A crRNA/Cas12a complex-driven rapid and visual detection method for four porcine diarrhea viruses

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This study introduces a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, with a molecular weight of approximately 144 kDa, enhances diagnostic accuracy through targeted gene editing. Incorporating fluorescence report probes and a lateral flow dipstick assay, this approach establishes a visual detection system capable of simultaneously identifying all four viruses. Results It enables the visualization of viral genomes from as low as 1 to 10 copies/µL without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.94% concordance with RT-qPCR for PoRV G9. Conclusions Offering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its critical role in enhancing biosecurity and disease management in veterinary contexts. Four porcine diarrhea viruses crRNA/Cas12a complex multiple LAMP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background In recent years, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) pandemic has triggered a global public health crisis with profound impact on economies, societies, and individuals [ 1 ]. Similarly, the widespread outbreaks of porcine corona-virus, causing neonatal diarrhea in piglets, have severely affected the swine industry, raising significant concerns about food safety. Notably, PEDV, TGEV, and PDCoV have spread worldwide, greatly reducing pork production efficiency and destabilizing the economic foundations of animal husbandry [ 2 ]. Four major prevalent porcine diarrhea viruses, PEDV and TGEV (both classified as α-coronavirus), PDCoV, and PoRV, are known to cause significant outbreaks [ 3 ]. PEDV, TGEV and PDCoV are single-stranded positive-sense RNA viruses with similar capsid structures. Their genomes include genes ORF1a, ORF1b, and ORF2 ~ ORF6, which are sequentially arranged and encode two nonstructural replicase proteins, four structural proteins: membrane protein (M), spike glycoprotein (S), envelope protein (E) and nucleocapsid protein (N), and a nonstructural helper protein encoded by ORF3, respectively [ 4 , 5 ]. PDCoV is unique among these vi-ruses due to its helper proteins NS6 and NS7/NS7a, located between the structural protein-coding genes, which may increase its zoonotic potential [ 6 ]. The highly con-served nature of the N and M coding genes makes them common targets for nucleic acid detection [ 7 ]. PoRV, a member of the Reoviridae family and Rotavirus genus, is a double-stranded RNA virus comprising 11 dsRNA segments that encode distinct proteins. These include six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and five non-structural proteins (NSP1-NSP5) [ 8 ]. PoRV was first reported in China in the 1980s after being isolated from pig diarrhea samples, confirming its presence in domestic swine populations [ 9 ]. PoRV genotypes are primarily classified based on the variations in the VP7 glycoprotein of the outer shell, with at least 18 identified G types. The P types are determined by sequence differences in the VP4 phosphatase functional region and include 6 distinct variants, resulting in at least 11 reported G-P combinations [ 10 ]. This complex serology and diverse genotypes of PoRV pose challenges for detection and control. Co-infection and secondary infections involving PEDV, TGEV, PDCoV, and PoRV are common, producing similar clinical signs, primarily characterized by watery diarrhea, which complicates clinical diagnosis [ 3 ]. Current detection methods for these four viruses fall into two categories: molecular biology techniques and serological diagnostic methods. These include virus isolation and electron microscopy, reverse transcription polymerase chain reaction (RT-PCR) [ 11 ], reverse transcription real-time quantitative PCR (RT-qPCR)[ 3 , 12 ], reverse transcription loop-mediated isothermal amplification (RT-LAMP)[ 13 – 16 ], immunofluorescence assay (IFA)[ 17 – 20 ], immunohistochemistry (IHC)[ 21 – 23 ], and enzyme-linked immunosorbent assay (ELISA)[ 24 – 27 ]. While these diagnostic methods are widely employed, molecular biology techniques often face limitations for real-time, on-site testing due to high equipment costs, complex operational procedures, the need for specialized personnel, and issues with sample processing and data analysis. Serological methods are constrained by longer detection times, reflect only the presence of antibodies rather than active infections, and their sensitivity and specificity may be influenced by immune status and viral mutations; moreover, they cannot distinguish whether elevated antibody levels result from natural infection with wild-type strains or from vaccination. The CRISPR/Cas system has recently emerged as a next-generation technology for pathogen or nucleic acid detection. This system, utilizing effector proteins such as Cas9, Cas12a, Cas12b, and Cas13, holds significant promise in diagnostics [ 28 ]. For instance, Pardee et al. developed a low-cost, paper-based sensor combining CRISPR/Cas9 technology with RNA amplification to distinguish Zika virus subtypes in 2017[ 29 ]. Zhang et al. created a detection system using dCas9 with a luciferase reporter for Mycobacterium tuberculosis [ 30 ], while Zhang Feng’ group integrated Cas13a with recombinase polymerase amplification (RPA) to develop the SHERLOCK system, a highly specific and sensitive detection platform capable of detecting single-copy viral genomes [ 31 ]. Beyond its genome-editing capability, CRISPR/Cas has demonstrated significant potential in nucleic acid detection. For example, Li et al. combined CRISPR/Cas12b with PCR to create HOLMES v2, a rapid and sensitive detection system that proved more effective and convenient than RT-qPCR and ELISA [ 32 ]. Similarly, CRISPR/Cas systems have been successfully applied to detect various porcine viruses, including porcine circovirus type 2 (PCV2) [ 33 ], porcine reproductive respiratory syndrome virus (PRRSV)[ 34 ], African swine fever virus (ASFV)[ 35 ], pseudorabies virus (PRV)[ 36 ], PEDV[ 37 ], and porcine parvovirus (PPV)[ 38 ]. CRISPR/Cas-based detection systems are characterized by high sensitivity, simplicity, and rapid response times, making them well-suited for on-site applications. This study integrates the advantages of loop-mediated isothermal amplification (LAMP), which does not require specialized equipment, with the collateral cleavage activity of huLbCas12a (namely, the capability to cleave fluorophore-quenched sin-gle-stranded DNA probe sensors) to establish a novel, visual, and effective method for simultaneously detecting four major porcine diarrhea viruses. Furthermore, the relia-bility and priority of this newly developed approach were confirmed with clinical samples and compared against traditional RT-(q)PCR method. Methods Viruses and clinical samples The cDNA of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus (PoRV) is preserved in the Veterinary Protein Engineering Vaccine Key Laboratory of Hunan Agricultural University. A total of 95 samples from different pig farms in Hunan, including fecal samples and rectal swabs, are stored at -80°C for future use. Design and preparation of LAMP primers, crRNA and probes Sequences of different subtypes of PEDV, TGEV, PDCoV, and PoRV G9 viruses were collected for analysis and comparison (Figure S1 ). Using the PrimerExplorer V5.0 online software and referring to the instructions on the website, LAMP primers for PEDV, TGEV, PDCoV, and PoRV G9 were designed. Conservative regions of the PEDV N gene, TGEV N gene, PDCoV N gene , and PoRV G9 VP7 gene were used to design detection sequences of 20–23 bp following the PAM using the CRISPR-DT online software ( http://bioinfolab.maimioh.edu ). The PoRV G9 VP7 gene sequence "AGTTGATGCTTCAGTAGG" is the only one available for use as a detection site. A T7 promoter-binding crRNA sequence was designed, enabling T7 polymerase (New England Biolabs) to recognize the annealed crRNA DNA duplex and perform in vitro transcription to produce a large amount of sgRNA. Based on the non-specific cleavage characteristics of huLbCas12a protein, an ssDNA probe rich in "A base" was designed, using FAM fluorescent group and BHQ1 quenching group, resulting in the FAM-BHQ1-ssDNA probe 1 . The above sequence is shown in Table S1 . All primers were synthesized by Tsingke (Beijing, China). Optimization of the CRISPR/Cas12a detection system combined with FAM-BHQ1-ssDNA probe The standard plasmids for PEDV N gene, TGEV N gene, PDCoV N gene, and PoRV G9 VP7 gene, as well as Cas12a protein, were prepared and stored by the Hunan Provincial Key Laboratory of Veterinary Protein Engineering Vaccine. The formula for calculating the number of copies is as follows: copy number = (Amount × 6.02 × 10 23 )/ (DNA length × 10 9 × 660 Da/bp). The number of copies of the standard plasmid was calculated according to the above formula and diluted as the detection template. The complete CRISPR-based 20 µL detection system includes Cas12a protein, sgRNA, 2 µM ssDNA-FQ reporter gene, 10× 2.1 NEBuffer (NEB, USA) and detection templates. This experiment optimized the components of the CRISPR system. In previous studies, the efficiency of the huLbCas12a-crRNA binary complex in targeting the desired sequence was related to the type and length of the crRNA codons. Therefore, a 20 bp sequence following PAM was selected as crRNA. Three crRNAs targeting conserved sequences were designed into nine groups for screening: crRNA1–9 (Table S1 ), with each group including an experimental and a control group. PoRV G9 VP7 has only one target site and thus was not screened. Additionally, the optimal ratio of huLbCas12a to crRNA was determined. Five control groups were designed for optimizing the ratio of huLbCas12a to crRNA, including positive and negative controls for each: 2:1 (250:125 nM), 1:1 (250:250 nM), 1:2 (250:500 nM), 1:3 (250:750 nM), and 1:4 (250:1000 nM). The other components were consistent with the CRISPR/Cas12a system. Each group was performed in triplicate, and the system was prepared in an eight-well tube. Real-time fluorescence data were collected using the ABI QuantStudio 5 RT-qPCR instrument. After the reaction, samples were observed under a gel imaging system with UV light. Optimization and evaluation of the LAMP To achieve efficient and high-yield enrichment of target sequences for four pig diarrhea viruses, this study optimized the components of the LAMP system. Each 25 µl LAMP reaction mixture includes: 2× LAMP Polymerase Buffer, 0.48 U Bst 3.0 DNA Polymerase, inner primers, outer primers, loop primers, 6 mM MgSO₄, 1.4 mM dNTP Mix, and target template. Evaluation and optimization of the LAMP system for detecting PEDV, TGEV, PDCoV, and PoRV G9 based on CRISPR/Cas12a are as follows: prepare the reaction mixture as described, adding 2.0 µL of DNA template and 3.5 µL of distilled water, then mixed and centrifuged. Optimize the final concentration ratio of inner to outer primers by setting the outer primer concentration to 0.2 mM as 1. Establish ratios of 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12, corresponding to inner primer concentrations of 0.4 µM, 0.8 µM, 1.2 µM, 1.6 µM, 2.0 µM, and 2.4 µM, respectively, with loop primers at a final concentration of 0.4 µM. The LAMP reaction was conducted at LAMP reactions were performed at varying temperatures (58, 60, 62, 63, 64, 65°C) and time intervals (30, 35, 40, 45, 50, 55, 60 minutes). Negative controls consisted of reactions conducted without nucleic acid templates. The optimization results indicate that the optimal reaction temperatures for the LAMP systems of PEDV, TGEV, PDCoV, and PoRV G9 are 63°C and 64°C. As time progresses, the brightness of the LAMP amplification product bands on the electrophoresis gel increases, indicating that the quantity of LAMP products also increases over time. The optimal reaction temperatures for gel electrophoresis bands are highlighted with red boxes (Figure S2). Results show that the LAMP reaction systems for PEDV, TGEV, and PoRV G9 reach maximum band brightness at 50 minutes, indicating that the LAMP reaction is complete; whereas the PDCoV LAMP system reaches completion at 45 minutes, suggesting slightly higher efficiency compared to the others (Figure S3). The optimization of the final concentrations of outer and inner primers is shown in Figure S4. Figure S4A, B, and C indicate that within the ratio range of 1:1 to 1:12 for outer to inner primers, the LAMP system performs adequately. As the ratio increases, the amplification effect becomes more pronounced until the template reaction is complete in the system. The brightest bands on the gel electrophoresis are observed at a ratio of 1:6 (0.2 µM:1.2 µM), indicating that the optimal outer to inner primer concentration ratio for PEDV, TGEV, and PDCoV LAMP systems are 0.2 µM:1.2 µM. In contrast, Figure S4D shows that no