Evaluation of Visual LAMP detection of Plasmodium falciparum and non-Plasmodium falciparum

Evaluation of Visual LAMP detection of Plasmodium falciparum and non-Plasmodium falciparum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Evaluation of Visual LAMP detection of Plasmodium falciparum and non- Plasmodium falciparum Shui Mao Zhou, Zu-Wu Tu, Yan Yang, Wei Feng Tang, Fang Chen, Xi Shuai Jia This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.17953/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: To detect the DNA of the malaria using Loop-mediated isothermal amplification (LAMP) and evaluate the effect. Method; According to the 18s rRNA sequence of the malaria, the LAMP primer of Plasmodium falciparum and non-Plasmodium falciparum were designed using Primer Explorer V5 software. The method of Visual LAMP detecting malaria was formation. The sensitivity, specificity and amplification efficiency were tested, compared with the Nest-PCR. Result: The filter papers of blood from 47 patients with Plasmodium falciparum and 49 with non-Plasmodium falciparum were detected using Visual LAMP. The patients with Plasmodium falciparum were all positive. In the patients with non-Plasmodium falciparum , false negative rate was 2.1%. In the 10 patients with Leishmaniasis, 8 with Schistosomiasis japonicum, 38 healthy human, none was positive. The results using visual LAMP were consistent with Nest-PCR (Kappa value = 0.956). All of the Tt in Visual LAMP are less than 60 min, especially the Tt of Plasmodium falciparum is 18.2 min. When adding calcium pyridoxine indicator during the Visual LAMP detection, the progress of the reaction to different substes of malaria delayed of about 9-23 min. Conclusion: LAMP is efficient and visible, deserved the prospect of application in the on-site and primary medical institutions. Infectious Diseases Malaria LAMP Nest-PCR Visualization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Malaria is one of the most important parasitic infectious diseases which threaten human life safety [1] . There are 4 kinds of malaria parasites, of which Plasmodium falciparum is the most harmful, toxic, and with the highest fatality rate [2,3] . According to The World Malaria Report 2018, $3.1 billion was invested in control and eradication of malaria in 2017, with a total of 219 million malaria cases worldwide and 435,000 deaths. In Africa, South-East Asia, the Eastern Mediterranean, the Western Pacific and China, Plasmodium falciparum accounted for 99.7%, 62.8%, 69%, 71.9% and 63.7% in all malaria cases, respectively [4,5] . Malaria control in China is at the elimination phase. Importation of malaria remains widespread due to increased travel and population movements to malaria-endemic areas. To reduce malaria complications, fatality and transmission, it is important to diagnose malaria correctly [1] . The common detection methods of malaria parasites are mainly optical microscope examination and RDT. In the chronic infection stage, due to the malaria parasite emias always maintain low density, optical microscope examination and RDT leak detection [3,6] . The RDT is insensitive to Plasmodium vivax and Plasmodium ovale. The patients with asymptomatic Plasmodium falciparum infection may be malaria-infected reservoir [7,8] . Therefore, a high sensitivity diagnostic methods able to detect low-density malaria parasite emias for the elimination of malaria is critical [6,9] .There is urgent need for extremely sensitive and field-based diagnostic system [10] . Molecular diagnosis is a highly sensitive method for detecting malaria infection [7,9,11] . For example, PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, but it requires expensive equipment [7] . The sensitivity of the ring-mediated so-mediated temperature amplification (LAMP) method is close to or exceeds the NEST-PCR [6,7,12,13] . LAMP method does not require expensive equipment and results can be judged by visual observation. The main purpose of this study is to evaluate the diagnostic performance of LAMP and compare it with the results of detection using microscope and PCR. Material and Method 1. Reagents and instruments: Bst DNA polymerase (large fragment, Biolabs, USA), dNTPs (TANGEN, China), calcium-yellow chlorophyll (Calcein, Sigma, USA), manganese tetrachloral chloride (MnCl2·4H2O; Sigma, USA), DNA Extraction Kit (OMEGA, USA), LA–500 Real-Time Turbidity Detection System (Loopamp, Japan), PTC–200 Peltier Thermal Cycler Gradient PCR (Bio-Rad, USA), Genius Bio-Imaging System Gel Imaging System (Syngene, UK). 2. Sample: This study was certified by the Ethics Committee of the Wuhan Center for Disease Control and Prevention. The informed consent of all patients and healthy people was obtained. 2.1 Malaria patients: Blood samples from malaria patients were clinically diagnosed in 2013–2018. The patients’ earlobe blood was collected, filter paper of blood spots was made(about 20μl), naturally dried, saved at 4℃. 2.2 Leishmaniasis patients: blood on glass, provided by the China Center for Disease Control and Prevention of Parasitic Diseases. 2.3 Schistosomiasis japonicum samples: bloodfrom rabbitinfected with Schistosomiasis japonicum, provided by the Hubei Provincial Center for Disease Control and Prevention schistosomiasis Research. 2.4 Healthy people: blood from the healthy volunteers were collected, filter paper blood spots were made, naturally dried, saved at 4℃. 3. DNA extraction: filter paper with dried blood were droplets by 3mmx3mm, according with The OMEGA D3096 micro-DNA extraction kit operation, extract DNA of malaria (50 μl), saved at –20℃. 4. Primer Synthesis: 3 groups of LAMP Primer was designed with Primer Explorer V5, including Plasmodium falciparum (AJ276844) and non- Plasmodium falciparum (NP.f, KY923424, LT594520, MF693428). For detection of malaria, 1 group of LAMP primers with high sensitivity, specific and amplification efficiency was screened by experimental. The Nest-PCR primer sequence (18S rRNA gene) is synthesized according to the references [6,14,15,16] , by Bioengineering Co., Ltd(Shanghai). 