centrifugation step at 2320 x g for 5 mins. Then, the supernatant was mixed with RNAlater ® solution (Ambion, Texas, United States) at ratio 1:1 and stored at -20 prior to RNA extraction. During postmortem, the collected kidney samples were immediately transferred into a sterile collection bottle containing RNAlater ® solution. For sample processing, kidney tissues of approximately 1 g were cut into small pieces and crushed by using the pestle and mortar along with sterile sand. Phosphate-buffer saline solution (Gibco, Massachusetts, United States) of 1g/mL was added into the homogenized kidney. Then, the mixture was transferred into a 15 mL tube to be centrifuged at 2320 x g for 5 mins to remove any large debris and sand. The kidney lysate was then subjected for total RNA extraction. Clinical samples evaluation using Taqman-based qRT-PCR and conventional RT-PCR Converted cDNA clinical samples were assessed by conventional RT-PCR using published primers (Table 6) with cDNA of UPM52 as a positive control together with no-template control following a modified protocol (Table 7). Taqman-based qRT-PCR was performed according to the developed protocol described above with each sample ran in triplicates along with NTC and cDNA of positive control (UPM52). Nuclease-free water (Promega, Wisconsin, United States) was used as the template in NTC in both conventional RT-PCR and qRT-PCR assays. Table 6 Primer sequences used to amplify two different regions of N gene Region Primer Sequence (5’-3’) Product size (bp) Source Middle region FN-2F GTTAGCTTAGGATTTGAGAACCC 680bp [9] FN-2R CACCATCTCTTGACCAAGTCT End region FN-3F GCTATGGAGTTATGCCATGGG 637bp FN-3R GTTGTGAACCTTGAGGTCCTAAG Table 7 PCR protocol applied for two different primer sets of N gene Step Temperature Time Cycle Initial denaturation 95 1 min 1x Denaturation Annealing 95 58 72 15 secs 1 min 35x Extension 1 min Final extension 72 5 mins 1x Hold 12 1x Platynosomum sp. detection from postmortem of shelter cats Liver, bile duct and faeces samples in rectum were collected in the postmortem investigation of shelter cats to detect the presence of Platynosomum sp. Liver samples were used to collect adult fluke while bile duct and faeces samples were used to detect ova. In order to allow for activation and collection of mature flukes, liver samples were extracted and immersed in warm water (38-40). Parasitic burden was calculated using the formula described in a previous study by applying the number of adult flukes collected per samples [22]. For ova collection from bile juice and faeces samples, any ova isolated from these clinical samples were pipetted into microcentrifuge tubes with normal saline and they were stored in -20 for further analysis. In faecal examination, two different methods were performed to detect fluke eggs: the simple floatation technique and centrifugal faecal sedimentation test in formal-ether solution [23, 24]. Adult fluke and ova were identified based on a previous study [25]. Abbreviations qRT-PCR Quantitative reverse transcription polymerase chain reaction FeMV Feline morbillivirus CDV Canine distemper virus NDV Newcastle disease MeV Measles virus FCoV Feline coronavirus FeLV Feline leukemia virus RT-PCR Reverse transcription polymerase chain reaction M Matrix protein F Fusion protein H Hemagglutinin protein L Large protein P Phosphoprotein N Nucleocapsid protein FURD Feline upper respiratory disease FLUTD Feline lower urinary tract disease IDT Integrated DNA Technologies IACUC Institutional animal care and use committee UVH University Veterinary Hospital IRIS International Renal Interest Society RdRp RNA-dependent RNA polymerase Declarations Ethics Approval and Consent to participate Animal ethics application was approved from the Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia (UPM/IACUC/AUP-R037/2018) prior to sample collection. Informed consent was obtained from the cats’ owners prior to sample collection. Hence, the use of cats in this study met the criteria of animal welfare regulations in Malaysia. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed in this study can be obtained within the tables and figures of the manuscript. Competing interest The authors declare that they have no competing interests. Funding This study was supported by the Universiti Putra Malaysia internal grant (Grant No: GP-IPS/2017/9536900). The funding body has no specific role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors’ contributions FMK contributed to the conception, design and revising the manuscript. STM contributed to the data collection, laboratory work, drafting and writing the manuscript. ARO and TSW contributed to the conception, data analyses and revising the manuscript. NAAA and NAN contributed to the data collection, laboratory work and revising the manuscript. All authors have read and approved the manuscript. Acknowledgements We would like to thank Dr. Zarirah Mohamed Zulperi for technical assistance and troubleshooting on the plasmid cloning and standard RNA preparation work. We would like to also thank all participating veterinary clinics, veterinary hospital and animal shelters especially the cats and their owners for allowing us to perform sample collection. References Techangamsuwan SCaS: First evidence of Feline morbillivirus detected in sheltered cats in Thailand . Thai J Vet Med Suppl 2017, 47 :127-128. Darold GM, Alfieri AA, Muraro LS, Amude AM, Zanatta R, Yamauchi KC, Alfieri AF, Lunardi M: First report of feline morbillivirus in South America . Arch Virol 2017, 162 (2):469-475. Yilmaz H, Tekelioglu BK, Gurel A, Bamac OE, Ozturk GY, Cizmecigil UY, Altan E, Aydin O, Yilmaz A, Berriatua E et al : Frequency, clinicopathological features and phylogenetic analysis of feline morbillivirus in cats in Istanbul, Turkey . J Feline Med Surg 2017, 19 (12):1206-1214. Sharp CR, Nambulli S, Acciardo AS, Rennick LJ, Drexler JF, Rima BK, Williams T, Duprex WP: Chronic Infection of Domestic Cats with Feline Morbillivirus, United States . Emerg Infect Dis 2016, 22 (4):760-762. Lorusso A, Di Tommaso M, Di Felice E, Zaccaria G, Luciani A, Marcacci M, Aste G, Boari A, Savini G: First report of feline morbillivirus in Europe . Vet Ital 2015, 51 (3):235-237. Furuya T, Sassa Y, Omatsu T, Nagai M, Fukushima R, Shibutani M, Yamaguchi T, Uematsu Y, Shirota K, Mizutani T: Existence of feline morbillivirus infection in Japanese cat populations . Arch Virol 2014, 159 (2):371-373. Woo PC, Lau SK, Wong BH, Fan RY, Wong AY, Zhang AJ, Wu Y, Choi GK, Li KS, Hui J et al : Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats . Proc Natl Acad Sci U S A 2012, 109 (14):5435-5440. Sourimant J, Plemper RK: Organization, Function, and Therapeutic Targeting of the Morbillivirus RNA-Dependent RNA Polymerase Complex . Viruses 2016, 8 (9). Park ES, Suzuki M, Kimura M, Maruyama K, Mizutani H, Saito R, Kubota N, Furuya T, Mizutani T, Imaoka K et al : Identification of a natural recombination in the F and H genes of feline morbillivirus . Virology 2014, 468-470 :524-531. Sieg M, Heenemann K, Ruckner A, Burgener I, Oechtering G, Vahlenkamp TW: Discovery of new feline paramyxoviruses in domestic cats with chronic kidney disease . Virus Genes 2015, 51 (2):294-297. Furuya T, Wachi A, Sassa Y, Omatsu T, Nagai M, Fukushima R, Shibutani M, Yamaguchi T, Uematsu Y, Shirota K et al : Quantitative PCR detection of feline morbillivirus in cat urine samples . J Vet Med Sci 2016, 77 (12):1701-1703. Mohd Isa NH, Selvarajah GT, Khor KH, Tan SW, Manoraj H, Omar NH, Omar AR, Mustaffa-Kamal F: Molecular detection and characterisation of feline morbillivirus in domestic cats in Malaysia . Vet Microbiol 2019, 236 :108382. De Luca E, Crisi PE, Di Domenico M, Malatesta D, Vincifori G, Di Tommaso M, Di Guardo G, Di Francesco G, Petrini A, Savini G et al : A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens . J Virol Methods 2018, 258 :24-28. Diallo A: Morbillivirus group genome organisation and protein.pdf> . Veterinary Microbiology 1990, 23 :155-163. Shabbir MZ, Rahman AU, Munir M: A comprehensive global perspective on phylogenomics and evolutionary dynamics of Small ruminant morbillivirus . Sci Rep 2020, 10 (1):17. Thakkar VD, Cox RM, Sawatsky B, da Fontoura Budaszewski R, Sourimant J, Wabbel K, Makhsous N, Greninger AL, von Messling V, Plemper RK: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity . J Virol 2018, 92 (8). Communie G, Ruigrok RW, Jensen MR, Blackledge M: Intrinsically disordered proteins implicated in paramyxoviral replication machinery . Curr Opin Virol 2014, 5 :72-81. Vetter TR, Schober P, Mascha EJ: Diagnostic Testing and Decision-Making: Beauty Is Not Just in the Eye of the Beholder . Anesth Analg 2018, 127 (4):1085-1091. Park ES, Suzuki M, Kimura M, Mizutani H, Saito R, Kubota N, Hasuike Y, Okajima J, Kasai H, Sato Y et al : Epidemiological and pathological study of feline morbillivirus infection in domestic cats in Japan . BMC Vet Res 2016, 12 (1):228. Stranieri