LAMP amplification product bands are observed within the ratio range of 1:1 to 1:4. Amplification begins at a ratio of 1:6, with the brightest bands observed at 1:10. Therefore, a ratio of 1:10 (0.2 µM:2.0 µM) is selected as the optimal concentration ratio for the outer to inner primers in the PoRV G9 LAMP reaction system. Evaluation of the sensitivity and specificity of four porcine diarrhea virus CRISPR-conjugated LAMP detection systems To evaluate the sensitivity of the LAMP-CRISPR detection systems for the four viruses, standard plasmids of these viruses were diluted in copy number gradients (ranging from 1×10 0 to 1×10 6 copies/µL) and used as templates according to section 2.3. Amplification was performed using the optimized LAMP systems and conditions. After the reaction was completed, amplification products with different copy numbers were introduced into CRISPR detection systems containing ssDNA probes. The reactions were conducted at 37°C for 30 minutes in an ABI QuantStudio 5, and fluorescence data were recorded. Following the reaction, the products were analyzed using a gel imager under UV light. Similarly, to determine whether LAMP-CRISPR is cross-detecting between detecting different porcine viruses, specificity was assessed for four porcine diarrhea virus LAMP-CRISPR detection systems. Referring to the optimized LAMP amplification system, the reaction master tubes were prepared and divided into nine groups with three replicates in each group, and a negative control group was set up. PCV2 and PPV DNA genomes and cDNA of CSFV, PRRSV, PEDV, TGEV, PDCoV and PoRV G9 were selected as amplification templates respectively. The amplified products were then subjected to CRISPR assay. Development of the CRISPR-based detection method combined with a quadruplex LAMP system for PEDV, TGEV, PDCoV, and PoRV To achieve simultaneous amplification of multiple target components, this study optimized dual, triple, and quadruple LAMP amplification systems. Initially, the reaction mixtures were prepared using optimized primer concentrations for TGEV and PoRV, with primer concentrations set at original, 1/2, and 1/3 of the original concentrations, and a negative control group was included. Based on the successful amplification of the TGEV-PoRV G9 LAMP system, PDCoV LAMP primers were added, similarly setting primer concentrations at original, 1/2, and 1/3, with sterilized water as the negative control template. Further, upon establishing a successful TGEV-PoRV-PDCoV LAMP system, PEDV LAMP primers were introduced. Unlike previous setups, the addition of four primer sets resulted in maintaining high primer concentrations in the reaction mixture, which increased the formation of primer dimers. Consequently, primer concentrations were further reduced, with experimental groups set at 1/2, 1/3, and 1/4 of the original concentration, and a negative control group was included. Using a cDNA mix of PEDV, TGEV, PDCoV, and PoRV G9 as templates, the reactions were performed at 64°C for 50 minutes, followed by heat inactivation at 80°C. A 5 µL aliquot of the reaction mixture was combined with one-fifth volume of loading buffer and subjected to nucleic acid electrophoresis on a 2% agarose gel to observe the results. Subsequently, the mixture was analyzed using the CRISPR detection system in the ABI QuantStudio 5 at 37°C for 30 minutes, with fluorescence data recorded. After the reaction, the results were visualized under a UV transilluminator for gel imaging. Combination of quadruple LAMP-CRISPR and lateral flow dipstick Lateral flow test strips were employed to alter the visual presentation of the detection results. The specific procedure is as follows: first, the LAMP-CRISPR detection system was prepared according to the optimized conditions, with the FAM-BHQ1-ssDNA reporter (Table S1 ) molecule substituted by FAM-Biotin-ssDNA. After the reaction is complete, the sample conjugation area of the lateral flow dipstick assay inserted into the CRISPR reaction tube, with approximately half of the conjugation zone submerged while maintaining a horizontal position. After the T line or C line develops, the test strip is removed and the results are interpreted within 10 minutes. For feasibility assessment, this study used a positive plasmid for PEDV detection. Feasibility analysis of porcine diarrhea sample detection based on LAMP-CRISPR In clinical cases, porcine diarrhea often involves mixed infections with PEDV, TGEV, PDCoV, and PoRV G9 in the field. A total of 95 samples were collected and initially tested for PEDV, TGEV, and PDCoV using RT-qPCR method. Subsequently, RT-PCR was employed to detect PoRV. The results were then compared with those obtained from the quadruple LAMP-CRISPR detection method developed in this study to validate the concordance rate of the testing approach. RT-RT-qPCR assays for PEDV, TGEV, and PDCoV were constructed by the laboratory to meet the detection limits of standard kits. Results Optimal crRNA selection for four porcine diarrhea viruses using CRISPR/Cas12a The selection of optimal crRNAs for the CRISPR/Cas12a detection system targeting the four porcine diarrhea viruses was based on fluorescence intensity measurements and UV transmittance images. The results for the PEDV CRISPR detection system are illustrated in Fig. 1 A and 1 B, which includes both UV transmittance images and bar charts displaying fluorescence values recorded by the ABI QuantStudio 5 after 30 minutes. Among the tested crRNAs, crRNA3 exhibited significantly higher fluorescence values compared to other groups, with statistically significant differences (p < 0.01). As a result, crRNA3 was selected as the optimal crRNA for the PEDV CRISPR detection system. Similarly, Fig. 1 C and 1 D present the results for the TGEV CRISPR detection system. Although the fluorescence intensities of all three crRNAs were comparable, crRNA5 had the lowest background fluorescence, making it the most distinguishable. Consequently, crRNA5 was chosen as the optimal crRNA for the TGEV CRISPR detection system. For PDCoV, Fig. 1 E and 1 F demonstrate that crRNA8 yielded the highest fluorescence intensity with significant differences compared to other groups (p < 0.001). Thus, crRNA8 was selected as the optimal crRNA for the PDCoV CRISPR detection. In the case of PoRV, which has only one available detection site, crRNA10 was automatically selected as the optimal detection crRNA. Optimal huLbCas12a and crRNA ratio selection for CRISPR/Cas12a system In theory, a 1:1 molar ratio of huLbCas12a to crRNA is expected to achieve the highest cleavage efficiency for the protein-RNA binary complex. However, factors such as the stability of huLbCas12a and crRNA, the assembly efficiency of the binary complex, and buffer conditions can affect the performance of CRISPR detection system for various pig diarrhea viruses. Therefore, the study optimized the huLbCas12a-to-crRNA ratio for multiple viruses. As shown in Fig. 2 A and 2 B, the fluorescence intensity for the PEDV CRISPR detection system was maximized at a huLbCas12a:crRNA ratio of 1:2 (250:500 nM), establishing this as the optimal ratio. Similarly, the highest fluorescence intensity for the TGEV and PDCoV CRISPR detection system was also achieved at a 1:2 ratio (Fig. 2 C and D), with the TGEV and PDCoV CRISPR detection group showing no intra-group variance (Fig. 2 E and F). For the PoRV G9 CRISPR detection system (Figs. 2 G and 2 H), fluorescence increased with the molar ratio, reaching a peak at 1:3 (250:750 nM). In summary, a huLbCas12a:crRNA ratio of 1:2 is optimal for the PEDV, TGEV, and PDCoV detection systems, while a 1:3 ratio is optimal for PoRV G9. This variation suggests that the assembly efficiency of the detection system varies across viruses, with higher crRNA concentrations-more than double the molar amount, maximizing the cleavage activity of huLbCas12a. Sensitivity test of the CRISPR-conjugated LAMP detection system for four porcine diarrhea viruses The sensitivity of the LAMP-CRISPR detection system for the four porcine diarrhea viruses was evaluated to assess the effectiveness in detecting viral limits. For the PEDV LAMP-CRISPR detection system, fluorescence values were significantly higher in viral samples than in the negative control (NC) group, although fluorescence values remained relatively constant across different copy numbers (Fig. 3 B). As shown in Fig. 3 A, only the NC group exhibited no visible fluorescence. For the TGEV LAMP-CRISPR detection system, Fig. 3 C and 3 D demonstrate that fluorescence was not visible in the NC group to the naked eye. Fluorescence values increased slightly with higher copy numbers, indicating that the system can detect even low viral copy numbers, including single-digit copies. In the PDCoV LAMP-CRISPR system, as illustrated in Fig. 3 E and 3 F, fluorescence became visible at 100 copies. However, the fluorescence value for the 10 3 copies group was slightly lower than that of other copy numbers. Finally, Figs. 3 G and H demonstrate a pronounced and intense green fluorescence starting at the 1 copy level in the PoRV G9 LAMP-CRISPR system. Overall, the LAMP-CRISPR detection system for the four porcine diarrhea viruses exhibit high sensitivity, with capability to visually detect viral genomes at concentrations as low as 1 copy/µL. These findings suggest that this established system is a highly effective tool for the sensitive detection of porcine diarrhea viruses. Specificity test of the CRISPR-conjugated LAMP detection system for four porcine diarrhea viruses The specificity of the LAMP-CRISPR detection system for the four porcine diarrhea viruses was evaluated for the accuracy in detecting the target viruses. As illustrated in Fig. 4 B, the PEDV LAMP-CRISPR detection system exhibited clear differences in fluorescence values between the target virus and test samples containing various viruses or the NC, as supported by direct visual observation (Fig. 4 A). Similarly, the remaining panels of Fig. 4 demonstrate that when the target sequence matched the genome of the intended virus, strong green fluorescence was observed under UV light. Consistently, the fluorescence intensity for the target virus was significantly higher than those of the non-target viruses or NC samples. Furthermore, no significant differences were observed between the NC group and the groups containing non-target viruses, indicating that the crRNA did not interact with the genomes of other viruses. These findings suggest that the LAMP-CRISPR detection system exhibits a high degree of specificity, reliably distinguishing between the target viruses and other porcine viruses. Application of the multiplex LAMP-Cas12a system in detecting four porcine diarrhea viruses Multiplex amplification enables the simultaneous amplification of multiple target genes in a single reaction, significantly enhancing the efficiency of the LAMP-CRISPR system. However, practical challenges such as primer dimer formation, non-specific amplification, and prolonged reaction times may arise due to interactions among multiple primer pairs and competitive amplification between individual primer sets. To achieve effective simultaneous amplification of multiple target sequences, this study optimized dual, triple, and quadruple LAMP amplification systems. Initially, using mixed cDNA templates, detectable electrophoretic bands were observed in lanes 2, 4, and 6, while no amplification occurred in lanes 1, 3, and 5, which served as negative controls (Figure S5A). Under UV transillumination, the TGEV and PoRV G9 CRISPR detection systems exhibited green fluorescence in the amplification groups, indicating that at the original, 1/2 and 1/3 primer concentrations, the LAMP primer sets specifically amplified the TGEV and PoRV G9 templates. These amplification products were subsequently recognized by crRNA and underwent non-specific cleavage. However, as primer concentrations decreased, band brightness also diminished, indicating that the LAMP amplification efficiency is dependent on primer concentration (Figure S5B and S5C). Building on the dual amplification system, the inclusion of PDCoV primer sets enabled amplification of template sequences in groups 2, 4, and 6, with visible green fluorescence under UV light (Figure S6B, S6C, and 6D). The results indicate that at the original, 1/2 and 1/3 primer concentrations, the CRISPR system successfully detected the triple amplification products (Figure S6A). To balance the primer ratios and optimize amplification in the quadruplex LAMP assay, primer concentrations were further reduced to one-fourth of the original concentration, as shown in Fig. 5 A. Under UV light, no fluorescence was detected in the negative controls for TGEV, PoRV, and PDCoV. However, strong green fluorescence appeared in the tubes containing quadruplex LAMP amplification products. Notably, fluorescence was observed only in tube 6 of