5. Visual LAMP: 10X Thermo pol Buffer 2.5μl, 10 mM dNTPs 3.5μl, 100 mM MgSO4 1.5μl, 40μM FIP, 40μl BIP, 5μM F3, 5μM B3, 10μM LpF, 10 m LpB each 1 sl, Bst DNA polymerase 1μl, 25μM Calcein 1μl, DNA extract 2μl, adding H 2 O to 25μl, 65℃ 60–90 min, observation. 6. Nest-PCR: reaction system and conditions and operation according to the reference [14,15,16] . 7. Results judge: LAMP results observed by eyes: green fluorescence is positive, orange is negative; To identified the subsets of malaria, the PCR amplification product was analysised by 1% agar sugar gel electrophoresis, and the Purpose Stripe of Plasmodium falciparum ( P.f), Plasmodium vivax (P.v), Plasmodium malaria ( P.m). and Plasmodium ovale curtisi P.oc) and Plasmodium ovale wallikeri (P.ow) are 205bp, 120bp, 144bp, 800bp and 780 bp respectively. 8. Statistic: Database was established using Excel 2003 software. The comparison between groups was analyzed with the test of χ 2 and the test level is α＝0.05 Results Judgment of Visual LAMP and Nest-PCR results: Adding calcium-yellow toludoline indicator, LAMP reaction liquid was observed by eyes. Changing into green as positive, unchanging (orange) as negative (Figure 1); The Nest-PCR amplification gel Imaging showed 205bp 、 120bp 、 144 bp 、 800 bp 和 780 bp bands , which are P.f, P.v, P.m, P.oc and P.ow positive each(Figure 2). Visual LAMP and Nest-PCR : 96 filter papers of blood from patients infected with malaria confirmed in 2013-2018 were tested by Visual LAMP and Nest-PCR simultaneously. 93 of them were positive (Table 1). Table 1 Visual LAMP and Nest-PCR test in malaria patients Method P.f P.v P.o P.m P.f +N P.f Total Microscope 47 27 18 4 0 96 Nest-PCR 46 25 17 4 1 93 LAMP 45 25 16 4 3 93 P =0.449 Consistency of Visual LAMP and Nest-PCR ： 96 filter papers of blood from patients infected with malaria and 38 blood sample from healthy human were tested by Visual LAMP and Nest-PCR. The consistent of the two method were analyzed. Kappa = 0.956 (Table 2). Table 2 The consistent of Visual LAMP and Nest PCR Method Classification LAMP Total P.f NP.f P.f+NP.f Negative Nest PCR P.f 45 0 1 0 46 NP.f 0 44 1 1 46 P.f+NP.f 0 0 1 0 1 Negative 0 1 0 40 41 Total 45 45 3 41 134 Kappa = 0.956 The specificity of the method: 10 patients with Leishmaniasis , 8 with Schistosomiasis japonicum , 38 healthy human were tested by Visual LAMP and Nest PCR , none was positive. The effect of calcium-yellow-green indicator on LAMP reaction: DNA from blood of 25000 P.f/μl, 3000 P.v/μl, 4000 P.o/μl, 2000 P.m/μl was extracted as template. The Tt of turbidity peak time are 18.2 min, 35.6min, 50.8min, 33.1min when the LAMP reaction fluid was without calcium-yellow-green indicator (A) (Figure 3-6). Tt plus are 29.1 min, 44.1min, 74.1min, 74.1min, 47.8min when calcium-yellow green oxalate indicator were added(B) (Figure 3-6). No turbidity showed in 90min when pure water is used as a template. Discussion Malaria is an infectious disease caused by different types of Plasmodium, which live in red blood cells (RBC) and destroy host erythrocyte, leading to fever and/or anemia, or even kidney failure, brain coma and death. Accurate diagnosis of malaria infection is critical. Microscope examination of blood smears is still considered the gold standard for the diagnosis of malaria, which was labor-intensive, time-consuming [7,10,16] , with detection limit of 100 malaria parasites/µl [10,16] . It was usually unable to detect low-density and mixed infections, the sensitivity and specificity of which depends on the skill of the technician, quality and staining quality of blood smear, and so on [3,17] . Compared with molecular diagnostic methods, in malaria endemic areas, only half of malaria patients were correctly diagnosed. The WHO external assessment of the malaria parasite’s mirror test capacity in China showed that the correct rates of negative, Plasmodium falciparum ( P.f), , Plasmodium vivax ( P.v) were 92.5%, 78.3%, 70.8% respectively [20] . RDT is facilitate and fast, with detection limit of 100 plasmids/μl [19,21] . It shows high sensitivity to plasmid infections of more than 100 plasmids/μl(median 94.3%), sensitivity of which decrease significantly with a density of less than 100 plasmids/μl (median 74.1%) [21] . Meantime it shows limited sensitivity when detecting low-density infection [16,22] , and less sensitivity to non- P.f， P.v as 66.0–88.0%, P.o as 5.5–86.7%, P.m as 21.4–45.2% [19] . PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, with a high sensitivity and specificity limit to 1–10 malaria parasites/μl [10,11] . However, it requires technical expertise and infrastructure which limit its wide range of applications. LAMP has been used in clinical diagnosis of a variety of infectious diseases, including malaria. It is considered to be faster, more operational and cost-effective than PCR. At the same time, it has been reported that in testing malaria, LAMP own a similar sensitivity and specificity to PCR [7,10,11,23,,24] . It is capable of identifying low-density malaria infections by LAMP, which cannot be detected by the microscope [7] . Sensitivity of LAMP is several orders of magnitude higher than by microscope and RDT [21] . At present, detecting malaria by LAMP is mainly to detect the genus malaria and Plasmodium falciparum, the primer is depend on 18s rRNA region of the malaria parasite species. In this study, 3 groups of LAMP Primer was designed including P.f and non- P.f. For detection of malaria, 1 group of LAMP primers with high sensitivity, specific and amplification efficiency was screened by experimental. By Visual LAMP, filter paper of blood from 47 cases of P.f and 49 non- P.f patients was detected. There was no significant difference with by Nest-PCR or by microscope examination ( P > 0.05), of which 2 non- P.f patients were both negative tested by Visual LAMP and Nest-PCR. It may because the blood on filter paper was kept too long, and the poor extraction quality of DNA or not extracted at all. 1 P.v case by Nest-PCR was positive, but by Visual LAMP was negative. 