A, Lauzi S, Dallari A, Gelain ME, Bonsembiante F, Ferro S, Paltrinieri S: Feline morbillivirus in Northern Italy: prevalence in urine and kidneys with and without renal disease . Vet Microbiol 2019, 233 :133-139. Javinsky E: Chapter 23 - Digestive System, Liver, and Abdominal Cavity . In: The Cat. edn. Edited by Little SE. Saint Louis: W.B. Saunders; 2012: 425-546. Margolis L, W.Esch G, Holmes JC, Kuris AM, Schad GA: The Use of Ecological Terms in Parasitology (Report of an Ad Hoc Committee of the American Society of Parasitologists) . The Journal of Parasitology 1982, 68 (1):131-133. Conboy AMZaGA: Veterinary Clinical Parasitology , 8th edn. Ames, Iowa: Blackwell Publishing Professional; 2012. Ritchie LS: An ether sedimentation technique for routine stool examinations . Bull U S Army Med Dep 1948, 8 (4):326. RA B, DI G, A J: Keys to the Trematode , vol. 3. Wallingford, UK and the Natural History Museum, London: CABI Publishing; 2008. Cite Share Download PDF Status: Published Journal Publication published 23 Mar, 2021 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Major revision 29 Dec, 2020 Reviews received at journal 28 Dec, 2020 Reviewers agreed at journal 28 Dec, 2020 Reviews received at journal 21 Dec, 2020 Reviewers agreed at journal 14 Dec, 2020 Reviewers invited by journal 10 Dec, 2020 Editor assigned by journal 10 Dec, 2020 Editor invited by journal 07 Dec, 2020 Submission checks completed at journal 07 Dec, 2020 First submitted to journal 03 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-121500","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":5962397,"identity":"323387d7-44fb-42f8-a93a-96c64fd84afe","order_by":0,"name":"Siti Tasnim Makhtar","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siti","middleName":"Tasnim","lastName":"Makhtar","suffix":""},{"id":5962398,"identity":"44d92c00-ac0c-4af6-a06a-c986e9f18cca","order_by":1,"name":"Sheau Wei Tan","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sheau","middleName":"Wei","lastName":"Tan","suffix":""},{"id":5962399,"identity":"3e6cadc0-4d3d-47d2-bea5-f2608df02162","order_by":2,"name":"Nur Amalina Nasruddin","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Amalina","lastName":"Nasruddin","suffix":""},{"id":5962400,"identity":"79d274d8-a68e-4bb9-a600-80cf2eb667b9","order_by":3,"name":"Nor Azlina Abdul Aziz","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nor","middleName":"Azlina Abdul","lastName":"Aziz","suffix":""},{"id":5962401,"identity":"93bf8d75-6bc2-4861-acbe-7aaf4b3dcfcf","order_by":4,"name":"Abdul Rahman Omar","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"Rahman","lastName":"Omar","suffix":""},{"id":5962402,"identity":"b4d29f3c-7d3d-4fc4-8997-12dcbdb2068d","order_by":5,"name":"Farina Mustaffa-Kamal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBAC9gYGhgMMDDYQHg8xWngOgLWkkagFCA6TooX97MPDBTXnE7eLHWB88LaNIdrgACEtPOkGh2ccu524c3YCs+HcNobcDYS02AO9cZiH7XbihtsJbNK8xGjh4X8G1PLvHEgL+2/itEgAbeFtOwC2hZlILUBbePuSjXfOTmyWnHNOIncmYYelMX/m+WYnu106+eCHN2U2uX2EtMCBAQNjA5CSYFAgQQsUyDcQq2UUjIJRMApGCgAATnND/I+qpIQAAAAASUVORK5CYII=","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Farina","middleName":"","lastName":"Mustaffa-Kamal","suffix":""}],"badges":[],"createdAt":"2020-12-04 03:44:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-121500/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-121500/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-021-02837-6","type":"published","date":"2021-03-23T15:00:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4164664,"identity":"611c2e55-8ebf-4f67-b158-6e036694b58c","added_by":"auto","created_at":"2020-12-10 16:54:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28983,"visible":true,"origin":"","legend":"The detection limit of qRT-PCR assay based on cRNA copy number. Amplification plot of 10-fold serial diluted cRNA ranging from 1.74 × 〖10〗^11 to 1.74 × 〖10〗^4 copies/μL.","description":"","filename":"Onlinefloatimage1.Png","url":"https://assets-eu.researchsquare.com/files/rs-121500/v1/8ed3c56dd294d3705476deab.Png"},{"id":4164665,"identity":"cd7114f7-40f9-4f14-8f8c-ab5acac3c8cb","added_by":"auto","created_at":"2020-12-10 16:54:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17400,"visible":true,"origin":"","legend":"Standard curve of qRT-PCR assay. Ten-fold dilutions of control cRNA were assessed with the qRT-PCR assay. Log copies per μL and quantitation cycle (Cq) are plotted on the x-axis and y-axis, respectively.","description":"","filename":"Onlinefloatimage2.Png","url":"https://assets-eu.researchsquare.com/files/rs-121500/v1/c9a25908832f36e6b42b898f.Png"},{"id":4164666,"identity":"a741fbdc-d363-4867-beb0-0efa4df58ec9","added_by":"auto","created_at":"2020-12-10 16:54:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16537,"visible":true,"origin":"","legend":"Specificity of N gene based on the qRT-PCR assay. Amplification plot detection of FeMV, FCoV, FeLV, CDV, MeV, NDV and no-template control (NTC). Amplification curve was only detected for FeMV (positive control).","description":"","filename":"Onlinefloatimage3.Png","url":"https://assets-eu.researchsquare.com/files/rs-121500/v1/b878f93ba0110ed5bc7c9242.Png"},{"id":13631986,"identity":"e4d0f489-df22-43be-aea2-5d139542248f","added_by":"auto","created_at":"2021-09-17 08:19:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1141463,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-121500/v1/edce57fb-b96a-46ed-b5e3-954cff6c055c.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDevelopment of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eFeline morbillivirus (FeMV) is a novel virus under the \u003cem\u003eParamyxoviridae\u003c/em\u003e family which has been associated with the occurrence of chronic kidney disease in cats. FeMV was first discovered in Hong Kong in 2012, followed by other countries, such as Japan, United States, Italy, Turkey, Thailand, Germany, and Brazil [1-7]. Similar to other morbilliviruses, the FeMV genome is composed of six genes (3\u0026rsquo;-\u003cem\u003eN\u003c/em\u003e-\u003cem\u003eP\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e-\u003cem\u003eM\u003c/em\u003e-\u003cem\u003eF\u003c/em\u003e-\u003cem\u003eH\u003c/em\u003e-\u003cem\u003eL\u003c/em\u003e-5\u0026rsquo;) which encode for six structural and two non-structural proteins [7]. The matrix (\u003cem\u003eM\u003c/em\u003e), fusion (\u003cem\u003eF\u003c/em\u003e), and hemagglutinin (\u003cem\u003eH\u003c/em\u003e) form the glycoprotein envelope surrounding the ribonucleocapsid [8]. The large (\u003cem\u003eL\u003c/em\u003e) and phosphoprotein (\u003cem\u003eP\u003c/em\u003e) form the RdRp complex responsible for all polymerase activities. The genomic or anti-genomic RNA is always encapsidated by the nucleoprotein (\u003cem\u003eN\u003c/em\u003e), thus making the production of N as a requirement before productive replication can occur. In addition to that, a putative recombination involving fusion (\u003cem\u003eF\u003c/em\u003e) and hemagglutinin (\u003cem\u003eH\u003c/em\u003e) genes has been documented for a Japanese strain, but whether or not this new virus strain represents interjumping virus from the wildlife population to domestic cats is yet to be determined [9].\u003c/p\u003e\n\u003cp\u003eSince the discovery of this novel virus, a number of publications describes utilizing conventional PCR assays to screen FeMV [5-7, 10]. However, the screening of FeMV by conventional PCR assays can be hampered by the low quantity of RNA in the samples [10, 11]. Furthermore, screening large number of samples and performing quantitative analysis are either laborious or impossible to be performed with conventional PCR assays. Our previous study has detected 82 FeMV-positive cats out of 208 cats in Malaysia using conventional PCR [12]. In addition to that, a high range of 85% - 99% nucleotide similarities were obtained from the analysis of partial nucleocapsid (\u003cem\u003eN\u003c/em\u003e) gene from these FeMV-positive cats. To our knowledge, there were only two published studies describing the development of quantitative-based assay to detect FeMV targeting the \u003cem\u003eL\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e gene [11, 13].\u003c/p\u003e\n\u003cp\u003eGiven the need to further characterize this virus and its pathogenesis in cat populations, this research aims to develop Taqman real-time RT-PCR (qRT-PCR) assay based on \u003cem\u003eN \u003c/em\u003egene. \u003cem\u003eN\u003c/em\u003e gene is an ideal target gene because it is a well-conserved gene and a major viral protein that folds and protects the viral RNA, playing a key role in virus replication [14]. The \u003cem\u003eN\u003c/em\u003e gene sequence was used to design primers to develop Taqman-based real-time PCR assay. Both specificity and sensitivity of the assay were assessed and further evaluated with clinical samples alongside the conventional RT-PCR. Ultimately, developing this specific and sensitive assay for FeMV will galvanize the subsequent detailed examination on the significance of this virus in domestic cat populations.