Figs. 5 B–F, indicating that at half and one-third primer concentrations, the quadruplex amplification system specifically targeted the TGEV, PoRV, and PDCoV templates, leading to competitive amplification. Based on these findings, a quadruplex LAMP-CRISPR detection system with 1/4 primer concentration was chosen for subsequent detections. Feasibility evaluation of the CRISPR detection system combined with lateral flow dipstick assay To further facilitate visual determination of detection results, the reporter molecules in the optimized quadruple CRISPR-based LAMP fluorescence detection system were tested using a lateral flow dipstick assay. This setup aimed to assess the feasibility of combining these methods. As shown in Fig. 6 , after immersing the strip into the PEDV LAMP-CRISPR detection solution, two red bands appeared on both the T and C lines across all test groups containing 10 0 to 10 3 viral copies/µL. This indicates non-specific cleavage of the FAM-Biotin-ssDNA probe by the huLbCas12a protein, while only a single red band appeared on the C line in the NC group. These results demonstrate that the LAMP-CRISPR detection system, when combined with lateral flow strip technology, exhibits high sensitivity, capable of detecting viral load as low as 1 copy/µL, thereby confirming the feasibility of coupling these two techniques for practical application. Comparison of the quadruple LAMP-CRISPR and RT-qPCR for detecting porcine diarrhea viruses in clinical samples To validate the reliability of the quadruple LAMP-conjugated CRISPR/Cas12a assay, nucleic acid extracted clinical samples were directly used as templates in a single-tube assay, as illustrated in Fig. 7 . First, RNA is extracted from clinical samples and added to a one-pot system containing both reverse transcription and LAMP reagents. The reaction is carried out at 64°C for 15–30 minutes. Subsequently, the mixture is heated at 80°C for 30 seconds to inactivate the reverse transcriptase and Bst DNA polymerase. Cas12a is then added to the reaction and incubated for an additional 30 minutes The results are visualized either under ultraviolet light or by direct inspection using a lateral flow strip. The cycle threshold (Ct) values for PEDV, TGEV, and PDCoV, as detected by RT-qPCR, are provided in Tables 1 , 2 , 3 and 4 , respectively. The detection results of the quadruple LAMP-CRISPR assay for PEDV, TGEV and PDCoV were compared with those of RT-qPCR, yielding a 100% compliance rate for these viruses. Additionally, the LAMP-CRISPR assay for PoRV G9 showed a 98.94% concordance rate compared with RT-qPCR. Analysis indicated that 31.57% of these clinical samples were positive for PEDV, 6.31% for TEDV, 21.00% for PDCoV, and 8.42% for PoRV (Table 5 ). Moreover, three mixed infections of TGEV and PEDV and one mixed infection of PoRV G9 and PDCoV were detected. Notably, the TGEV detection rate was somewhat inflated due to potential cross-reactions from the use of live TGEV vaccines. In summary, the quadruple LAMP-CRISPR system demonstrated high reliability for detecting porcine diarrhea diseases and could be effectively applied to clinical diagnostics. Discussion Molecular diagnostic methods for PEDV, TGEV, PDCoV, and PoRV G9 play a crucial role in epidemic prevention and control, enabling early detection, accurate monitoring of epidemic of outbreak dynamics, and timely intervention to mitigate the spread of these viruses. However, there is an ongoing need for more cost-effective, highly sensitive, and precise viral detection methods that can be widely deployed. These four Porcine diarrhea viruses frequently appear in single or mixed infections, posing significant challenges for outbreak control and subsequent treatment [ 3 ]. Currently, molecular and serological diagnostics remain the primary approaches for detecting these viruses. For instance, Chen et al. established a multiplex RT-qPCR detection method for PEDV, TGEV, PDCoV, and PoRV, but it only achieved a sensitivity of 27 copies/µL [ 3 ], limiting its application in real-time clinical diagnostics. Additionally, serological detection methods cannot simultaneously detect four porcine diarrhea pathogens in mixed infections, nor can they accurately diagnose infections in their early stages [ 39 , 40 ]. There are also no reports of multiplex detection using IFA and IHC [ 17 – 20 ]. Moreover, these methods impose stringent requirements regarding sample sources, equipment, and the qualifications of testing personnel. While a CRISPR-based detection system for multiplex detection of porcine coronaviruses has previously been described [ 41 ], comprehensive validation using multiple clinical samples and direct comparison with traditional detection methods were not conducted. This study, informed by epidemiological surveys of porcine diarrhea viruses in China [ 9 , 42 ], aims to provide a robust detection tool for fundamental research on porcine coronaviruses and rotaviruses. To meet the demand for faster and more accessible diagnostic tools, we developed a nucleic acid detection approach that combines the CRISPR/Cas12a system with a multiplex LAMP amplification platform and displays results though a lateral flow test strip for easy visualization. This method offers a practical, real-time alternative for clinical settings. To validate the feasibility of the multiplex LAMP amplification system, we initially used TGEV and PoRV G9 as models to establish amplification methods with varying primer concentrations, which were then paired with the CRISPR system for product detection. The LAMP-CRISPR system demonstrated high specificity, even with mixed virus samples. Building on this, we optimized the system for triple and quadruple LAMP amplification setups. However, as the number of primers sets increased, competitive amplification was observed, especially at high primer concentrations, with TGEV, PDCoV, and PoRV G9 amplifying more strongly than PEDV. By reducing the primer concentrations, we found that the quadruple LAMP system, even at 1/4 of the optimal primer set concentration, allowed the CRISPR system to detect all four viruses efficiently, significantly enhancing detection sensitivity to as low as 1–10 copies, com-pared to the traditional PCR methods. A potential issue encountered in the study was the spontaneous quenching of fluorescent probes during storage, which reduced visibility under UV excitation. To address this, the fluorescent reporter probe in the LAMP-CRISPR system was replaced with a FAM-Biotin-ssDNA reporter molecule and paired with lateral flow dipstick assay. This modification enabled detection of even low levels of target sequences, where the cleavage of FAM-Biotin-ssDNA resulted in two visible red lines on the dipstick. This system, which operates at a constant temperature using a water bath, breaks the limitations imposed by traditional molecular diagnostic methods, offering true real-time clinical detection without the need for complex equipment. In summary, the quadruple LAMP-CRISPR detection system was successfully ap-plied to clinical sample testing and compared with traditional nucleic acid detection methods. The results for PEDV, TGEV, and PDCoV were consistent between the two detection methods, with a 100% overall agreement. In detail, the positive rates were 31.57% for PEDV, 6.31% for TGEV, and 21% for PDCoV. For PoRV, the positivity rate was 8.42%, with an overall agreement of 98.94% compared to RT-qPCR, detecting one additional positive sample, likely due to the higher sensitivity of the quadruple LAMP-CRISPR system. Furthermore, the system successfully detected multiple viral genomes within the same sample, providing experimental evidence of mixed infections involving PEDV, TGEV, PDCoV, and PoRV. Conclusions This study establishes a multiplex nucleic acid detection platform that integrates CRISPR/Cas12a with LAMP to enable rapid, cost-effective, and highly sensitive simultaneous detection of PEDV, TGEV, PDCoV, and PoRV G9. Through systematic molecular and functional characterization of the crRNA/Cas12a complex, we defined crRNA specificity and optimized the crRNA:Cas12a stoichiometry, thereby markedly improving diagnostic specificity and accuracy—advances that align with the biological study of natural macromolecules. The assay is field-deployable and readily scalable, supporting early and precise pathogen detection in veterinary settings, mitigating economic losses in the swine industry, strengthening biosecurity and disease management, and providing a transferable framework and practical basis for broader application of CRISPR/Cas systems in molecular diagnostics. Declarations Author Contributions: YY, ABW, and LL contributed to conception and design of the study. LL and ZJH conducted experiments. TL, YJC, XYP, YGW, FL, YMY, YYC, KXW, XJH, YZ and NDW analyzed data. LL and ABW wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. Funding: This study was funded by “Research and development of protein particle vaccines for African Swine Fever and other major viral diseases” (2021NK1030) and Support was also provided by “Furong” Scholar funding to Aibing Wang. Ethics statement: The ethical approval and usage plan for this study have been approved by the Biomedical Research Ethics Committee of Hunan Agricultural University (No. 2025156). We have adhered to the ARRIVE guidelines. Disclosure statement : No potential conflict of interest was reported by the author(s). Consent for publication Not applicable. Data availability statement: The data that support the findings of this study are openly available at https://doi.org/10.6084/m9.figshare.29556512.v1 . Conflicts of Interest: The authors declare no conflict of interest. Supplementary Materials: The following material is available online. References Lu S, Tong X, Han Y, et al. Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a. Nat Biomed Eng. 2022;6(3):286–97. Wen F, Yang J, Li A, et al. Genetic characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Guangdong, China, between 2018 and 2019. PLoS ONE. 2021;16(6):e0253622. Zhu H, Wang G, Liu X, et al. Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA. Microb Pathog. 2024;191:106646. Jung K, Saif LJ. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J. 2015;204(2):134–43. Song D, Park B. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes. 2012;44(2):167–75. Lednicky JA, Tagliamonte MS, White SK, et al. Independent infections of porcine deltacoronavirus among Haitian children. Nature. 2021;600(7887):133–7. Bigault L, Brown P, Bernard C, Blanchard Y, Grasland B. Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR. J Virol Methods. 2020;283:113906. Vlasova AN, Amimo JO, Saif LJ. Porcine Rotaviruses: Epidemiology, Immune Responses and Control Strategies. Viruses. 2017;9(3):48. Zhang F, Luo Y, Lin C, et al. Epidemiological monitoring and genetic variation analysis of pathogens associated with porcine viral diarrhea in southern China from 2021 to 2023. Front Microbiol. 2024;15:1303915. Xue R, Tian Y, Zhang Y, et al. Diversity of group A rotavirus of porcine rotavirus in Shandong province China. Acta Virol. 2018;62(3):229–34. Hou W, Fan M, Zhu Z, Li X. Establishment and Application of a Triplex Real-Time RT-PCR Assay for Differentiation of PEDV, PoRV, and PDCoV. Viruses. 2023;15(6):1238. Marthaler D, Homwong N, Rossow K, et al. Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples. J Virol Methods. 2014;209:30–4. Li C, Liang J, Yang D, et al. Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method. Anim (Basel). 2022;12(19):2712. Li P, Ren X. Reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus. Curr Microbiol. 2011;62(3):1074–80. Zhang F, Ye Y, Song D, et al. A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification. Biol Res. 2017;50(1):30. Hu X, Zhang S, et al. Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine. Chi J Virol. 2016;32(6):740–6. Chinese. Cong Y, Li X, Bai Y, et al. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells. Virol. 2015;478:1–8. Trincone A, Schwegmann-Weßels C. Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein. Virus Res. 2015;202:3–11. Okda F, Lawson S, Liu X, et al. Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus. BMC Vet Res. 2016;12:95. Wang J, Zhou J, Zhu X, et al. Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China. Microb Pathog. 2024;190:106612. Jung K, Miyazaki A, Saif LJ. Immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (Gn) pigs infected with porcine epidemic diarrhea virus (PEDV) and serum cytokine responses of Gn pigs to acute PEDV infection. Res Vet Sci. 2018;119:99–108. Whitworth KM, Rowland RRR, Petrovan V, et al. Resistance to coronavirus infection in amino peptidase N-deficient pigs. Transgenic Res. 2019;28(1):21–32. 