1 P.f case by visual LAMP was positive, but by Nest-PCR was negative. The false negative rate of visual LAMP of non- P.f is 2.1%. 47 cases of P.f were all positive by Visual LAMP and Nest-PCR, of which 2 cases of P.f and non- P.f mixture infection were found by Visual LAMP, only 1 case of mixture infection by Nest-PCR, and 1 case of P.o by microscope examination. It may be due to the false positive or the more sensitivity of Visual LAMP, that the P.f and non- P.f mixture infection could be discovered by Visual LAMP but microscope examination.Detected by Visual LAMP and Nest PCR simultaneously, the results of blood in filter paper from 96 malaria patient and 38 healthy men showed extremely consistent (Kappa = 0.956). In the results of 10 patients with Leishmaniasis, 8 with Schistosomiasis japonicum, 38 healthy human, none was positive，which indicated that the Visual LAMP primer in this study had high specificity. Visual LAMP is a method that require no expensive instruments or special laboratories, with simple and convenient operation, suitable for on-site inspection. In LAMP of this study, LA–500 real-time turbidity detection system were used and DNA from blood of 25000 P.f/μl, 3000 P.v/μl, 4000 P.o/μl, 2000 P.m/μl was extracted as template. When the LAMP reaction was not added with calcium-yellow green indicator, the turbidity peak time(Tt) is 18.2 min, 35.6 min, 50.8min, 33.1min. Mean Tt was less than 60min, which represented a higher amplification efficiency. LAMP Tt of P.v is 18.2min in particular, the amplification efficiency of which was very high. It could meet the requirements of LAMP. When adding the calcium-yellow green indicator in LAMP, Tt is 29.1min, 44.1 min, 74.1 min, 47.8min. The delay effect of adding calcite-yellow-green-oxidant in malaria subsets was different, the mean of which was about 9–23min. It is convenient for the visual observation of the results when adding calcium-yellow green indicator, suitable for on-site application. As worldwide countries set targets of malaria elimination, accurate diagnosis, optimal treatment, stop of transmission, and reducing mortality of malaria are becoming critical [25,26] . As a result of the sudden decline in local patients and the gradual elimination of malaria, there are a trend of degradation in the clinical diagnosis and screening capacity of malaria. The prevalence of infrequent diagnosis, with some cases ranging from onset to the determination of diagnosis for more than 30 days. At the same time, due to increasing travel and population movements to malaria-endemic areas, imported malaria is still widespread [21] . Particularly, asymptomatic malaria infection and low-density malaria parasite emias are hard to be detected by microscope examination and RDT due to below the detection threshold [11,24] , causing misdiagnosis or delayed diagnosis [3,21] . But they could maintaining transmission of malaria as infected intermediate host [24,26] . It has been reported that subclinical malaria accounts for 70–80% of all malaria infections [11] . Therefore, it is a challenge to detect the asymptomatic malaria parasite infection and low density malaria parasite emias in malaria prevention and control [8,24] . Stopping transmission of malaria requires detecting methods with high sensitive. The LAMP shows high sensitivity and specificity, by which asymptomatic and asymptomatic malaria infections could be diagnosed accurately, and low-density infections that cannot be detected by conventional diagnosis methods could be identified [26] . At the same time, LAMP is fast, easy to operate and effective which provides the possibility for clinical diagnosis and monitoring of malaria. Conclusion Visual LAMP is highly sensitive and specific to the diagnosis of malaria,and the results using visual LAMP are consistent with Nest-PCR.During the malaria elimination stage,visual LAMP provides a portable, sensitive and specific method for on-site inspection. Declarations Ethics approval and consent to participate This study was certified by the Ethics Committee of the Wuhan Center for Disease Control and Prevention. The informed consent of all patients and healthy people was obtained. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study is financially supported by the Health Bureau of Wuhan (award number: WG18Q05),and the the Health Bureau of Wuhan(award number: WG17B04). Authors’ contributions ZHOU Shui-mao and JIA Xi-shuai conceived the study. ZHOU Shui-mao,TU Zu-wu and JIA Xi-shuai undertook statistical analysis,and drafted the manuscript. YANG Yan,TANG Wei-feng, CHEN Fang undertook epidemiological investigations and sample collection.All authors contributed to the writing of the manuscript and approved the submitted version of themanuscript. Acknowledgements Not applicable References [1]Study on the stability and specificity of the kit for rapid detection of Plasmodium falciparum and Plasmodium vivax by Colloidal gold method［J］.Straits Science,2019,147（3）:3-6 [2]Wang ZH,Wei CY,Wang H.Research development on non-coding RNA of Plasmodium falciparum ［J］. Chin J Parasitol Parasit Dis.2018,36（4）:409-412 [3] Hashimoto M , Sakamoto H , Ido Y ,Iet al. n situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species in wide-range thin blood smears［J］. Malar J . 2018; 17: 235，doi: 10.1186/s12936-018-2381-7 [4]World Health Organization. World malaria report 2017[R].Geneva: WHO, 2017. 