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eOptimization of the Taqman-based real-time RT-PCR (qRT-PCR) targeting N gene of FeMV\u003c/h2\u003e\n\u003cp\u003eThe Taqman-based qRT-PCR was optimized by determining the optimal primers and probe concentration of which 0.8 L of 10 M primers and 0.2 L of 10 M probe with a final concentration of 400 nM and 100 nM, respectively were used. Therefore, the final reaction volume of 20 \u0026nbsp;consisted of 10 L 2x SensiFAST Probe No-ROX Mix, 0.8 L of each forward and reverse primers, 0.2 L probe, 4 L converted cDNA template, and 4.2 L nuclease-free water.\u003c/p\u003e\n\u003ch2\u003eSensitivity of Taqman-based real-time RT-PCR (qRT-PCR) targeting N gene of FeMV\u003c/h2\u003e\n\u003cp\u003eThe concentration of the cRNA was 12 ng/L, which was equivalent to 1.74 x \u0026nbsp;copies/L. A series of 10-fold dilution of the cRNA, ranging from 1.74 x \u0026nbsp;to 1.74 x \u0026nbsp;was reversed transcribed and then used to determine the sensitivity of the real-time PCR assay. The lower detection limit based on the cRNA copy number was achieved at 1.74 x \u0026nbsp;copies/L, with a corresponding Cq value of 34.32 \u0026nbsp;0.5 (Figure 1a). The standard curve of the qRT-PCR assay based on the cRNA copy number was plotted to be linear with the coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) of 0.999, the slope of -3.402 and the efficiency of 96.8% (Figure 1b).\u003c/p\u003e\n\u003ch2\u003eSpecificity of Taqman-based real-time RT-PCR (qRT-PCR) targeting N gene of FeMV\u003c/h2\u003e\n\u003cp\u003eSpecificity of the qRT-PCR assay was assessed against the viral strains from the same \u003cem\u003eParamyxoviridae\u003c/em\u003e family, which were the Newcastle disease virus (NDV), canine distemper virus (CDV) and measles virus (MeV), and other feline viruses, such as feline leukemia virus (FeLV) and feline coronavirus (FCoV). None of the RNA viruses showed amplification signals, proving the high specificity of the assay (Figure 2). Besides that, the no-template control did not show any amplification signal.\u003c/p\u003e\n\u003ch2\u003eReproducibility of Taqman-based real-time RT-PCR (qRT-PCR) targeting the N gene of FeMV\u003c/h2\u003e\n\u003cp\u003eThe reproducibility of the qRT-PCR was determined by the intra- and inter-assay evaluation of the Cq value using RNAs from three different positive samples: UPM23, UPM52 and UPM202. The intra-assay was analyzed by using the RNAs from these three samples in triplicates per run whereas for the inter-assay assessment, the RNAs were amplified in triplicates in three different consecutive runs. The calculated SD and CV values for the intra-assay variations ranged from 0.10 to 0.17, and from 0.34% to 0.53%, respectively (Table 1). Meanwhile, for the inter-assay evaluation, the calculated SD and CV values ranged from 0.45 to 0.66 and 1.38% to 2.03%, respectively (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Intra-assay variation\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" width=\"283\"\u003e\n\u003cp\u003e\u003cstrong\u003eCq value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e\u003cstrong\u003eMean Cq\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eS.D.\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eCV%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eReplicate 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003eReplicate 2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003eReplicate 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003eUPM23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e28.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e28.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e27.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e27.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.36\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003eUPM52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e32.92\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e32.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e32.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e32.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.34\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003eUPM202\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e32.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e32.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e32.49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e32.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.53\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 Inter-assay variation\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" width=\"258\"\u003e\n\u003cp\u003e\u003cstrong\u003eCq value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eMean Cq\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eS.D.\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eCV%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003eAssay 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003eAssay 2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003eAssay 3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003eUPM23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e27.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e27.66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e28.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e27.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e1.87\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003eUPM52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e32.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e32.57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e33.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e32.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e1.38\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003eUPM202\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e31.90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e32.44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e33.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e32.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e2.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003e\u003cbr /\u003eClinical samples evaluation using Taqman-based qRT-PCR and comparison with conventional RT-PCR Assay\u003c/h2\u003e\n\u003cp\u003eUrine samples (n = 55) and kidney samples (n = 16) were evaluated to determine the performance of the newly developed qRT-PCR assay (Table 3). Of these 71 samples, 22/55 (40.0%) urine and 3/16 (18.8%) kidney samples were positive for FeMV with log copy number ranging from 3.54 \u0026nbsp;0.01 to 6.60 \u0026nbsp;0.12. Besides that, of the 16 cats that had both kidney and urine samples collected, none of these cats had detectable FeMV RNA in both samples concurrently. For example, the three cats showing FeMV-positive kidney samples (UPM304, UPM312 and UPM321) had no detectable FeMV RNA in their urine samples. Conversely, one of the cats (UPM305) showing detectable FeMV RNA in its urine sample had no FeMV RNA in its kidney samples. In order to determine the sensitivity of Taqman qRT-PCR assay in detecting FeMV from the clinical samples, conventional RT-PCR using two different sets of published primers targeting \u003cem\u003eN\u003c/em\u003e gene [9] was conducted on all 71 samples, of which only 11/55 (20%) urine samples were positive while no (0/16; 0%) kidney samples were detected positive.\u003c/p\u003e\n\u003cp\u003eInterestingly, the cats deemed as healthy with their health status available had a higher positive rate of detection (13/20) compared to the cats diagnosed with a disease (3/10). The source of the cats, whether they came from shelters or were pet cats, might also be important in the FeMV-positive detection rate. A higher percentage of positive detection rate was observed among client-owned cats (46%; 18/39) compared to the shelter cats (43%; 7/16).\u003c/p\u003e\n\u003cp\u003eBiological samples taken from the cats in shelters were also part of other study looking at the presence of the \u003cem\u003ePlatynosomum \u003c/em\u003esp., a hepatic trematode found in the gall bladder and biliary ducts of cats. Incidentally, \u003cem\u003ePlatynosomum \u003c/em\u003esp. was detected in 7 out of 16 (43.8%) shelter cats, of which 4 (57.1%) of them were also FeMV-positive.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 Detection of FeMV using conventional RT-PCR and qRT-PCR quantification from clinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cstrong\u003eSample ID\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003eRT-PCR \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(\u003cem\u003eN\u003c/em\u003e gene)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003eLog copy number (mean\u003c/strong\u003e \u003cstrong\u003e\u0026nbsp;S.D.