10.1007/s11248-018-0100-3 . Li B, Zheng L, Li H, Ding Q, Wang Y, Wei Z. Porcine deltacoronavirus causes diarrhea in various ages of field-infected pigs in China. Biosci Rep. 2019;39(9):BSR20190676. Sozzi E, Moreno A, Lelli D, et al. Development and validation of a monoclonal antibody-based competitive ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV). Res Vet Sci. 2018;121:106–10. Sestak K, Zhou Z, Shoup DI, Saif LJ. Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs. J Vet Diagn Invest. 1999;11(3):205–14. Han F, Shan F, Hou J, et al. Establishment and application of PDCoV antigen-specific DAS-ELISA detection method. BMC Vet Res. 2024;20(1):342. Memon AM, Chen F, Khan SB, et al. Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus. Anim Biotechnol. 2023;34(5):1807–14. Li Y, Li S, Wang J, Liu G. CRISPR/Cas Systems towards Next-Generation Biosensing. Trends Biotechnol. 2019;37(7):730–43. Pardee K, Green AA, Takahashi MK, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 2016;165(5):1255–66. Zhang Y, Wang Y, Xu L, Lou C, Ouyang Q, Qian L. Paired dCas9 design as a nucleic acid detection platform for pathogenic strains. Methods. 2022;203:70–7. Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438–42. Li L, Li S, Wu N, et al. HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation. ACS Synth Biol. 2019;8(10):2228–37. Lei L, Liao F, Tan L, et al. LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2. Anim (Basel). 2022;12(18):2413. Liu S, Tao D, Liao Y, et al. Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus. ACS Synth Biol. 2021;10(10):2499–507. He Q, Yu D, Bao M, et al. High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system. Biosens Bioelectron. 2020;154:112068. Wang H, Li H, Tang B, et al. Fast and sensitive differential diagnosis of pseudorabies virus-infected versus pseudorabies virus-vaccinated swine using CRISPR-Cas12a. Microbiol Spectr. 2024;12(1):e0261723. Yang K, Liang Y, Li Y, et al. Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains. Anal Bioanal Chem. 2021;413(30):7521–9. Wei J, Li Y, Cao Y, et al. Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay. Front Cell Infect Microbiol. 2022;12:879887. Gerber PF, Gong Q, Huang YW, Wang C, Holtkamp D, Opriessnig T. Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA. Vet J. 2014;202(1):33–6. Tabatabaei MS, Ahmed M. Enzyme-Linked Immunosorbent Assay (ELISA). Methods Mol Biol. 2022;2508:115–34. Liu J, Tao D, Chen X, et al. Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay. Viruses. 2022;14(4):833. Zhao J, Shi BJ, Huang XG, et al. A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012. J Virol Methods. 2013;194(1–2):107–12. Tables Table 1 Ct values of PoRV G9-positive samples detected by RT-qPCR. Number 1 2 3 4 5 6 7 Ct value 22.5 27.47 25.43 20.45 15.08 27.47 33.13 Table 2 Ct values of PEDV-positive samples detected by RT-qPCR. Number 1 2 3 4 5 6 7 8 9 10 Ct value 21.79 19.80 17.63 21.10 23.68 27.28 24.14 24.06 24.32 21.20 Number 11 12 13 14 15 16 17 18 19 20 Ct value 22.79 30.91 30.43 31.61 30.93 30.90 30.83 30.88 30.72 23.75 Number 21 22 23 24 25 26 27 28 29 30 Ct value 34.26 33.89 32.84 33.50 33.18 30.94 33.69 33.69 35.10 34.05 Table 3 Ct values of TGEV-positive samples detected by RT-qPCR. Number 1 2 3 4 5 6 Ct value 27.80 25.32 31.01 35.50 29.40 28.96 Table 4 Ct values of PDCoV-positive samples detected by RT-qPCR. Number 1 2 3 4 5 6 7 8 9 10 Ct value 4.67 27.64 27.47 28.88 29.40 28.96 31.33 29.85 26.63 26.73 Number 11 12 13 14 15 16 17 18 19 20 Ct value 32.01 31.46 33.97 30.18 28.84 31.41 30.54 32.36 30.51 25.98 Table 5 Comparison and compliance validation of the quadruple LAMP-CRISPR assay system with conventional nucleic acid detection methods. Detection Positive Negative Positive coincidence rate Negative coincidence rate Overall coincidence rate PEDV RT-qPCR 30 65 100% 100% 100% PEDV LAMP-CRSPR 30 65 PDCoV RT-qPCR 28 67 100% 100% 100% PDCoV LAMP-CRSPR 28 67 TGEV RT-qPCR 6 89 100% 100% 100% TGEV LAMP-CRISPR 6 89 PoRV G9 RT-qPCR 7 88 100% 98.86% 98.94% PoRV G9 LAMP-CRISPR 8 87 Additional Declarations No competing interests reported. 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10:06:56","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145472,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/18dbd14f2d24a5cf0d6ed583.html"},{"id":95392114,"identity":"512c7506-f215-4c1d-b9b2-530302417a3c","added_by":"auto","created_at":"2025-11-07 14:12:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":295051,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence CRISPR assay results. The red rectangle denotes the optimal selection. A, C and E present the visual outcomes of crRNA screening for PEDV, TGEV and PDCoV, respectively. B, D and F display histograms showing the fluorescence intensity for PEDV, TGEV and PDCoV. ns,** and *** denote not significant, p\u0026lt;0.01 and 0.001, respectively(n=3 technical replicates, value represents mean ± SEM).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/62b777773c31c402be8e708b.png"},{"id":95392115,"identity":"f449dae4-6776-4e6a-b3cc-35d7b6cdac78","added_by":"auto","created_at":"2025-11-07 14:12:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":376479,"visible":true,"origin":"","legend":"\u003cp\u003eOptimal huLbCas12a-to-crRNA ratio results. A, C, E and G display the visual outcomes of different huLbCas12a-to-crRNA ratios for PEDV, TGEV, PDCoV and PoRV, respectively.\u003cstrong\u003e \u003c/strong\u003eB, D, F and H demonstrate histograms of fluorescence intensity corresponding to each ratio for PEDV, TGEV, PDCoV and PoRV. ns,** and *** denote not significant p\u0026lt;0.01 and 0.001, respectively(n=3 technical replicates, value represents mean ± SEM).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/6cfe32e153d697e6717cdcde.png"},{"id":95392117,"identity":"eaa2dde3-0b4e-44e3-9fea-a24e4ecde2e8","added_by":"auto","created_at":"2025-11-07 14:12:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":249085,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity evaluation of the LAMP-CRISPR assay for porcine diarrhea-associated viruses. (A, C, E, and G) Visual sensitivity evaluation of the CRISPR-coupled LAMP detection system for PEDV, TGEV, PDCoV and PoRV G9 using varying concentrations of standard plasmids. (B, D, F, and H) Fluorescence intensity histograms depicting sensitivity analysis of the CRISPR-coupled LAMP assay system for PEDV, TGEV, PDCoV, and PoRV. NC: Negative control. *** represents p \u0026lt; 0.001 for the indicated comparisons (n=3 technical replicates, value represents mean ± SEM).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/04a78e26fbcd77bfb42e25e7.png"},{"id":95392118,"identity":"6a2c2785-b085-421f-b806-415e5c386c31","added_by":"auto","created_at":"2025-11-07 14:12:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":248762,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity evaluation of LAMP-CRISPR assay for four porcine diarrhea-associated viruses. (A, C, E and G) Visual specificity evaluation of the CRISPR-coupled LAMP detection system for PEDV, TGEV, PDCoV and PoRV G9 using the genetic DNAs of various porcine diarrhea-associated viruses. (B, D, E and F) Fluorescence intensity histograms depicting the specificity analysis of the CRISPR-coupled LAMP detection system for PEDV, TGEV, PDCoV and PoRV G9. NC: Negative control. *** represents p \u0026lt; 0.001 for the indicated comparisons (n=3 technical replicates, value represents mean ± SEM).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/b1e9b4dea71475258bbdb927.png"},{"id":95525678,"identity":"15d49162-c3bf-4d02-8e34-cd852998148a","added_by":"auto","created_at":"2025-11-10 10:05:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285956,"visible":true,"origin":"","legend":"\u003cp\u003eApplication of the quadruple LAMP-CRISPR detection system. (A) Gel electrophoresis results of quadruple LAMP amplification products. (B, C, E and F) Visual verification of the quadruple LAMP-CRISPR detection. Lanes 1, 3, and 5 represent negative controls, while 2, 4, and 6 are template test groups with quadruple LAMP amplification products; M represents DL3000 DNA marker.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/d5ccb6b9b82d6337813db769.png"},{"id":95526778,"identity":"865a7928-ffa9-4600-b26a-c45b4affd00c","added_by":"auto","created_at":"2025-11-10 10:07:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":362132,"visible":true,"origin":"","legend":"\u003cp\u003eLAMP/CRISPR-coupled lateral flow dipstick assay. Following the LAMP-CRISPR detection, the resulting products were subjected to a lateral flow dipstick assay. The T-line was conjugated with biotin antibodies, and the C-line with FAM antibodies. The appearance of red bands on both the quality control line (C line) and the detection line (T line) indicated a positive detection of the target gene, while a single band on the C line represented a negative result. NC: Negative control. Standard plasmid concentrations of the PEDV N gene at 10\u003csup\u003e0\u003c/sup\u003e, 10\u003csup\u003e1\u003c/sup\u003e, 10\u003csup\u003e2\u003c/sup\u003e, and 10\u003csup\u003e3\u003c/sup\u003e copies/µL were used as LAMP templates.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/c39f3d05fdd99b0f0ece1a63.png"},{"id":95526667,"identity":"9cb2759b-8468-4568-a944-573d31528cd4","added_by":"auto","created_at":"2025-11-10 10:07:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104834,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the single-tube quadruple LAMP-Cas12a assay for detecting four porcine diarrhea viruses.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/5413f6ec1d72867a33fb594c.png"},{"id":107351047,"identity":"42f4ba21-50a7-4075-9946-c235117e238d","added_by":"auto","created_at":"2026-04-20 16:08:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3283774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/3cfd1c3f-658b-4e6e-9b4d-a07de251f7fa.pdf"},{"id":95392120,"identity":"2b31621a-2083-45f4-a727-20a6e76a4780","added_by":"auto","created_at":"2025-11-07 14:12:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2651105,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7675377/v1/46d43e7a4786e97c11f73395.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A crRNA/Cas12a complex-driven rapid and visual detection method for four porcine diarrhea viruses","fulltext":[{"header":"Background","content":"\u003cp\u003eIn recent years, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) pandemic has triggered a global public health crisis with profound impact on economies, societies, and individuals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Similarly, the widespread outbreaks of porcine corona-virus, causing neonatal diarrhea in piglets, have severely affected the swine industry, raising significant concerns about food safety. Notably, PEDV, TGEV, and PDCoV have spread worldwide, greatly reducing pork production efficiency and destabilizing the economic foundations of animal husbandry [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Four major prevalent porcine diarrhea viruses, PEDV and TGEV (both classified as α-coronavirus), PDCoV, and PoRV, are known to cause significant outbreaks [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. PEDV, TGEV and PDCoV are single-stranded positive-sense RNA viruses with similar capsid structures. Their genomes include genes ORF1a, ORF1b, and ORF2\u0026thinsp;~\u0026thinsp;ORF6, which are sequentially arranged and encode two nonstructural replicase proteins, four structural proteins: membrane protein (M), spike glycoprotein (S), envelope protein (E) and nucleocapsid protein (N), and a nonstructural helper protein encoded by ORF3, respectively [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. PDCoV is unique among these vi-ruses due to its helper proteins NS6 and NS7/NS7a, located between the structural protein-coding genes, which may increase its zoonotic potential [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The highly con-served nature of the N and M coding genes makes them common targets for nucleic acid detection [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePoRV, a member of the Reoviridae family and Rotavirus genus, is a double-stranded RNA virus comprising 11 dsRNA segments that encode distinct proteins. These include six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and five non-structural proteins (NSP1-NSP5) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. PoRV was first reported in China in the 1980s after being isolated from pig diarrhea samples, confirming its presence in domestic swine populations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PoRV genotypes are primarily classified based on the variations in the VP7 glycoprotein of the outer shell, with at least 18 identified G types. The P types are determined by sequence differences in the VP4 phosphatase functional region and include 6 distinct variants, resulting in at least 11 reported G-P combinations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This complex serology and diverse genotypes of PoRV pose challenges for detection and control.