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13:01:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.17953/v1","doiUrl":"https://doi.org/10.21203/rs.2.17953/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":198608,"identity":"152427d4-1820-4330-9b55-a5afdf3c09fc","added_by":"auto","created_at":"2019-12-03 20:54:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":145450,"visible":true,"origin":"","legend":"LAMP reaction\n1:P.f, 2:P.v, 3:P.m, 4:P.o, 5:Leishmaniasis, 6:S.j, 7:Healthy Man, 8:Blank Control","description":"","filename":"1.PNG","url":"https://assets-eu.researchsquare.com/files/0ac8146a-cabd-4378-ad98-688201cd575b/v1/1.PNG"},{"id":198610,"identity":"bcb3c433-923c-4e2c-9a88-c67240685869","added_by":"auto","created_at":"2019-12-03 20:54:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71095,"visible":true,"origin":"","legend":"PCR Amplifier Gel Imaging\n1,2:P.f, 3,4:P.v, 5,6:P.m, 7,8:P.oc, 9,10:P.ow, M:Marker","description":"","filename":"2.PNG","url":"https://assets-eu.researchsquare.com/files/0ac8146a-cabd-4378-ad98-688201cd575b/v1/2.PNG"},{"id":198611,"identity":"a24af282-b19b-4a9c-bde7-429cefc4baba","added_by":"auto","created_at":"2019-12-03 20:54:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18558,"visible":true,"origin":"","legend":"Effect of the indicator on the LAMP of P.f","description":"","filename":"3.PNG","url":"https://assets-eu.researchsquare.com/files/0ac8146a-cabd-4378-ad98-688201cd575b/v1/3.PNG"},{"id":198612,"identity":"8ab601bf-0208-48f4-a337-71c54271adeb","added_by":"auto","created_at":"2019-12-03 20:54:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17918,"visible":true,"origin":"","legend":"Effect of the indicator on the LAMP of P.v","description":"","filename":"4.PNG","url":"https://assets-eu.researchsquare.com/files/0ac8146a-cabd-4378-ad98-688201cd575b/v1/4.PNG"},{"id":198613,"identity":"205a02e5-1726-4af5-ba84-b5373422a872","added_by":"auto","created_at":"2019-12-03 20:54:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17630,"visible":true,"origin":"","legend":"Effect of the indicator on the LAMP of P.o","description":"","filename":"5.PNG","url":"https://assets-eu.researchsquare.com/files/0ac8146a-cabd-4378-ad98-688201cd575b/v1/5.PNG"},{"id":198614,"identity":"ff89b593-6546-4e4f-8d3f-5c18a3816a0a","added_by":"auto","created_at":"2019-12-03 20:54:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16577,"visible":true,"origin":"","legend":"Effect of the indicator on the LAMP of P.m","description":"","filename":"6.PNG","url":"https://assets-eu.researchsquare.com/files/0ac8146a-cabd-4378-ad98-688201cd575b/v1/6.PNG"},{"id":15665695,"identity":"e6c2c054-ced6-4a4e-9706-41c82bf902d9","added_by":"auto","created_at":"2021-11-18 13:32:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":463384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8582/v1/a4a79b03-e039-4082-ad51-383883a2786f.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEvaluation of Visual LAMP detection of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and non-\u003cem\u003ePlasmodium falciparum\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\n\u003cp\u003eMalaria is one of the most important parasitic infectious diseases which threaten human life safety\u003csup\u003e[1]\u003c/sup\u003e. There are 4 kinds of malaria parasites, of which \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is the most harmful, toxic, and with the highest fatality rate\u003csup\u003e[2,3]\u003c/sup\u003e. According to The World Malaria Report 2018, $3.1 billion was invested in control and eradication of malaria in 2017, with a total of 219 million malaria cases worldwide and 435,000 deaths. In Africa, South-East Asia, the Eastern Mediterranean, the Western Pacific and China, \u003cem\u003ePlasmodium falciparum\u003c/em\u003e accounted for 99.7%, 62.8%, 69%, 71.9% and 63.7% in all malaria cases, respectively\u003csup\u003e[4,5]\u003c/sup\u003e. Malaria control in China is at the elimination phase. Importation of malaria remains widespread due to increased travel and population movements to malaria-endemic areas. To reduce malaria complications, fatality and transmission, it is important to diagnose malaria correctly \u003csup\u003e[1]\u003c/sup\u003e. The common detection methods of malaria parasites are mainly optical microscope examination and RDT. In the chronic infection stage, due to the malaria parasite emias always maintain low density, optical microscope examination and RDT leak detection \u003csup\u003e[3,6]\u003c/sup\u003e. The RDT is insensitive to \u003cem\u003ePlasmodium vivax and Plasmodium ovale.\u003c/em\u003e The patients with asymptomatic \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infection may be malaria-infected reservoir\u003csup\u003e[7,8]\u003c/sup\u003e. Therefore, a high sensitivity diagnostic methods able to detect low-density malaria parasite emias for the elimination of malaria is critical\u003csup\u003e[6,9]\u003c/sup\u003e.There is urgent need for extremely sensitive and field-based diagnostic system \u003csup\u003e[10]\u003c/sup\u003e. Molecular diagnosis is a highly sensitive method for detecting malaria infection \u003csup\u003e[7,9,11]\u003c/sup\u003e. For example, PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, but it requires expensive equipment \u003csup\u003e[7]\u003c/sup\u003e. The sensitivity of the ring-mediated so-mediated temperature amplification (LAMP) method is close to or exceeds the NEST-PCR \u003csup\u003e[6,7,12,13]\u003c/sup\u003e. LAMP method does not require expensive equipment and results can be judged by visual observation. The main purpose of this study is to evaluate the diagnostic performance of LAMP and compare it with the results of detection using microscope and PCR.\u003c/p\u003e\n\n"},{"header":"Material and Method","content":"\n\u003cp\u003e\u003cem\u003e1. \u003c/em\u003eReagents and instruments: Bst DNA polymerase (large fragment, Biolabs, USA), dNTPs (TANGEN, China), calcium-yellow chlorophyll (Calcein, Sigma, USA), manganese tetrachloral chloride (MnCl2·4H2O; Sigma, USA), DNA Extraction Kit (OMEGA, USA), LA–500 Real-Time Turbidity Detection System (Loopamp, Japan), PTC–200 Peltier Thermal Cycler Gradient PCR (Bio-Rad, USA), Genius Bio-Imaging System Gel Imaging System (Syngene, UK).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e2. \u003c/em\u003eSample: This study was certified by the Ethics Committee of the Wuhan Center for Disease Control and Prevention. The informed consent of all patients and healthy people was obtained.