\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003e\u003cstrong\u003eSource of cat\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003eHealth status \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e6.600.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.940.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.930.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM202\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.770.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM203\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM204\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.860.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM205\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.550.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM207\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.670.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM210\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.660.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM219\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.800.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eCKD stage IV\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM220\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.110.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM221\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.660.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM224\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM225\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.540.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM226\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.710.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM227\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM228\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM229\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM231\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.290.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM232\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eCKD Stage II\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM240\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM241\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM271\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHeart disease\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM272\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eCKD Stage II\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM273\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM274\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eSuspected CKD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM275\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eFURD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM276\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.050.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM277\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM278\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM279\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM280\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eSuspected CKD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM281\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM282\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.630.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eFLUTD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM283\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM284\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eHealthy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.740.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM286\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.880.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eObstructive FLUTD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM287\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eClient-owned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eSuspected CKD\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM304\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM305\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.340.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM306\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.760.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM307\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM308\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM310\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM311\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM312\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM313\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM314\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.450.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM315\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e4.920.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM316\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM317\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM318\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM320\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUrine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM321\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM304\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.800.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM305\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM306\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM307\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM308\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM310\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM311\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM312\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.700.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM313\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM314\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM315\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM316\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cem\u003ePlatynosomum \u003c/em\u003esp.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM317\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM318\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM320\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eKidney\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eUPM321\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e3.890.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"116\"\u003e\n\u003cp\u003eShelter\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBLD denotes below limit of detection of qRT-PCR\u003c/p\u003e\n\u003cp\u003eN/A denotes not available\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFeline morbillivirus (FeMV) has been speculated to cause chronic kidney disease (CKD) and it is commonly detected in urine and kidney tissue samples. Previously, conventional assays such as RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP) have been developed targeting the \u003cem\u003eL\u003c/em\u003e gene of FeMV [4, 6, 7, 15, 16]. However, these conventional assays were qualitative and required additional step of gel electrophoresis to visualize the PCR product. In this study, a two-step Taqman-based qRT-PCR diagnostic assay for the detection of FeMV-\u003cem\u003eN\u003c/em\u003e gene was developed. This quantitative assay was designed to detect the \u003cem\u003eN\u003c/em\u003e gene, one of the most conserved regions in Morbilliviruses. Other quantitative-based assays specifically qRT-PCR has also been developed to detect FeMV, but all were designed to either target the \u003cem\u003eL\u003c/em\u003e gene and \u003cem\u003eP\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e gene [11, 13]. Besides, these qRT-PCR assays had low positive rate of detection which could be due to limited availability of FeMV sequences at that time resulting in less specific primers and probe. Hence, it is crucial to design the primers and probe based on the latest sequences available. In addition, \u003cem\u003eP\u003c/em\u003e gene has been shown to have the highest rates of nucleotide polymorphisms, ranging from 88.6% to 99.3% [9]. Besides that, a study conducted on \u003cem\u003ePeste des petits ruminant virus\u003c/em\u003e (PPRV), another Morbillivirus member revealed that the \u003cem\u003eL\u003c/em\u003e gene had the highest evolution rate at 9.75 x 10\u003csup\u003e-4\u003c/sup\u003e site per year while the \u003cem\u003eN\u003c/em\u003e gene showed the lowest evolution rate at 1.1 x 10\u003csup\u003e-3\u003c/sup\u003e site per year [15].