\u003c/p\u003e\u003cp\u003eCo-infection and secondary infections involving PEDV, TGEV, PDCoV, and PoRV are common, producing similar clinical signs, primarily characterized by watery diarrhea, which complicates clinical diagnosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Current detection methods for these four viruses fall into two categories: molecular biology techniques and serological diagnostic methods. These include virus isolation and electron microscopy, reverse transcription polymerase chain reaction (RT-PCR) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], reverse transcription real-time quantitative PCR (RT-qPCR)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], reverse transcription loop-mediated isothermal amplification (RT-LAMP)[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], immunofluorescence assay (IFA)[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], immunohistochemistry (IHC)[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and enzyme-linked immunosorbent assay (ELISA)[\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. While these diagnostic methods are widely employed, molecular biology techniques often face limitations for real-time, on-site testing due to high equipment costs, complex operational procedures, the need for specialized personnel, and issues with sample processing and data analysis.\u003c/p\u003e\u003cp\u003eSerological methods are constrained by longer detection times, reflect only the presence of antibodies rather than active infections, and their sensitivity and specificity may be influenced by immune status and viral mutations; moreover, they cannot distinguish whether elevated antibody levels result from natural infection with wild-type strains or from vaccination.\u003c/p\u003e\u003cp\u003eThe CRISPR/Cas system has recently emerged as a next-generation technology for pathogen or nucleic acid detection. This system, utilizing effector proteins such as Cas9, Cas12a, Cas12b, and Cas13, holds significant promise in diagnostics [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For instance, Pardee et al. developed a low-cost, paper-based sensor combining CRISPR/Cas9 technology with RNA amplification to distinguish Zika virus subtypes in 2017[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Zhang et al. created a detection system using dCas9 with a luciferase reporter for Mycobacterium tuberculosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], while Zhang Feng\u0026rsquo; group integrated Cas13a with recombinase polymerase amplification (RPA) to develop the SHERLOCK system, a highly specific and sensitive detection platform capable of detecting single-copy viral genomes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Beyond its genome-editing capability, CRISPR/Cas has demonstrated significant potential in nucleic acid detection. For example, Li et al. combined CRISPR/Cas12b with PCR to create HOLMES v2, a rapid and sensitive detection system that proved more effective and convenient than RT-qPCR and ELISA [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, CRISPR/Cas systems have been successfully applied to detect various porcine viruses, including porcine circovirus type 2 (PCV2) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], porcine reproductive respiratory syndrome virus (PRRSV)[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], African swine fever virus (ASFV)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], pseudorabies virus (PRV)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], PEDV[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and porcine parvovirus (PPV)[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. CRISPR/Cas-based detection systems are characterized by high sensitivity, simplicity, and rapid response times, making them well-suited for on-site applications.\u003c/p\u003e\u003cp\u003eThis study integrates the advantages of loop-mediated isothermal amplification (LAMP), which does not require specialized equipment, with the collateral cleavage activity of huLbCas12a (namely, the capability to cleave fluorophore-quenched sin-gle-stranded DNA probe sensors) to establish a novel, visual, and effective method for simultaneously detecting four major porcine diarrhea viruses. Furthermore, the relia-bility and priority of this newly developed approach were confirmed with clinical samples and compared against traditional RT-(q)PCR method.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eViruses and clinical samples\u003c/h2\u003e\u003cp\u003eThe cDNA of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus (PoRV) is preserved in the Veterinary Protein Engineering Vaccine Key Laboratory of Hunan Agricultural University. A total of 95 samples from different pig farms in Hunan, including fecal samples and rectal swabs, are stored at -80\u0026deg;C for future use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDesign and preparation of LAMP primers, crRNA and probes\u003c/h3\u003e\n\u003cp\u003eSequences of different subtypes of PEDV, TGEV, PDCoV, and PoRV G9 viruses were collected for analysis and comparison (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using the PrimerExplorer V5.0 online software and referring to the instructions on the website, LAMP primers for PEDV, TGEV, PDCoV, and PoRV G9 were designed. Conservative regions of the PEDV N gene, TGEV N gene, PDCoV \u003cem\u003eN\u003c/em\u003e \u003cb\u003egene\u003c/b\u003e, and PoRV G9 \u003cem\u003eVP7\u003c/em\u003e gene were used to design detection sequences of 20\u0026ndash;23 bp following the PAM using the CRISPR-DT online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfolab.maimioh.edu\u003c/span\u003e\u003cspan address=\"http://bioinfolab.maimioh.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The PoRV G9 \u003cem\u003eVP7\u003c/em\u003e gene sequence \"AGTTGATGCTTCAGTAGG\" is the only one available for use as a detection site. A T7 promoter-binding crRNA sequence was designed, enabling T7 polymerase (New England Biolabs) to recognize the annealed crRNA DNA duplex and perform in vitro transcription to produce a large amount of sgRNA. Based on the non-specific cleavage characteristics of huLbCas12a protein, an ssDNA probe rich in \"A base\" was designed, using FAM fluorescent group and BHQ1 quenching group, resulting in the FAM-BHQ1-ssDNA probe\u003csup\u003e1\u003c/sup\u003e. The above sequence is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All primers were synthesized by Tsingke (Beijing, China).\u003c/p\u003e\n\u003ch3\u003eOptimization of the CRISPR/Cas12a detection system combined with FAM-BHQ1-ssDNA probe\u003c/h3\u003e\n\u003cp\u003eThe standard plasmids for PEDV N gene, TGEV N gene, PDCoV \u003cem\u003eN\u003c/em\u003e gene, and PoRV G9 \u003cem\u003eVP7\u003c/em\u003e gene, as well as Cas12a protein, were prepared and stored by the Hunan Provincial Key Laboratory of Veterinary Protein Engineering Vaccine. The formula for calculating the number of copies is as follows: copy number = (Amount \u0026times; 6.02 \u0026times; 10\u003csup\u003e23\u003c/sup\u003e)/ (DNA length \u0026times; 10\u003csup\u003e9\u003c/sup\u003e \u0026times; 660 Da/bp). The number of copies of the standard plasmid was calculated according to the above formula and diluted as the detection template. The complete CRISPR-based 20 \u0026micro;L detection system includes Cas12a protein, sgRNA, 2 \u0026micro;M ssDNA-FQ reporter gene, 10\u0026times; 2.1 NEBuffer (NEB, USA) and detection templates.\u003c/p\u003e\u003cp\u003eThis experiment optimized the components of the CRISPR system. In previous studies, the efficiency of the huLbCas12a-crRNA binary complex in targeting the desired sequence was related to the type and length of the crRNA codons. Therefore, a 20 bp sequence following PAM was selected as crRNA. Three crRNAs targeting conserved sequences were designed into nine groups for screening: crRNA1\u0026ndash;9 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), with each group including an experimental and a control group. PoRV G9 VP7 has only one target site and thus was not screened. Additionally, the optimal ratio of huLbCas12a to crRNA was determined. Five control groups were designed for optimizing the ratio of huLbCas12a to crRNA, including positive and negative controls for each: 2:1 (250:125 nM), 1:1 (250:250 nM), 1:2 (250:500 nM), 1:3 (250:750 nM), and 1:4 (250:1000 nM). The other components were consistent with the CRISPR/Cas12a system. Each group was performed in triplicate, and the system was prepared in an eight-well tube. Real-time fluorescence data were collected using the ABI QuantStudio 5 RT-qPCR instrument. After the reaction, samples were observed under a gel imaging system with UV light.\u003c/p\u003e\n\u003ch3\u003eOptimization and evaluation of the LAMP\u003c/h3\u003e\n\u003cp\u003eTo achieve efficient and high-yield enrichment of target sequences for four pig diarrhea viruses, this study optimized the components of the LAMP system. Each 25 \u0026micro;l LAMP reaction mixture includes: 2\u0026times; LAMP Polymerase Buffer, 0.48 U Bst 3.0 DNA Polymerase, inner primers, outer primers, loop primers, 6 mM MgSO₄, 1.4 mM dNTP Mix, and target template.\u003c/p\u003e\u003cp\u003eEvaluation and optimization of the LAMP system for detecting PEDV, TGEV, PDCoV, and PoRV G9 based on CRISPR/Cas12a are as follows: prepare the reaction mixture as described, adding 2.0 \u0026micro;L of DNA template and 3.5 \u0026micro;L of distilled water, then mixed and centrifuged. Optimize the final concentration ratio of inner to outer primers by setting the outer primer concentration to 0.2 mM as 1. Establish ratios of 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12, corresponding to inner primer concentrations of 0.4 \u0026micro;M, 0.8 \u0026micro;M, 1.2 \u0026micro;M, 1.6 \u0026micro;M, 2.0 \u0026micro;M, and 2.4 \u0026micro;M, respectively, with loop primers at a final concentration of 0.4 \u0026micro;M. The LAMP reaction was conducted at LAMP reactions were performed at varying temperatures (58, 60, 62, 63, 64, 65\u0026deg;C) and time intervals (30, 35, 40, 45, 50, 55, 60 minutes). Negative controls consisted of reactions conducted without nucleic acid templates.\u003c/p\u003e\u003cp\u003eThe optimization results indicate that the optimal reaction temperatures for the LAMP systems of PEDV, TGEV, PDCoV, and PoRV G9 are 63\u0026deg;C and 64\u0026deg;C. As time progresses, the brightness of the LAMP amplification product bands on the electrophoresis gel increases, indicating that the quantity of LAMP products also increases over time. The optimal reaction temperatures for gel electrophoresis bands are highlighted with red boxes (Figure S2). Results show that the LAMP reaction systems for PEDV, TGEV, and PoRV G9 reach maximum band brightness at 50 minutes, indicating that the LAMP reaction is complete; whereas the PDCoV LAMP system reaches completion at 45 minutes, suggesting slightly higher efficiency compared to the others (Figure S3). The optimization of the final concentrations of outer and inner primers is shown in Figure S4. Figure S4A, B, and C indicate that within the ratio range of 1:1 to 1:12 for outer to inner primers, the LAMP system performs adequately. As the ratio increases, the amplification effect becomes more pronounced until the template reaction is complete in the system. The brightest bands on the gel electrophoresis are observed at a ratio of 1:6 (0.2 \u0026micro;M:1.2 \u0026micro;M), indicating that the optimal outer to inner primer concentration ratio for PEDV, TGEV, and PDCoV LAMP systems are 0.2 \u0026micro;M:1.2 \u0026micro;M. In contrast, Figure S4D shows that no LAMP amplification product bands are observed within the ratio range of 1:1 to 1:4. Amplification begins at a ratio of 1:6, with the brightest bands observed at 1:10. Therefore, a ratio of 1:10 (0.2 \u0026micro;M:2.0 \u0026micro;M) is selected as the optimal concentration ratio for the outer to inner primers in the PoRV G9 LAMP reaction system.\u003c/p\u003e\n\u003ch3\u003eEvaluation of the sensitivity and specificity of four porcine diarrhea virus CRISPR-conjugated LAMP detection systems\u003c/h3\u003e\n\u003cp\u003eTo evaluate the sensitivity of the LAMP-CRISPR detection systems for the four viruses, standard plasmids of these viruses were diluted in copy number gradients (ranging from 1\u0026times;10\u003csup\u003e0\u003c/sup\u003e to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e copies/\u0026micro;L) and used as templates according to section 2.3. Amplification was performed using the optimized LAMP systems and conditions. After the reaction was completed, amplification products with different copy numbers were introduced into CRISPR detection systems containing ssDNA probes. The reactions were conducted at 37\u0026deg;C for 30 minutes in an ABI QuantStudio 5, and fluorescence data were recorded. Following the reaction, the products were analyzed using a gel imager under UV light. Similarly, to determine whether LAMP-CRISPR is cross-detecting between detecting different porcine viruses, specificity was assessed for four porcine diarrhea virus LAMP-CRISPR detection systems. Referring to the optimized LAMP amplification system, the reaction master tubes were prepared and divided into nine groups with three replicates in each group, and a negative control group was set up. PCV2 and PPV DNA genomes and cDNA of CSFV, PRRSV, PEDV, TGEV, PDCoV and PoRV G9 were selected as amplification templates respectively. The amplified products were then subjected to CRISPR assay.