\u003c/p\u003e\n\u003cp\u003e2.1 Malaria patients: Blood samples from malaria patients were clinically diagnosed in 2013–2018. The patients’ earlobe blood was collected, filter paper of blood spots was made(about 20μl), naturally dried, saved at 4℃.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2 \u003c/em\u003e\u003cem\u003eLeishmaniasis \u003c/em\u003epatients: blood on glass, provided by the China Center for Disease Control and Prevention of Parasitic Diseases.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3 \u003c/em\u003e\u003cem\u003eSchistosomiasis japonicum\u003c/em\u003e samples: bloodfrom rabbitinfected with\u003cem\u003e Schistosomiasis japonicum,\u003c/em\u003e provided by the Hubei Provincial Center for Disease Control and Prevention schistosomiasis Research.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4 \u003c/em\u003eHealthy people: blood from the healthy volunteers were collected, filter paper blood spots were made, naturally dried, saved at 4℃.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e3. \u003c/em\u003eDNA extraction: filter paper with dried blood were droplets by 3mmx3mm, according with The OMEGA D3096 micro-DNA extraction kit operation, extract DNA of malaria (50 μl), saved at –20℃.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e4. \u003c/em\u003ePrimer Synthesis: 3 groups of LAMP Primer was designed with Primer Explorer V5, including \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (AJ276844) and non-\u003cem\u003ePlasmodium falciparum\u003c/em\u003e (NP.f, KY923424, LT594520, MF693428). For detection of malaria, 1 group of LAMP primers with high sensitivity, specific and amplification efficiency was screened by experimental. The Nest-PCR primer sequence (18S rRNA gene) is synthesized according to the references \u003csup\u003e[6,14,15,16]\u003c/sup\u003e, by Bioengineering Co., Ltd(Shanghai).\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e5. \u003c/em\u003eVisual LAMP: 10X Thermo pol Buffer 2.5μl, 10 mM dNTPs 3.5μl, 100 mM MgSO4 1.5μl, 40μM FIP, 40μl BIP, 5μM F3, 5μM B3, 10μM LpF, 10 m LpB each 1 sl, Bst DNA polymerase 1μl, 25μM Calcein 1μl, DNA extract 2μl, adding H\u003csub\u003e2\u003c/sub\u003eO to 25μl, 65℃ 60–90 min, observation.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e6. \u003c/em\u003eNest-PCR: reaction system and conditions and operation according to the reference\u003csup\u003e[14,15,16]\u003c/sup\u003e.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e7.\u003c/em\u003e Results judge: LAMP results observed by eyes: green fluorescence is positive, orange is negative; To identified the subsets of malaria, the PCR amplification product was analysised by 1% agar sugar gel electrophoresis, and the Purpose Stripe of \u003cem\u003ePlasmodium falciparum (\u003c/em\u003eP.f), \u003cem\u003ePlasmodium vivax\u003c/em\u003e (P.v), \u003cem\u003ePlasmodium malaria (\u003c/em\u003eP.m). \u003cem\u003eand Plasmodium ovale\u003c/em\u003e \u003cem\u003ecurtisi\u003c/em\u003eP.oc) and \u003cem\u003ePlasmodium ovale\u003c/em\u003e \u003cem\u003ewallikeri\u003c/em\u003e (P.ow) are 205bp, 120bp, 144bp, 800bp and 780 bp respectively.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e8.\u003c/em\u003e Statistic: Database was established using Excel 2003 software. The comparison between groups was analyzed with the test of χ 2 and the test level is α＝0.05\u003c/p\u003e\n\n"},{"header":"Results","content":"\u003col\u003e\n\u003cli\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e Judgment of Visual LAMP and Nest-PCR results: Adding calcium-yellow toludoline indicator, LAMP reaction liquid was observed by eyes. Changing into green as positive, unchanging (orange) as negative (Figure 1); The Nest-PCR amplification gel Imaging showed 205bp\u003c/span\u003e、\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e120bp\u003c/span\u003e、\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e144 bp\u003c/span\u003e、\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e800 bp\u003c/span\u003e和\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e780 bp bands , which are P.f, P.v, P.m, P.oc and P.ow positive each(Figure 2).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003col start=\"2\"\u003e\n\u003cli\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e Visual LAMP and Nest-PCR : 96 filter papers of blood from patients infected with malaria confirmed in 2013-2018 were tested by Visual LAMP and Nest-PCR simultaneously. 93 of them were positive (Table 1).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eTable 1 Visual LAMP and Nest-PCR test in malaria patients\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse;\" width=\"504\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 13.5pt;\"\u003e\n\u003ctd style=\"width: 56.7pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eMethod\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 51.3pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"68\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eP.v\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eP.o\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eP.m\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eP.f\u003c/span\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e+N\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: left; tab-stops: 13.8pt; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Total\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 13.5pt;\"\u003e\n\u003ctd style=\"width: 56.7pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eMicroscope\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 51.3pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"68\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e47\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; 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padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e96\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 