\u003c/p\u003e\n\u003cp\u003eNucleoprotein (\u003cem\u003eN\u003c/em\u003e) comprises of two regions: core domain (\u003cem\u003eN\u003c/em\u003e\u003csub\u003eCORE\u003c/sub\u003e) and C-terminal domain (also known as \u003cem\u003eN\u003c/em\u003e\u003csub\u003eTAIL\u003c/sub\u003e) [16]. \u003cem\u003eN\u003c/em\u003e\u003csub\u003eCORE\u003c/sub\u003e covers the first 400 amino acids of the \u003cem\u003eN\u003c/em\u003e protein, and it is responsible for the RNA encapsidation prior to viral replication and translation. This core domain also consists of three previously identified conserved motifs. \u003cem\u003eN\u003c/em\u003e protein also possesses nuclear export signal, nuclear localization signal (NLS) and RNA binding motifs. Both nuclear export signal and NLS are believed to transport the \u003cem\u003eN\u003c/em\u003e protein to the nucleus of the host cell while the RNA binding site is considered to be involved in \u003cem\u003eN\u003c/em\u003e-\u003cem\u003eN\u003c/em\u003e self-interaction and interaction of \u003cem\u003eN\u003c/em\u003e-\u003cem\u003eN\u003c/em\u003e RNA monomers during the genomic RNA binding. \u003cem\u003eN\u003c/em\u003e-\u003csub\u003eTAIL\u003c/sub\u003e region comprises of structurally variable region of approximately 120 to 150 amino acids which mediate the interaction with \u003cem\u003eP\u003c/em\u003e gene [17]. Besides that, \u003cem\u003eN\u003c/em\u003e gene is integral during the initial infection as the RNA synthesis by RNA-dependent RNA polymerase (RdRp) complex will only recognize and synthesize the viral RNA encapsidated by \u003cem\u003eN\u003c/em\u003e protein as a template [8]. Given that the \u003cem\u003eN\u003c/em\u003e gene is conserved compared to other genes of FeMV and detectable during the early phase of infection, a quantitative assay targeting the \u003cem\u003eN\u003c/em\u003e gene of FeMV was developed in this study. In order to develop this assay, a partial sequence of 1.5kb \u003cem\u003eN\u003c/em\u003e gene was obtained from seven local isolates. Upon obtaining the 1.5kb of FeMV-\u003cem\u003eN\u003c/em\u003e gene sequences of these isolates, primers and probe for Taqman-based qRT-PCR assay were designed specifically to target 122bp of \u003cem\u003eN\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003eFor the development of this assay, a standard curve was generated to accurately quantify FeMV. The sensitivity of the developed assay was done by quantifying 10-fold serial dilutions of standard complementary RNA (cRNA). The results showed the assay could detect up to 10,000 copy number of FeMV. Even though the resulting sensitivity of this newly developed qRT-PCR assay was low, this assay has done none of the other established assays had to date, which was utilizing newly designed primers and probe set targeting \u003cem\u003eN\u003c/em\u003e gene. The specificity test of the assay was performed by running the assay against other viral strains from the same family, which were NDV, CDV and MeV, and two feline viruses, FeLV and FCoV. Absence of cross-reaction proved high specificity of the developed assay. In addition to that, the low sensitivity reported for this assay could be due to the highly specific designed primers based on stringent parameters as evident by the absence of amplification in other tested viruses. In a diagnostic test, a highly specific assay may compromise the sensitivity of the assay, resulting in an inverse relationship [18]. Furthermore, incorporating probe known for its specificity in this assay might also contribute to a highly specific qRT-PCR assay, compromising its sensitivity. In order to evaluate the reproducibility of the assay, intra- and inter-assay assessments were done by testing three different positive samples (UPM23, UPM52 and UPM202). The value of standard deviation calculated for both intra- and inter-assay indicated that the developed qRT-PCR assay was low in variability and had high reproducibility. Besides that, comparing the coefficient of variation (CV) of both intra- and inter-assay with previously reported study, the value of CV recorded in this study was relatively low, confirming the reproducibility of this assay [13].\u003c/p\u003e\n\u003cp\u003eIn order to further evaluate the sensitivity of the qRT-PCR assay, clinical samples (n = 71) were tested to detect FeMV by targeting the \u003cem\u003eN\u003c/em\u003e gene and compared with conventional RT-PCR assay. All samples which were detected positive by the conventional RT-PCR (11/71) were also detected by the qRT-PCR assay. However, there were 15 qRT-PCR FeMV-positive samples that were not detected by the conventional RT-PCR. Some of these samples had copy number of ~1000. Based on the standard curve, even though the resulted lowest limit of detection was ~10,000 copy number, the quantification on clinical samples proved that the newly designed qRT-PCR assay was able to amplify samples which had lower copy number.\u003c/p\u003e\n\u003cp\u003eEvaluation of the clinical samples showed that healthy cats had a higher positive rate compared to cats diagnosed with a disease. Since this study was a cross-sectional study, it was difficult to interpret whether this finding was significant. Nevertheless, a prospective follow up of these cats may provide an insight on the clinical relationship of the cats with FeMV infection. Besides that, a higher rate of detection was observed among client-owned cats compared to shelter cats which was consistent with our previous study, but in contrast with other studies [1, 12]. This observation could be due to the differences in the shelter management among different countries. Shelter cats are usually placed in an enclosed area, limiting their interactions with free-roaming cats, and hence, reducing transmission from one cat to another. Furthermore, the transmission among client-owned cats may occur if the cats are living outdoors or if they are semi-roamer cats as they can interact with other free-roaming cats [19]. Besides that, a multi-cat household may have a higher chance to transmit the virus to other healthy cats already living within the household or newly introduced animals [2]. It was also noted that all cats were not detected positive for both urine and kidney samples as also observed by many studies [3, 20]. This observation may be caused by the early-stage infection, whereby the virus has yet to travel to the kidney. Consequently, this finding would suggest for a further study to observe the viral shedding and specifically, viral load over time accompanied with serum urea and creatinine level among FeMV-positive cats to determine the relationship of the severity of kidney disease and FeMV pathogenesis.\u003c/p\u003e\n\u003cp\u003eThe cats recruited in this study was also part of a study to detect the presence of \u003cem\u003ePlatynosomum \u003c/em\u003esp., a fluke living in pancreatic ducts, bile ducts and gall bladder. This parasite requires two intermediate hosts, a snail and followed by a second intermediate host, such as skink, gecko, lizard or toad. A cat can be infected with \u003cem\u003ePlatynosomum \u003c/em\u003esp. after ingesting an infected second intermediate host [21]. The clinical signs of \u003cem\u003ePlatynosomum \u003c/em\u003esp. infection vary from asymptomatic to progressive disease, and it can also cause death due to hepatic failure and biliary tract obstruction. Biological samples of the liver, bile ducts and feces were obtained from the shelter cats in this study in which \u003cem\u003ePlatynosomum \u003c/em\u003esp. was detected in 4 out of 7 FeMV-positive cats. Although this finding was incidental, co-infection of FeMV and \u003cem\u003ePlatynosomum \u003c/em\u003esp. should be further explored, especially in shelter cat populations given that this cohort of cats could potentially harbor many pathogens.\u003c/p\u003e\n\u003cp\u003eThe developed qRT-PCR assay can be utilized for field samples given its high specificity and novelty of targeting the \u003cem\u003eN\u003c/em\u003e gene of FeMV. Although there has yet a clear indication on whether or not this virus is involved in the pathogenesis of chronic kidney disease in cats, further studies are needed to determine the extent of infection among cats and its clinical significance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, a probe-based qRT-PCR assay targeting the \u003cem\u003eN\u003c/em\u003e gene of FeMV has been successfully optimized and validated. This study is the first quantitative assay targeting the FeMV-\u003cem\u003eN\u003c/em\u003e gene which is integral in RNA synthesis by RNA-dependent RNA polymerase (RdRp) complex. Even though the developed assay demonstrated a low sensitivity compared to previously designed qRT-PCR assay targeting \u003cem\u003eL\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e gene, compared to the conventional RT-PCR, it can detect samples that has low viral load. Hence, the developed qRT-PCR assay can be applied in diagnostic and quantification of the FeMV viral load, especially during the early stage of infection.