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDevelopment of the CRISPR-based detection method combined with a quadruplex LAMP system for PEDV, TGEV, PDCoV, and PoRV\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo achieve simultaneous amplification of multiple target components, this study optimized dual, triple, and quadruple LAMP amplification systems. Initially, the reaction mixtures were prepared using optimized primer concentrations for TGEV and PoRV, with primer concentrations set at original, 1/2, and 1/3 of the original concentrations, and a negative control group was included. Based on the successful amplification of the TGEV-PoRV G9 LAMP system, PDCoV LAMP primers were added, similarly setting primer concentrations at original, 1/2, and 1/3, with sterilized water as the negative control template. Further, upon establishing a successful TGEV-PoRV-PDCoV LAMP system, PEDV LAMP primers were introduced. Unlike previous setups, the addition of four primer sets resulted in maintaining high primer concentrations in the reaction mixture, which increased the formation of primer dimers. Consequently, primer concentrations were further reduced, with experimental groups set at 1/2, 1/3, and 1/4 of the original concentration, and a negative control group was included. Using a cDNA mix of PEDV, TGEV, PDCoV, and PoRV G9 as templates, the reactions were performed at 64\u0026deg;C for 50 minutes, followed by heat inactivation at 80\u0026deg;C. A 5 \u0026micro;L aliquot of the reaction mixture was combined with one-fifth volume of loading buffer and subjected to nucleic acid electrophoresis on a 2% agarose gel to observe the results. Subsequently, the mixture was analyzed using the CRISPR detection system in the ABI QuantStudio 5 at 37\u0026deg;C for 30 minutes, with fluorescence data recorded. After the reaction, the results were visualized under a UV transilluminator for gel imaging.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCombination of quadruple LAMP-CRISPR and lateral flow dipstick\u003c/h2\u003e\u003cp\u003eLateral flow test strips were employed to alter the visual presentation of the detection results. The specific procedure is as follows: first, the LAMP-CRISPR detection system was prepared according to the optimized conditions, with the FAM-BHQ1-ssDNA reporter (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) molecule substituted by FAM-Biotin-ssDNA. After the reaction is complete, the sample conjugation area of the lateral flow dipstick assay inserted into the CRISPR reaction tube, with approximately half of the conjugation zone submerged while maintaining a horizontal position. After the T line or C line develops, the test strip is removed and the results are interpreted within 10 minutes. For feasibility assessment, this study used a positive plasmid for PEDV detection.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFeasibility analysis of porcine diarrhea sample detection based on LAMP-CRISPR\u003c/h3\u003e\n\u003cp\u003eIn clinical cases, porcine diarrhea often involves mixed infections with PEDV, TGEV, PDCoV, and PoRV G9 in the field. A total of 95 samples were collected and initially tested for PEDV, TGEV, and PDCoV using RT-qPCR method. Subsequently, RT-PCR was employed to detect PoRV. The results were then compared with those obtained from the quadruple LAMP-CRISPR detection method developed in this study to validate the concordance rate of the testing approach. RT-RT-qPCR assays for PEDV, TGEV, and PDCoV were constructed by the laboratory to meet the detection limits of standard kits.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eOptimal crRNA selection for four porcine diarrhea viruses using CRISPR/Cas12a\u003c/h2\u003e\u003cp\u003eThe selection of optimal crRNAs for the CRISPR/Cas12a detection system targeting the four porcine diarrhea viruses was based on fluorescence intensity measurements and UV transmittance images. The results for the PEDV CRISPR detection system are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, which includes both UV transmittance images and bar charts displaying fluorescence values recorded by the ABI QuantStudio 5 after 30 minutes. Among the tested crRNAs, crRNA3 exhibited significantly higher fluorescence values compared to other groups, with statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As a result, crRNA3 was selected as the optimal crRNA for the PEDV CRISPR detection system. Similarly, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD present the results for the TGEV CRISPR detection system. Although the fluorescence intensities of all three crRNAs were comparable, crRNA5 had the lowest background fluorescence, making it the most distinguishable. Consequently, crRNA5 was chosen as the optimal crRNA for the TGEV CRISPR detection system. For PDCoV, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF demonstrate that crRNA8 yielded the highest fluorescence intensity with significant differences compared to other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Thus, crRNA8 was selected as the optimal crRNA for the PDCoV CRISPR detection. In the case of PoRV, which has only one available detection site, crRNA10 was automatically selected as the optimal detection crRNA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eOptimal huLbCas12a and crRNA ratio selection for CRISPR/Cas12a system\u003c/h2\u003e\u003cp\u003eIn theory, a 1:1 molar ratio of huLbCas12a to crRNA is expected to achieve the highest cleavage efficiency for the protein-RNA binary complex. However, factors such as the stability of huLbCas12a and crRNA, the assembly efficiency of the binary complex, and buffer conditions can affect the performance of CRISPR detection system for various pig diarrhea viruses. Therefore, the study optimized the huLbCas12a-to-crRNA ratio for multiple viruses. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the fluorescence intensity for the PEDV CRISPR detection system was maximized at a huLbCas12a:crRNA ratio of 1:2 (250:500 nM), establishing this as the optimal ratio. Similarly, the highest fluorescence intensity for the TGEV and PDCoV CRISPR detection system was also achieved at a 1:2 ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D), with the TGEV and PDCoV CRISPR detection group showing no intra-group variance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). For the PoRV G9 CRISPR detection system (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), fluorescence increased with the molar ratio, reaching a peak at 1:3 (250:750 nM). In summary, a huLbCas12a:crRNA ratio of 1:2 is optimal for the PEDV, TGEV, and PDCoV detection systems, while a 1:3 ratio is optimal for PoRV G9. This variation suggests that the assembly efficiency of the detection system varies across viruses, with higher crRNA concentrations-more than double the molar amount, maximizing the cleavage activity of huLbCas12a.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSensitivity test of the CRISPR-conjugated LAMP detection system for four porcine diarrhea viruses\u003c/h2\u003e\u003cp\u003eThe sensitivity of the LAMP-CRISPR detection system for the four porcine diarrhea viruses was evaluated to assess the effectiveness in detecting viral limits. For the PEDV LAMP-CRISPR detection system, fluorescence values were significantly higher in viral samples than in the negative control (NC) group, although fluorescence values remained relatively constant across different copy numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, only the NC group exhibited no visible fluorescence. For the TGEV LAMP-CRISPR detection system, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD demonstrate that fluorescence was not visible in the NC group to the naked eye. Fluorescence values increased slightly with higher copy numbers, indicating that the system can detect even low viral copy numbers, including single-digit copies. In the PDCoV LAMP-CRISPR system, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, fluorescence became visible at 100 copies. However, the fluorescence value for the 10\u003csup\u003e3\u003c/sup\u003e copies group was slightly lower than that of other copy numbers. Finally, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H demonstrate a pronounced and intense green fluorescence starting at the 1 copy level in the PoRV G9 LAMP-CRISPR system. Overall, the LAMP-CRISPR detection system for the four porcine diarrhea viruses exhibit high sensitivity, with capability to visually detect viral genomes at concentrations as low as 1 copy/\u0026micro;L. These findings suggest that this established system is a highly effective tool for the sensitive detection of porcine diarrhea viruses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSpecificity test of the CRISPR-conjugated LAMP detection system for four porcine diarrhea viruses\u003c/h2\u003e\u003cp\u003eThe specificity of the LAMP-CRISPR detection system for the four porcine diarrhea viruses was evaluated for the accuracy in detecting the target viruses. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the PEDV LAMP-CRISPR detection system exhibited clear differences in fluorescence values between the target virus and test samples containing various viruses or the NC, as supported by direct visual observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, the remaining panels of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e demonstrate that when the target sequence matched the genome of the intended virus, strong green fluorescence was observed under UV light. Consistently, the fluorescence intensity for the target virus was significantly higher than those of the non-target viruses or NC samples. Furthermore, no significant differences were observed between the NC group and the groups containing non-target viruses, indicating that the crRNA did not interact with the genomes of other viruses. These findings suggest that the LAMP-CRISPR detection system exhibits a high degree of specificity, reliably distinguishing between the target viruses and other porcine viruses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eApplication of the multiplex LAMP-Cas12a system in detecting four porcine diarrhea viruses\u003c/h2\u003e\u003cp\u003eMultiplex amplification enables the simultaneous amplification of multiple target genes in a single reaction, significantly enhancing the efficiency of the LAMP-CRISPR system. However, practical challenges such as primer dimer formation, non-specific amplification, and prolonged reaction times may arise due to interactions among multiple primer pairs and competitive amplification between individual primer sets. To achieve effective simultaneous amplification of multiple target sequences, this study optimized dual, triple, and quadruple LAMP amplification systems. Initially, using mixed cDNA templates, detectable electrophoretic bands were observed in lanes 2, 4, and 6, while no amplification occurred in lanes 1, 3, and 5, which served as negative controls (Figure S5A). Under UV transillumination, the TGEV and PoRV G9 CRISPR detection systems exhibited green fluorescence in the amplification groups, indicating that at the original, 1/2 and 1/3 primer concentrations, the LAMP primer sets specifically amplified the TGEV and PoRV G9 templates. These amplification products were subsequently recognized by crRNA and underwent non-specific cleavage. However, as primer concentrations decreased, band brightness also diminished, indicating that the LAMP amplification efficiency is dependent on primer concentration (Figure S5B and S5C).\u003c/p\u003e\u003cp\u003eBuilding on the dual amplification system, the inclusion of PDCoV primer sets enabled amplification of template sequences in groups 2, 4, and 6, with visible green fluorescence under UV light (Figure S6B, S6C, and 6D). The results indicate that at the original, 1/2 and 1/3 primer concentrations, the CRISPR system successfully detected the triple amplification products (Figure S6A).