13.5pt;\"\u003e\n\u003ctd style=\"width: 56.7pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eNest-PCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 51.3pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"68\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e46\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e25\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e17\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e93\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 13.5pt;\"\u003e\n\u003ctd style=\"width: 56.7pt; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eLAMP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 51.3pt; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"68\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e45\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e25\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e16\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt .75pt 0in .75pt; height: 13.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e93\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan style=\"color: #000000;\"\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman', serif;\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e\u003cspan style=\"color: #000000;\"\u003e=0.449\u0026nbsp;\u003c/span\u003e \u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003col start=\"3\"\u003e\n\u003cli\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eConsistency of Visual LAMP and Nest-PCR\u003c/span\u003e：\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e 96 filter papers of blood from patients infected with malaria and 38 blood sample from healthy human were tested by Visual LAMP and Nest-PCR. The consistent of the two method were analyzed. Kappa = 0.956 (Table 2). \u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"text-align: left;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eTable 2 The consistent of Visual LAMP and Nest PCR\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse;\" width=\"511\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 16.5pt;\"\u003e\n\u003ctd style=\"width: .75in; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"2\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eMethod\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"2\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eClassification\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 223.05pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" colspan=\"4\" width=\"297\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eLAMP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 52.5pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"2\" width=\"70\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eTotal\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 15.75pt;\"\u003e\n\u003ctd style=\"width: 35.35pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 15.75pt;\" width=\"47\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 15.75pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eNP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89.2pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 15.75pt;\" width=\"119\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eP.f+NP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 15.75pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eNegative\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 16.5pt;\"\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"4\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eNest PCR\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 35.35pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"47\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e45\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89.2pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"119\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 52.5pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"70\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e46\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 16.5pt;\"\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eNP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 35.35pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"47\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e44\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89.2pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"119\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 52.5pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"70\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e46\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 16.5pt;\"\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003eP.f+NP.f\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 35.35pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"47\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89.2pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"119\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 52.5pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"70\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 14.25pt;\"\u003e\n\u003ctd style=\"width: .75in; padding: .75pt .75pt 0in .75pt; height: 14.25pt;\" width=\"72\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eNegative\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 35.35pt; padding: .75pt .75pt 0in .75pt; height: 14.25pt;\" width=\"47\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 14.25pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89.2pt; padding: .75pt .75pt 0in .75pt; height: 14.25pt;\" width=\"119\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; padding: .75pt .75pt 0in .75pt; height: 14.25pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e40\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 52.5pt; padding: .75pt .75pt 0in .75pt; height: 14.25pt;\" width=\"70\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e41\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 