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003eTotal RNA extraction and cDNA synthesis\u003c/h2\u003e\n\u003cp\u003eThe total RNA extraction for collected samples was performed by using Direct-zol\u003csup\u003e\u0026trade;\u003c/sup\u003e RNA MiniPrep Plus kit (Zymo Research, California, United States) following the manufacturer\u0026rsquo;s guidelines. Extracted RNA was first subjected to cDNA synthesis prior to conventional RT-PCR and qRT-PCR assays. cDNA synthesis was performed by using the SensiFAST\u003csup\u003e\u0026trade;\u003c/sup\u003e cDNA Synthesis Kit (Bioline, London, UK) with 20 L reaction consisting of 10 L purified RNA, 4 L of 5x TransAmp Buffer, 1 L reverse transcriptase and 5 L nuclease-free water. The reaction was carried out at 25\u0026nbsp;for 10 mins, 42\u0026nbsp;for 15 mins, 48\u0026nbsp;for 15 mins and 85\u0026nbsp;for 5 mins. The cDNA was stored in -20\u0026nbsp;until use.\u003c/p\u003e\n\u003ch2\u003eTaqman-based real-time RT-PCR (qRT-PCR) assay\u003c/h2\u003e\n\u003cp\u003ePrimer set and probe were designed based on partial \u003cem\u003eN\u003c/em\u003e gene sequences of FeMV-Malaysia isolates (UPM23, UPM52, UPM53, UPM210, UPM231, UPM305 and UPM315) using Integrated DNA Technologies (IDT) software (Table 4). The designed probe and primer set were then compared with the alignment of closely-related FeMV from other countries\u0026rsquo; isolates: Japan (SS3, MiJP003, ChJP073), China (M252A) and Thailand (Thai-U16) (Table 4). Specific sequences for primers and probe used were as follows to yield 122bp PCR amplicons: forward primer, GGTCAAGAGATGGTGAGAAGAT; reverse primer, CCAGATTCACCTCCCGAATTA; and probe, TTTGCGCGAGAACTTGGGCTATCT. The FeMV Taqman probe was labeled with 6-FAM at 5\u0026rsquo;-end, and fluorescence quencher, ZEN and IBFQ at an internal site and 3\u0026rsquo;-end, respectively. Real-time RT-PCR was performed using CFX96 machine (Bio-Rad, California, USA) utilizing SensiFAST\u003csup\u003eTM\u003c/sup\u003e Probe NO-ROX Kit (Bioline, London, UK) following the manufacturer\u0026rsquo;s suggestion. Briefly, each reaction well was consisted of 4 L cDNA, 10 L of 2x SensiFAST Probe No-ROX Mix, a final concentration of 400 nM for each forward and reverse FeMV primers, 100 nM probe and nuclease-free water up to the final volume of 20 L. The assay was performed at 95\u0026nbsp;for 2 mins, followed by 35 cycles of 95\u0026nbsp;for 15 secs and 54\u0026nbsp;for 45 secs and each sample was run in triplicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4 GenBank accession number \u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003e\u003cstrong\u003eIsolate\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003e\u003cstrong\u003eAccession Number\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eSS3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eLC036587\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMiJP003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eAB924121\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eChJP073\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eAB924122\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eM252A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eJQ411016\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eThai-U16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMF627832\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN264638\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN264639\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN264640\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM210\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN264641\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM231\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN264642\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM305\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN792827\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eUPM315\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"301\"\u003e\n\u003cp\u003eMN792828\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003e\u003cbr /\u003eStandard RNA preparation\u003c/h2\u003e\n\u003cp\u003eGeneration of standard RNA was prepared by sub-cloning the partial sequence of 1.5kb FeMV-\u003cem\u003eN\u003c/em\u003e gene into a pSC-A-amp/kan plasmid vector by using StrataClone PCR cloning kit (Agilent Technologies, California, United States) according to the manufacturer\u0026rsquo;s instruction. After linearization, RNA transcription was performed using the Riboprobe\u003csup\u003e\u0026reg;\u003c/sup\u003e \u003cem\u003ein vitro\u003c/em\u003e Transcription Systems (Promega, Wisconsin, United States) with the T7 RNA polymerase promoter site available in vector pSC-A-amp/kan following the manufacturer\u0026rsquo;s direction. After DNase treatment, the transcript was purified by phenol:chloroform purification and quantified by using spectrophotometer (Bio-Rad, California, USA). The copy number of cRNA calculated was 1.74 x 10\u003csup\u003e11\u003c/sup\u003e copies/L. cRNA was diluted in 10-fold serial dilution and converted to cDNA prior to standard curve generation.\u003c/p\u003e\n\u003ch2\u003eSpecificity and sensitivity\u003c/h2\u003e\n\u003cp\u003eThe specificity of the developed assay was assessed by testing on viral RNA from same genus, which were canine distemper virus (CDV) and measles virus (MeV), from same family, Newcastle disease virus (NDV), and from other feline viruses, which were feline leukemia virus (FeLV) and feline coronavirus (FCoV) (Table 5).\u003c/p\u003e\n\u003cp\u003eThe sensitivity of qRT-PCR assay was assessed by running 10-fold serial dilution of cRNA standard (1.74 x 10\u003csup\u003e11\u003c/sup\u003e to 1.74 x 10\u003csup\u003e2\u003c/sup\u003e copies/L) to detect the threshold limit of the assay. Ten different 10-fold serial dilution was performed by adding 1 L of RNA into 9 L of nuclease-free water. The mixture was then vortexed for at least 10 secs before the same step was repeated for the next dilution. Each of the diluted RNA along with the stock RNA control was converted into cDNA and subjected for qRT-PCR in triplicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5 List of viruses used in this study and its sources\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003e\u003cstrong\u003eVirus\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"374\"\u003e\n\u003cp\u003e\u003cstrong\u003eSources\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eCanine distemper virus (CDV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"374\"\u003e\n\u003cp\u003eNobivac Puppy DP, Intervet\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eMeasles virus (MeV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"374\"\u003e\n\u003cp\u003eSerum Institute of India LTD, Pune\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eNewcastle disease virus (NDV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"374\"\u003e\n\u003cp\u003eDepartment of Vet. Pathology and Microbiology, UPM\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eFeline leukemia virus (FeLV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"374\"\u003e\n\u003cp\u003eDepartment of Vet. Pathology and Microbiology, UPM\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"227\"\u003e\n\u003cp\u003eFeline coronavirus (FCoV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"374\"\u003e\n\u003cp\u003eDepartment of Vet. Pathology and Microbiology, UPM\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003e\u003cbr /\u003eReproducibility\u003c/h2\u003e\n\u003cp\u003eIn order to assess reproducibility of the developed assay, three different positive samples were selected which were then subjected for intra- and inter-assay. Three different positive samples (UPM23, UPM52, UPM202) were assayed in the same run in triplicates to evaluate the intra-assay variations. In order to assess for inter-assay variations, the same three samples were subjected for three different consecutive runs in triplicates. Values for mean, standard deviation (SD) and coefficient of variations (CV) for both variation assays were calculated by using Microsoft Excel Software (version 2016, USA).\u003c/p\u003e\n\u003ch2\u003eClinical samples collection\u003c/h2\u003e\n\u003cp\u003eAnimal ethics application was approved by the Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia (UPM/IACUC/AUP-R037/2018). Convenient sampling was performed whereby urine (n = 55) and kidney samples (n = 16) were collected from veterinary hospital, private veterinary clinics around Klang Valley, Malaysia and animal shelters. The cats presented to the veterinary hospital or private veterinary clinics were either presented for annual health examination, neutering procedure or having health-related issues, such as kidney-related and heart diseases. Cats\u0026rsquo; samples which their serum urea-creatinine data were available were further sub-grouped into cats with presence or absence of kidney-related disease based on the International Renal Interest Society (IRIS) Guidelines. The owner\u0026rsquo;s consent was requested prior to samples collection. When available, kidney samples (n = 16) from post-mortem and corresponding urine samples (n = 16) were collected from animal shelters.