\u003c/p\u003e\u003cp\u003eTo balance the primer ratios and optimize amplification in the quadruplex LAMP assay, primer concentrations were further reduced to one-fourth of the original concentration, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Under UV light, no fluorescence was detected in the negative controls for TGEV, PoRV, and PDCoV. However, strong green fluorescence appeared in the tubes containing quadruplex LAMP amplification products. Notably, fluorescence was observed only in tube 6 of Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;F, indicating that at half and one-third primer concentrations, the quadruplex amplification system specifically targeted the TGEV, PoRV, and PDCoV templates, leading to competitive amplification. Based on these findings, a quadruplex LAMP-CRISPR detection system with 1/4 primer concentration was chosen for subsequent detections.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eFeasibility evaluation of the CRISPR detection system combined with lateral flow dipstick assay\u003c/h2\u003e\u003cp\u003eTo further facilitate visual determination of detection results, the reporter molecules in the optimized quadruple CRISPR-based LAMP fluorescence detection system were tested using a lateral flow dipstick assay. This setup aimed to assess the feasibility of combining these methods. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, after immersing the strip into the PEDV LAMP-CRISPR detection solution, two red bands appeared on both the T and C lines across all test groups containing 10\u003csup\u003e0\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e viral copies/\u0026micro;L. This indicates non-specific cleavage of the FAM-Biotin-ssDNA probe by the huLbCas12a protein, while only a single red band appeared on the C line in the NC group. These results demonstrate that the LAMP-CRISPR detection system, when combined with lateral flow strip technology, exhibits high sensitivity, capable of detecting viral load as low as 1 copy/\u0026micro;L, thereby confirming the feasibility of coupling these two techniques for practical application.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eComparison of the quadruple LAMP-CRISPR and RT-qPCR for detecting porcine diarrhea viruses in clinical samples\u003c/h2\u003e\u003cp\u003eTo validate the reliability of the quadruple LAMP-conjugated CRISPR/Cas12a assay, nucleic acid extracted clinical samples were directly used as templates in a single-tube assay, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. First, RNA is extracted from clinical samples and added to a one-pot system containing both reverse transcription and LAMP reagents. The reaction is carried out at 64\u0026deg;C for 15\u0026ndash;30 minutes. Subsequently, the mixture is heated at 80\u0026deg;C for 30 seconds to inactivate the reverse transcriptase and Bst DNA polymerase. Cas12a is then added to the reaction and incubated for an additional 30 minutes The results are visualized either under ultraviolet light or by direct inspection using a lateral flow strip. The cycle threshold (Ct) values for PEDV, TGEV, and PDCoV, as detected by RT-qPCR, are provided in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, respectively. The detection results of the quadruple LAMP-CRISPR assay for PEDV, TGEV and PDCoV were compared with those of RT-qPCR, yielding a 100% compliance rate for these viruses. Additionally, the LAMP-CRISPR assay for PoRV G9 showed a 98.94% concordance rate compared with RT-qPCR. Analysis indicated that 31.57% of these clinical samples were positive for PEDV, 6.31% for TEDV, 21.00% for PDCoV, and 8.42% for PoRV (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, three mixed infections of TGEV and PEDV and one mixed infection of PoRV G9 and PDCoV were detected. Notably, the TGEV detection rate was somewhat inflated due to potential cross-reactions from the use of live TGEV vaccines. In summary, the quadruple LAMP-CRISPR system demonstrated high reliability for detecting porcine diarrhea diseases and could be effectively applied to clinical diagnostics.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMolecular diagnostic methods for PEDV, TGEV, PDCoV, and PoRV G9 play a crucial role in epidemic prevention and control, enabling early detection, accurate monitoring of epidemic of outbreak dynamics, and timely intervention to mitigate the spread of these viruses. However, there is an ongoing need for more cost-effective, highly sensitive, and precise viral detection methods that can be widely deployed. These four Porcine diarrhea viruses frequently appear in single or mixed infections, posing significant challenges for outbreak control and subsequent treatment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, molecular and serological diagnostics remain the primary approaches for detecting these viruses. For instance, Chen et al. established a multiplex RT-qPCR detection method for PEDV, TGEV, PDCoV, and PoRV, but it only achieved a sensitivity of 27 copies/\u0026micro;L [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], limiting its application in real-time clinical diagnostics. Additionally, serological detection methods cannot simultaneously detect four porcine diarrhea pathogens in mixed infections, nor can they accurately diagnose infections in their early stages [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. There are also no reports of multiplex detection using IFA and IHC [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, these methods impose stringent requirements regarding sample sources, equipment, and the qualifications of testing personnel.\u003c/p\u003e\u003cp\u003eWhile a CRISPR-based detection system for multiplex detection of porcine coronaviruses has previously been described [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], comprehensive validation using multiple clinical samples and direct comparison with traditional detection methods were not conducted. This study, informed by epidemiological surveys of porcine diarrhea viruses in China [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], aims to provide a robust detection tool for fundamental research on porcine coronaviruses and rotaviruses. To meet the demand for faster and more accessible diagnostic tools, we developed a nucleic acid detection approach that combines the CRISPR/Cas12a system with a multiplex LAMP amplification platform and displays results though a lateral flow test strip for easy visualization. This method offers a practical, real-time alternative for clinical settings.\u003c/p\u003e\u003cp\u003eTo validate the feasibility of the multiplex LAMP amplification system, we initially used TGEV and PoRV G9 as models to establish amplification methods with varying primer concentrations, which were then paired with the CRISPR system for product detection. The LAMP-CRISPR system demonstrated high specificity, even with mixed virus samples. Building on this, we optimized the system for triple and quadruple LAMP amplification setups. However, as the number of primers sets increased, competitive amplification was observed, especially at high primer concentrations, with TGEV, PDCoV, and PoRV G9 amplifying more strongly than PEDV. By reducing the primer concentrations, we found that the quadruple LAMP system, even at 1/4 of the optimal primer set concentration, allowed the CRISPR system to detect all four viruses efficiently, significantly enhancing detection sensitivity to as low as 1\u0026ndash;10 copies, com-pared to the traditional PCR methods.\u003c/p\u003e\u003cp\u003eA potential issue encountered in the study was the spontaneous quenching of fluorescent probes during storage, which reduced visibility under UV excitation. To address this, the fluorescent reporter probe in the LAMP-CRISPR system was replaced with a FAM-Biotin-ssDNA reporter molecule and paired with lateral flow dipstick assay. This modification enabled detection of even low levels of target sequences, where the cleavage of FAM-Biotin-ssDNA resulted in two visible red lines on the dipstick. This system, which operates at a constant temperature using a water bath, breaks the limitations imposed by traditional molecular diagnostic methods, offering true real-time clinical detection without the need for complex equipment.\u003c/p\u003e\u003cp\u003eIn summary, the quadruple LAMP-CRISPR detection system was successfully ap-plied to clinical sample testing and compared with traditional nucleic acid detection methods. The results for PEDV, TGEV, and PDCoV were consistent between the two detection methods, with a 100% overall agreement. In detail, the positive rates were 31.57% for PEDV, 6.31% for TGEV, and 21% for PDCoV. For PoRV, the positivity rate was 8.42%, with an overall agreement of 98.94% compared to RT-qPCR, detecting one additional positive sample, likely due to the higher sensitivity of the quadruple LAMP-CRISPR system. Furthermore, the system successfully detected multiple viral genomes within the same sample, providing experimental evidence of mixed infections involving PEDV, TGEV, PDCoV, and PoRV.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study establishes a multiplex nucleic acid detection platform that integrates CRISPR/Cas12a with LAMP to enable rapid, cost-effective, and highly sensitive simultaneous detection of PEDV, TGEV, PDCoV, and PoRV G9. Through systematic molecular and functional characterization of the crRNA/Cas12a complex, we defined crRNA specificity and optimized the crRNA:Cas12a stoichiometry, thereby markedly improving diagnostic specificity and accuracy\u0026mdash;advances that align with the biological study of natural macromolecules. The assay is field-deployable and readily scalable, supporting early and precise pathogen detection in veterinary settings, mitigating economic losses in the swine industry, strengthening biosecurity and disease management, and providing a transferable framework and practical basis for broader application of CRISPR/Cas systems in molecular diagnostics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYY, ABW, and LL contributed to conception and design of the study. LL and ZJH conducted experiments. TL, YJC, XYP, YGW, FL, YMY, YYC, KXW, XJH, YZ and NDW\u0026nbsp;analyzed data. LL and ABW wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was funded by \u0026ldquo;Research and development of protein particle vaccines for African Swine Fever and other major viral diseases\u0026rdquo; (2021NK1030) and Support was also provided by \u0026ldquo;Furong\u0026rdquo; Scholar funding to Aibing Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u0026nbsp;\u003c/strong\u003eThe ethical approval and usage plan for this study have been approved by the Biomedical Research Ethics Committee of Hunan Agricultural University (No. 2025156).\u0026nbsp;We have adhered to the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e: No potential conflict of interest was reported by the author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e The data that support the findings of this study are openly available at\u0026nbsp;\u003cstrong\u003ehttps://doi.org/10.6084/m9.figshare.29556512.v1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u003c/strong\u003e The following material is available online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLu S, Tong X, Han Y, et al. Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a. Nat Biomed Eng. 2022;6(3):286\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWen F, Yang J, Li A, et al. Genetic characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Guangdong, China, between 2018 and 2019. PLoS ONE. 2021;16(6):e0253622.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu H, Wang G, Liu X, et al. Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA. Microb Pathog. 2024;191:106646.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJung K, Saif LJ. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J. 2015;204(2):134\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong D, Park B. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes. 2012;44(2):167\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLednicky JA, Tagliamonte MS, White SK, et al. Independent infections of porcine deltacoronavirus among Haitian children. Nature. 2021;600(7887):133\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBigault L, Brown P, Bernard C, Blanchard Y, Grasland B. Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR. J Virol Methods. 2020;283:113906.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVlasova AN, Amimo JO, Saif LJ. Porcine Rotaviruses: Epidemiology, Immune Responses and Control Strategies. Viruses. 2017;9(3):48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang F, Luo Y, Lin C, et al. Epidemiological monitoring and genetic variation analysis of pathogens associated with porcine viral diarrhea in southern China from 2021 to 2023. Front Microbiol. 2024;15:1303915.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXue R, Tian Y, Zhang Y, et al. Diversity of group A rotavirus of porcine rotavirus in Shandong province China. Acta Virol. 