17.85pt;\"\u003e\n\u003ctd style=\"width: 1.5in; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.85pt;\" colspan=\"2\" width=\"144\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eTotal\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 35.35pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.85pt;\" width=\"47\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e45\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.85pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e45\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 89.2pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.85pt;\" width=\"119\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.25pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.85pt;\" width=\"66\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e41\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 52.5pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.85pt;\" width=\"70\"\u003e\n\u003cp style=\"text-align: center; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 11.0pt; line-height: 107%; font-family: 'Times New Roman',serif; color: black;\"\u003e134\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eKappa = 0.956\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003col start=\"4\"\u003e\n\u003cli\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e The specificity of the method: 10 patients with \u003cem\u003eLeishmaniasis\u003c/em\u003e, 8 with \u003cem\u003eSchistosomiasis japonicum\u003c/em\u003e, 38 healthy human were tested by Visual LAMP and Nest PCR , none was positive. \u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003col start=\"5\"\u003e\n\u003cli\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e The effect of calcium-yellow-green indicator on LAMP reaction: DNA from blood of 25000 P.f/\u0026mu;l, 3000 P.v/\u0026mu;l, 4000 P.o/\u0026mu;l, 2000 P.m/\u0026mu;l was extracted as template. The \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 107%; font-family: 'Times New Roman',serif;\"\u003eTt \u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eof turbidity peak time are 18.2 min, 35.6min, 50.8min, 33.1min when the LAMP reaction fluid was without calcium-yellow-green indicator (A) (Figure 3-6). Tt plus are 29.1 min, 44.1min, 74.1min, 74.1min, 47.8min when calcium-yellow green oxalate indicator were added(B) (Figure 3-6). No turbidity showed in 90min when pure water is used as a template.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Discussion","content":"\n\u003cp\u003eMalaria is an infectious disease caused by different types of \u003cem\u003ePlasmodium,\u003c/em\u003e which live in red blood cells (RBC) and destroy host erythrocyte, leading to fever and/or anemia, or even kidney failure, brain coma and death. Accurate diagnosis of malaria infection is critical. Microscope examination of blood smears is still considered the gold standard for the diagnosis of malaria, which was labor-intensive, time-consuming\u003csup\u003e[7,10,16]\u003c/sup\u003e, with detection limit of 100 malaria parasites/µl \u003csup\u003e[10,16]\u003c/sup\u003e. It was usually unable to detect low-density and mixed infections, the sensitivity and specificity of which depends on the skill of the technician, quality and staining quality of blood smear, and so on \u003csup\u003e[3,17]\u003c/sup\u003e. Compared with molecular diagnostic methods, in malaria endemic areas, only half of malaria patients were correctly diagnosed. The WHO external assessment of the malaria parasite’s mirror test capacity in China showed that the correct rates of negative, \u003cem\u003ePlasmodium falciparum (\u003c/em\u003e\u003cem\u003eP.f),\u003c/em\u003e, \u003cem\u003ePlasmodium vivax\u003c/em\u003e (\u003cem\u003eP.v)\u003c/em\u003e were 92.5%, 78.3%, 70.8% respectively\u003csup\u003e[20]\u003c/sup\u003e. RDT is facilitate and fast, with detection limit of 100 plasmids/μl\u003csup\u003e[19,21]\u003c/sup\u003e. It shows high sensitivity to plasmid infections of more than 100 plasmids/μl(median 94.3%), sensitivity of which decrease significantly with a density of less than 100 plasmids/μl (median 74.1%) \u003csup\u003e[21]\u003c/sup\u003e. Meantime it shows limited sensitivity when detecting low-density infection \u003csup\u003e[16,22]\u003c/sup\u003e, and less sensitivity to non-\u003cem\u003eP.f，\u003c/em\u003e\u003cem\u003eP.v as \u003c/em\u003e66.0–88.0%, \u003cem\u003eP.o as \u003c/em\u003e5.5–86.7%, \u003cem\u003eP.m as\u003c/em\u003e 21.4–45.2% \u003csup\u003e[19]\u003c/sup\u003e. PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, with a high sensitivity and specificity limit to 1–10 malaria parasites/μl \u003csup\u003e[10,11]\u003c/sup\u003e. However, it requires technical expertise and infrastructure which limit its wide range of applications. LAMP has been used in clinical diagnosis of a variety of infectious diseases, including malaria. It is considered to be faster, more operational and cost-effective than PCR. At the same time, it has been reported that in testing malaria, LAMP own a similar sensitivity and specificity to PCR \u003csup\u003e[7,10,11,23,,24]\u003c/sup\u003e. It is capable of identifying low-density malaria infections by LAMP, which cannot be detected by the microscope\u003csup\u003e[7]\u003c/sup\u003e. Sensitivity of LAMP is several orders of magnitude higher than by microscope and RDT \u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAt present, detecting malaria by LAMP is mainly to detect the \u003cem\u003egenus malaria\u003c/em\u003e and \u003cem\u003ePlasmodium falciparum,\u003c/em\u003e the primer is depend on 18s rRNA region of the malaria parasite species. In this study, 3 groups of LAMP Primer was designed including \u003cem\u003eP.f \u003c/em\u003eand non-\u003cem\u003eP.f.