\u003c/p\u003e\n\u003ch2\u003eSample processing\u003c/h2\u003e\n\u003cp\u003eA urine sample was collected into a sterile sample collection bottle either by cystocentesis or manual compression. The supernatant of urine was obtained after a centrifugation step at 2320 x g for 5 mins. Then, the supernatant was mixed with RNAlater\u003csup\u003e\u0026reg;\u003c/sup\u003e solution (Ambion, Texas, United States) at ratio 1:1 and stored at -20\u0026nbsp;prior to RNA extraction. During postmortem, the collected kidney samples were immediately transferred into a sterile collection bottle containing RNAlater\u003csup\u003e\u0026reg;\u003c/sup\u003e solution. For sample processing, kidney tissues of approximately 1 g were cut into small pieces and crushed by using the pestle and mortar along with sterile sand. Phosphate-buffer saline solution (Gibco, Massachusetts, United States) of 1g/mL was added into the homogenized kidney. Then, the mixture was transferred into a 15 mL tube to be centrifuged at 2320 x g for 5 mins to remove any large debris and sand. The kidney lysate was then subjected for total RNA extraction.\u003c/p\u003e\n\u003ch2\u003eClinical samples evaluation using Taqman-based qRT-PCR and conventional RT-PCR\u003c/h2\u003e\n\u003cp\u003eConverted cDNA clinical samples were assessed by conventional RT-PCR using published primers (Table 6) with cDNA of UPM52 as a positive control together with no-template control following a modified protocol (Table 7).\u003c/p\u003e\n\u003cp\u003eTaqman-based qRT-PCR was performed according to the developed protocol described above with each sample ran in triplicates along with NTC and cDNA of positive control (UPM52). Nuclease-free water (Promega, Wisconsin, United States) was used as the template in NTC in both conventional RT-PCR and qRT-PCR assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6 Primer sequences used to amplify two different regions of \u003cem\u003eN\u003c/em\u003e gene\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e\u003cstrong\u003eRegion\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e\u003cstrong\u003ePrimer\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"264\"\u003e\n\u003cp\u003e\u003cstrong\u003eSequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u003cstrong\u003eProduct size (bp)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"71\"\u003e\n\u003cp\u003e\u003cstrong\u003eMiddle region\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eFN-2F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"264\"\u003e\n\u003cp\u003eGTTAGCTTAGGATTTGAGAACCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"94\"\u003e\n\u003cp\u003e680bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" width=\"85\"\u003e\n\u003cp\u003e[9]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eFN-2R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"264\"\u003e\n\u003cp\u003eCACCATCTCTTGACCAAGTCT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"71\"\u003e\n\u003cp\u003e\u003cstrong\u003eEnd region\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eFN-3F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"264\"\u003e\n\u003cp\u003eGCTATGGAGTTATGCCATGGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"94\"\u003e\n\u003cp\u003e637bp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003eFN-3R\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"264\"\u003e\n\u003cp\u003eGTTGTGAACCTTGAGGTCCTAAG\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr /\u003eTable 7 PCR protocol applied for two different primer sets of \u003cem\u003eN\u003c/em\u003e gene \u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eStep\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"246\"\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003eCycle\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eInitial denaturation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"246\"\u003e\n\u003cp\u003e95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e1 min\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e1x\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eDenaturation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnealing\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"246\"\u003e\n\u003cp\u003e95\u003c/p\u003e\n\u003cp\u003e58\u003c/p\u003e\n\u003cp\u003e72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e15 secs\u003c/p\u003e\n\u003cp\u003e1 min\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e35x\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eExtension\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e1 min\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eFinal extension\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"246\"\u003e\n\u003cp\u003e72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e5 mins\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e1x\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eHold\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"246\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e1x\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003e\u003cbr /\u003ePlatynosomum sp. detection from postmortem of shelter cats\u003c/h2\u003e\n\u003cp\u003eLiver, bile duct and faeces samples in rectum were collected in the postmortem investigation of shelter cats to detect the presence of \u003cem\u003ePlatynosomum sp.\u003c/em\u003e Liver samples were used to collect adult fluke while bile duct and faeces samples were used to detect ova. In order to allow for activation and collection of mature flukes, liver samples were extracted and immersed in warm water (38-40). Parasitic burden was calculated using the formula described in a previous study by applying the number of adult flukes collected per samples [22]. For ova collection from bile juice and faeces samples, any ova isolated from these clinical samples were pipetted into microcentrifuge tubes with normal saline and they were stored in -20\u0026nbsp;for further analysis. In faecal examination, two different methods were performed to detect fluke eggs: the simple floatation technique and centrifugal faecal sedimentation test in formal-ether solution [23, 24]. Adult fluke and ova were identified based on a previous study [25].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eqRT-PCR Quantitative reverse transcription polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eFeMV Feline morbillivirus\u003c/p\u003e\n\u003cp\u003eCDV Canine distemper virus\u003c/p\u003e\n\u003cp\u003eNDV Newcastle disease\u003c/p\u003e\n\u003cp\u003eMeV Measles virus\u003c/p\u003e\n\u003cp\u003eFCoV Feline coronavirus\u003c/p\u003e\n\u003cp\u003eFeLV Feline leukemia virus\u003c/p\u003e\n\u003cp\u003eRT-PCR Reverse transcription polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eM Matrix protein\u003c/p\u003e\n\u003cp\u003eF Fusion protein\u003c/p\u003e\n\u003cp\u003eH Hemagglutinin protein\u003c/p\u003e\n\u003cp\u003eL Large protein\u003c/p\u003e\n\u003cp\u003eP Phosphoprotein\u003c/p\u003e\n\u003cp\u003eN Nucleocapsid protein\u003c/p\u003e\n\u003cp\u003eFURD Feline upper respiratory disease\u003c/p\u003e\n\u003cp\u003eFLUTD Feline lower urinary tract disease\u003c/p\u003e\n\u003cp\u003eIDT Integrated DNA Technologies\u003c/p\u003e\n\u003cp\u003eIACUC Institutional animal care and use committee\u003c/p\u003e\n\u003cp\u003eUVH University Veterinary Hospital\u003c/p\u003e\n\u003cp\u003eIRIS International Renal Interest Society\u003c/p\u003e\n\u003cp\u003eRdRp RNA-dependent RNA polymerase\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics Approval and Consent to participate\u003c/h2\u003e\n\u003cp\u003eAnimal ethics application was approved from the Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia (UPM/IACUC/AUP-R037/2018) prior to sample collection. Informed consent was obtained from the cats\u0026rsquo; owners prior to sample collection. Hence, the use of cats in this study met the criteria of animal welfare regulations in Malaysia.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed in this study can be obtained within the tables and figures of the manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was supported by the Universiti Putra Malaysia internal grant (Grant No: GP-IPS/2017/9536900). The funding body has no specific role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eFMK contributed to the conception, design and revising the manuscript. STM contributed to the data collection, laboratory work, drafting and writing the manuscript. ARO and TSW contributed to the conception, data analyses and revising the manuscript. NAAA and NAN contributed to the data collection, laboratory work and revising the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Dr. Zarirah Mohamed Zulperi for technical assistance and troubleshooting on the plasmid cloning and standard RNA preparation work. We would like to also thank all participating veterinary clinics, veterinary hospital and animal shelters especially the cats and their owners for allowing us to perform sample collection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTechangamsuwan SCaS: \u003cstrong\u003eFirst evidence of Feline morbillivirus detected in sheltered cats in Thailand\u003c/strong\u003e. \u003cem\u003eThai J Vet Med Suppl \u003c/em\u003e2017, \u003cstrong\u003e47\u003c/strong\u003e:127-128.