2018;62(3):229\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHou W, Fan M, Zhu Z, Li X. Establishment and Application of a Triplex Real-Time RT-PCR Assay for Differentiation of PEDV, PoRV, and PDCoV. Viruses. 2023;15(6):1238.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarthaler D, Homwong N, Rossow K, et al. Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples. J Virol Methods. 2014;209:30\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi C, Liang J, Yang D, et al. Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method. Anim (Basel). 2022;12(19):2712.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi P, Ren X. Reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus. Curr Microbiol. 2011;62(3):1074\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang F, Ye Y, Song D, et al. A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification. Biol Res. 2017;50(1):30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu X, Zhang S, et al. Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine. Chi J Virol. 2016;32(6):740\u0026ndash;6. Chinese.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCong Y, Li X, Bai Y, et al. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells. Virol. 2015;478:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrincone A, Schwegmann-We\u0026szlig;els C. Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein. Virus Res. 2015;202:3\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkda F, Lawson S, Liu X, et al. Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus. BMC Vet Res. 2016;12:95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Zhou J, Zhu X, et al. Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China. Microb Pathog. 2024;190:106612.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJung K, Miyazaki A, Saif LJ. Immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (Gn) pigs infected with porcine epidemic diarrhea virus (PEDV) and serum cytokine responses of Gn pigs to acute PEDV infection. Res Vet Sci. 2018;119:99\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhitworth KM, Rowland RRR, Petrovan V, et al. Resistance to coronavirus infection in amino peptidase N-deficient pigs. Transgenic Res. 2019;28(1):21\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11248-018-0100-3\u003c/span\u003e\u003cspan address=\"10.1007/s11248-018-0100-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi B, Zheng L, Li H, Ding Q, Wang Y, Wei Z. Porcine deltacoronavirus causes diarrhea in various ages of field-infected pigs in China. Biosci Rep. 2019;39(9):BSR20190676.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSozzi E, Moreno A, Lelli D, et al. Development and validation of a monoclonal antibody-based competitive ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV). Res Vet Sci. 2018;121:106\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSestak K, Zhou Z, Shoup DI, Saif LJ. Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs. J Vet Diagn Invest. 1999;11(3):205\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan F, Shan F, Hou J, et al. Establishment and application of PDCoV antigen-specific DAS-ELISA detection method. BMC Vet Res. 2024;20(1):342.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMemon AM, Chen F, Khan SB, et al. Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus. Anim Biotechnol. 2023;34(5):1807\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Li S, Wang J, Liu G. CRISPR/Cas Systems towards Next-Generation Biosensing. Trends Biotechnol. 2019;37(7):730\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePardee K, Green AA, Takahashi MK, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 2016;165(5):1255\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Wang Y, Xu L, Lou C, Ouyang Q, Qian L. Paired dCas9 design as a nucleic acid detection platform for pathogenic strains. Methods. 2022;203:70\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi L, Li S, Wu N, et al. HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation. ACS Synth Biol. 2019;8(10):2228\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLei L, Liao F, Tan L, et al. LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2. Anim (Basel). 2022;12(18):2413.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu S, Tao D, Liao Y, et al. Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus. ACS Synth Biol. 2021;10(10):2499\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe Q, Yu D, Bao M, et al. High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system. Biosens Bioelectron. 2020;154:112068.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang H, Li H, Tang B, et al. Fast and sensitive differential diagnosis of pseudorabies virus-infected versus pseudorabies virus-vaccinated swine using CRISPR-Cas12a. Microbiol Spectr. 2024;12(1):e0261723.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang K, Liang Y, Li Y, et al. Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains. Anal Bioanal Chem. 2021;413(30):7521\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei J, Li Y, Cao Y, et al. Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay. Front Cell Infect Microbiol. 2022;12:879887.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGerber PF, Gong Q, Huang YW, Wang C, Holtkamp D, Opriessnig T. Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA. Vet J. 2014;202(1):33\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTabatabaei MS, Ahmed M. Enzyme-Linked Immunosorbent Assay (ELISA). Methods Mol Biol. 2022;2508:115\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu J, Tao D, Chen X, et al. Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay. Viruses. 2022;14(4):833.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao J, Shi BJ, Huang XG, et al. A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012. J Virol Methods. 2013;194(1\u0026ndash;2):107\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\" style=\"margin-right: calc(44%); width: 56%;\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCt values of PoRV G9-positive samples detected by RT-qPCR.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 18.3183%;\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 9.6096%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 18.3183%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCt value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 9.6096%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e22.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.43\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e20.45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.08\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 12.012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCt values of PEDV-positive samples detected by RT-qPCR.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCt value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e21.79\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e19.80\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e17.63\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e21.10\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e23.68\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e27.28\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e24.14\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e24.06\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e24.32\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e21.20\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCt value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e22.79\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.91\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.43\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.93\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.83\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.88\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.72\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e23.75\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCt value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e34.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.84\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.94\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.69\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.69\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e34.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\" style=\"margin-right: calc(50%); width: 50%;\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCt values of TGEV-positive samples detected by RT-qPCR.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 20.4082%;\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 20.4082%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCt value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e29.40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.96\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCt values of PDCoV-positive samples detected by RT-qPCR.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCt value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e4.67\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e27.64\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e27.47\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e28.88\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e29.40\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e28.96\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e31.33\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e29.85\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e26.63\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e26.73\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCt value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.84\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.54\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.98\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison and compliance validation of the quadruple LAMP-CRISPR assay system with conventional nucleic acid detection methods.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDetection\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePositive coincidence rate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNegative coincidence rate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOverall coincidence rate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePEDV RT-qPCR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePEDV LAMP-CRSPR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePDCoV RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePDCoV LAMP-CRSPR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGEV RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGEV LAMP-CRISPR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePoRV G9 RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e88\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e98.86%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e98.94%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePoRV G9 LAMP-CRISPR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e87\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\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":"Four porcine diarrhea viruses, crRNA/Cas12a complex, multiple LAMP","lastPublishedDoi":"10.21203/rs.3.rs-7675377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7675377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePorcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus-A (PoRV) G9 are major swine pathogens primarily responsible for gastrointestinal diseases, particularly affecting lactating piglets and resulting in significant economic losses, especially in China. This study introduces a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, with a molecular weight of approximately 144 kDa, enhances diagnostic accuracy through targeted gene editing. Incorporating fluorescence report probes and a lateral flow dipstick assay, this approach establishes a visual detection system capable of simultaneously identifying all four viruses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIt enables the visualization of viral genomes from as low as 1 to 10 copies/\u0026micro;L without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.94% concordance with RT-qPCR for PoRV G9.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOffering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its critical role in enhancing biosecurity and disease management in veterinary contexts.\u003c/p\u003e","manuscriptTitle":"A crRNA/Cas12a complex-driven rapid and visual detection method for four porcine diarrhea viruses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 14:12:30","doi":"10.21203/rs.3.rs-7675377/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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