\u003c/li\u003e\n\u003cli\u003eDarold GM, Alfieri AA, Muraro LS, Amude AM, Zanatta R, Yamauchi KC, Alfieri AF, Lunardi M: \u003cstrong\u003eFirst report of feline morbillivirus in South America\u003c/strong\u003e. \u003cem\u003eArch Virol \u003c/em\u003e2017, \u003cstrong\u003e162\u003c/strong\u003e(2):469-475.\u003c/li\u003e\n\u003cli\u003eYilmaz H, Tekelioglu BK, Gurel A, Bamac OE, Ozturk GY, Cizmecigil UY, Altan E, Aydin O, Yilmaz A, Berriatua E\u003cem\u003e et al\u003c/em\u003e: 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morbillivirus in cat urine samples\u003c/strong\u003e. \u003cem\u003eJ Vet Med Sci \u003c/em\u003e2016, \u003cstrong\u003e77\u003c/strong\u003e(12):1701-1703.\u003c/li\u003e\n\u003cli\u003eMohd Isa NH, Selvarajah GT, Khor KH, Tan SW, Manoraj H, Omar NH, Omar AR, Mustaffa-Kamal F: \u003cstrong\u003eMolecular detection and characterisation of feline morbillivirus in domestic cats in Malaysia\u003c/strong\u003e. \u003cem\u003eVet Microbiol \u003c/em\u003e2019, \u003cstrong\u003e236\u003c/strong\u003e:108382.\u003c/li\u003e\n\u003cli\u003eDe Luca E, Crisi PE, Di Domenico M, Malatesta D, Vincifori G, Di Tommaso M, Di Guardo G, Di Francesco G, Petrini A, Savini G\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eA real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens\u003c/strong\u003e. \u003cem\u003eJ Virol Methods \u003c/em\u003e2018, \u003cstrong\u003e258\u003c/strong\u003e:24-28.\u003c/li\u003e\n\u003cli\u003eDiallo A: \u003cstrong\u003eMorbillivirus group genome organisation and protein.pdf\u0026gt;\u003c/strong\u003e. \u003cem\u003eVeterinary Microbiology \u003c/em\u003e1990, \u003cstrong\u003e23\u003c/strong\u003e:155-163.\u003c/li\u003e\n\u003cli\u003eShabbir MZ, Rahman AU, Munir M: \u003cstrong\u003eA comprehensive global perspective on phylogenomics and evolutionary dynamics of Small ruminant morbillivirus\u003c/strong\u003e. \u003cem\u003eSci Rep \u003c/em\u003e2020, \u003cstrong\u003e10\u003c/strong\u003e(1):17.\u003c/li\u003e\n\u003cli\u003eThakkar VD, Cox RM, Sawatsky B, da Fontoura Budaszewski R, Sourimant J, Wabbel K, Makhsous N, Greninger AL, von Messling V, Plemper RK: \u003cstrong\u003eThe Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity\u003c/strong\u003e. \u003cem\u003eJ Virol \u003c/em\u003e2018, \u003cstrong\u003e92\u003c/strong\u003e(8).\u003c/li\u003e\n\u003cli\u003eCommunie G, Ruigrok RW, Jensen MR, Blackledge M: \u003cstrong\u003eIntrinsically disordered proteins implicated in paramyxoviral replication machinery\u003c/strong\u003e. \u003cem\u003eCurr Opin Virol \u003c/em\u003e2014, \u003cstrong\u003e5\u003c/strong\u003e:72-81.\u003c/li\u003e\n\u003cli\u003eVetter TR, Schober P, Mascha EJ: \u003cstrong\u003eDiagnostic Testing and Decision-Making: Beauty Is Not Just in the Eye of the Beholder\u003c/strong\u003e. \u003cem\u003eAnesth Analg \u003c/em\u003e2018, \u003cstrong\u003e127\u003c/strong\u003e(4):1085-1091.\u003c/li\u003e\n\u003cli\u003ePark ES, Suzuki M, Kimura M, Mizutani H, Saito R, Kubota N, Hasuike Y, Okajima J, Kasai H, Sato Y\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eEpidemiological and pathological study of feline morbillivirus infection in domestic cats in Japan\u003c/strong\u003e. \u003cem\u003eBMC Vet Res \u003c/em\u003e2016, \u003cstrong\u003e12\u003c/strong\u003e(1):228.\u003c/li\u003e\n\u003cli\u003eStranieri A, Lauzi S, Dallari A, Gelain ME, Bonsembiante F, Ferro S, Paltrinieri S: \u003cstrong\u003eFeline morbillivirus in Northern Italy: prevalence in urine and kidneys with and without renal disease\u003c/strong\u003e. \u003cem\u003eVet Microbiol \u003c/em\u003e2019, \u003cstrong\u003e233\u003c/strong\u003e:133-139.\u003c/li\u003e\n\u003cli\u003eJavinsky E: \u003cstrong\u003eChapter 23 - Digestive System, Liver, and Abdominal Cavity\u003c/strong\u003e. In: \u003cem\u003eThe Cat.\u003c/em\u003e edn. Edited by Little SE. Saint Louis: W.B. Saunders; 2012: 425-546.\u003c/li\u003e\n\u003cli\u003eMargolis L, W.Esch G, Holmes JC, Kuris AM, Schad GA: \u003cstrong\u003eThe Use of Ecological Terms in Parasitology (Report of an Ad Hoc Committee of the American Society of Parasitologists)\u003c/strong\u003e. \u003cem\u003eThe Journal of Parasitology \u003c/em\u003e1982, \u003cstrong\u003e68\u003c/strong\u003e(1):131-133.\u003c/li\u003e\n\u003cli\u003eConboy AMZaGA: \u003cstrong\u003eVeterinary Clinical Parasitology\u003c/strong\u003e, 8th edn. Ames, Iowa: Blackwell Publishing Professional; 2012.\u003c/li\u003e\n\u003cli\u003eRitchie LS: \u003cstrong\u003eAn ether sedimentation technique for routine stool examinations\u003c/strong\u003e. \u003cem\u003eBull U S Army Med Dep \u003c/em\u003e1948, \u003cstrong\u003e8\u003c/strong\u003e(4):326.\u003c/li\u003e\n\u003cli\u003eRA B, DI G, A J: \u003cstrong\u003eKeys to the Trematode\u003c/strong\u003e, vol. 3. Wallingford, UK and the Natural History Museum, London: CABI Publishing; 2008.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Feline Morbillivirus, Taqman-based real-time RT-PCR, N gene","lastPublishedDoi":"10.21203/rs.3.rs-121500/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-121500/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eFeline morbillivirus (FeMV) is a member of genus Morbillivirus which has been associated with the chronic kidney disease in cats even though a definite relationship is still unclear. Morbilliviruses are associated with severe diseases such as Peste des petits ruminants, canine distemper and measles. FeMV has been detected in many countries including Malaysia. This study aims to develop a Taqman real-time RT-PCR (qRT-PCR) assay targeting the \u003cem\u003eN\u003c/em\u003e gene of FeMV in clinical samples to detect early phase of FeMV infection.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eA specific assay was developed since no amplification was observed in viral strains from the same \u003cem\u003eParamyxoviridae\u003c/em\u003e family, such as canine distemper virus (CDV), Newcastle disease virus (NDV) and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 x 10\u003csup\u003e4\u003c/sup\u003e copies/\u003c/p\u003e\u003cp\u003eL with Cq value of 34.32 \u003c/p\u003e\u003cp\u003e\u0026nbsp;0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34% - 0.53% and 1.38% - 2.03%, respectively. Besides that, the clinical sample evaluation using this assay showed a higher detection rate, with 26 (37%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe Taqman-based real-time RT PCR assay targeting the \u003cem\u003eN\u003c/em\u003e gene described here is more sensitive, specific, rapid and reproducible compared to the conventional RT-PCR assay targeting the \u003cem\u003eN\u003c/em\u003e gene and it could be used to detect early infection in cats.\u003c/p\u003e","manuscriptTitle":"Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-10 16:54:00","doi":"10.21203/rs.3.rs-121500/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-12-30T04:56:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-29T03:28:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e9269435-3834-47c0-a71c-1e3de87d0335","date":"2020-12-29T00:31:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-21T17:35:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1617a855-625d-40f2-936b-1423d0c9b9e2","date":"2020-12-14T12:32:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-11T04:33:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-11T04:01:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-07T14:33:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-07T08:51:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2020-12-04T03:35:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"059d8266-f921-4ebe-b577-6b326e443bb5","owner":[],"postedDate":"December 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1422171,"name":"Small Animal Medicine"}],"tags":[],"updatedAt":"2021-03-29T15:00:45+00:00","versionOfRecord":{"articleIdentity":"rs-121500","link":"https://doi.org/10.1186/s12917-021-02837-6","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2021-03-23 15:00:30","publishedOnDateReadable":"March 23rd, 2021"},"versionCreatedAt":"2020-12-10 16:54:00","video":"","vorDoi":"10.1186/s12917-021-02837-6","vorDoiUrl":"https://doi.org/10.1186/s12917-021-02837-6","workflowStages":[]},"version":"v1","identity":"rs-121500","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-121500","identity":"rs-121500","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}