\u003c/em\u003e For detection of malaria, 1 group of LAMP primers with high sensitivity, specific and amplification efficiency was screened by experimental. By Visual LAMP, filter paper of blood from 47 cases of \u003cem\u003eP.f\u003c/em\u003e and 49 non-\u003cem\u003eP.f\u003c/em\u003e patients was detected. There was no significant difference with by Nest-PCR or by microscope examination (\u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05), of which 2 non-\u003cem\u003eP.f\u003c/em\u003e patients were both negative tested by Visual LAMP and Nest-PCR. It may because the blood on filter paper was kept too long, and the poor extraction quality of DNA or not extracted at all. 1 \u003cem\u003eP.v\u003c/em\u003e case by Nest-PCR was positive, but by Visual LAMP was negative. 1 \u003cem\u003eP.f\u003c/em\u003e case by visual LAMP was positive, but by Nest-PCR was negative. The false negative rate of visual LAMP of non-\u003cem\u003eP.f\u003c/em\u003e is 2.1%. 47 cases of \u003cem\u003eP.f\u003c/em\u003e were all positive by Visual LAMP and Nest-PCR, of which 2 cases of \u003cem\u003eP.f\u003c/em\u003e and non-\u003cem\u003eP.f\u003c/em\u003e mixture infection were found by Visual LAMP, only 1 case of mixture infection by Nest-PCR, and 1 case of \u003cem\u003eP.o \u003c/em\u003eby microscope examination. It may be due to the false positive or the more sensitivity of Visual LAMP, that the \u003cem\u003eP.f\u003c/em\u003e and non-\u003cem\u003eP.f\u003c/em\u003e mixture infection could be discovered by Visual LAMP but microscope examination.Detected by Visual LAMP and Nest PCR simultaneously, the results of blood in filter paper from 96 malaria patient and 38 healthy men showed extremely consistent (Kappa = 0.956). In the results of 10 patients with \u003cem\u003eLeishmaniasis,\u003c/em\u003e 8 with \u003cem\u003eSchistosomiasis japonicum,\u003c/em\u003e 38 healthy human, none was positive，which indicated that the Visual LAMP primer in this study had high specificity. Visual LAMP is a method that require no expensive instruments or special laboratories, with simple and convenient operation, suitable for on-site inspection. In LAMP of this study, LA–500 real-time turbidity detection system were used and DNA from blood of 25000 P.f/μl, 3000 P.v/μl, 4000 P.o/μl, 2000 P.m/μl was extracted as template. When the LAMP reaction was not added with calcium-yellow green indicator, the turbidity peak time(Tt) is 18.2 min, 35.6 min, 50.8min, 33.1min. Mean Tt was less than 60min, which represented a higher amplification efficiency. LAMP Tt of \u003cem\u003eP.v\u003c/em\u003e is 18.2min in particular, the amplification efficiency of which was very high. It could meet the requirements of LAMP. When adding the calcium-yellow green indicator in LAMP, Tt is 29.1min, 44.1 min, 74.1 min, 47.8min. The delay effect of adding calcite-yellow-green-oxidant in malaria subsets was different, the mean of which was about 9–23min. It is convenient for the visual observation of the results when adding calcium-yellow green indicator, suitable for on-site application.\u003c/p\u003e\n\u003cp\u003eAs worldwide countries set targets of malaria elimination, accurate diagnosis, optimal treatment, stop of transmission, and reducing mortality of malaria are becoming critical \u003csup\u003e[25,26]\u003c/sup\u003e. As a result of the sudden decline in local patients and the gradual elimination of malaria, there are a trend of degradation in the clinical diagnosis and screening capacity of malaria. The prevalence of infrequent diagnosis, with some cases ranging from onset to the determination of diagnosis for more than 30 days. At the same time, due to increasing travel and population movements to malaria-endemic areas, imported malaria is still widespread \u003csup\u003e[21]\u003c/sup\u003e. Particularly, asymptomatic malaria infection and low-density malaria parasite emias are hard to be detected by microscope examination and RDT due to below the detection threshold \u003csup\u003e[11,24]\u003c/sup\u003e, causing misdiagnosis or delayed diagnosis\u003csup\u003e[3,21]\u003c/sup\u003e. But they could maintaining transmission of malaria as infected intermediate host\u003csup\u003e[24,26]\u003c/sup\u003e. It has been reported that subclinical malaria accounts for 70–80% of all malaria infections\u003csup\u003e[11]\u003c/sup\u003e. Therefore, it is a challenge to detect the asymptomatic malaria parasite infection and low density malaria parasite emias in malaria prevention and control \u003csup\u003e[8,24]\u003c/sup\u003e. Stopping transmission of malaria requires detecting methods with high sensitive. The LAMP shows high sensitivity and specificity, by which asymptomatic and asymptomatic malaria infections could be diagnosed accurately, and low-density infections that cannot be detected by conventional diagnosis methods could be identified \u003csup\u003e[26]\u003c/sup\u003e. At the same time, LAMP is fast, easy to operate and effective which provides the possibility for clinical diagnosis and monitoring of malaria.\u003c/p\u003e\n\n"},{"header":"Conclusion","content":"\u003cp\u003eVisual LAMP is highly sensitive and specific to the diagnosis of malaria,and the results using visual LAMP are consistent with Nest-PCR.During the malaria elimination stage,visual LAMP provides a portable, sensitive and specific method for on-site inspection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis study was certified by the Ethics Committee of the Wuhan Center for Disease Control and Prevention. The informed consent of all patients and healthy people was obtained.\u003c/p\u003e\n\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study is financially supported by the Health Bureau of Wuhan (award number: WG18Q05),and the the Health Bureau of Wuhan(award number: WG17B04).\u003c/p\u003e\n\n\u003cp\u003eAuthors’ contributions\u003c/p\u003e\n\u003cp\u003eZHOU Shui-mao and JIA Xi-shuai conceived the study. ZHOU Shui-mao,TU Zu-wu and JIA Xi-shuai undertook statistical analysis,and drafted the manuscript. YANG Yan,TANG Wei-feng, CHEN Fang undertook epidemiological investigations and sample collection.All authors contributed to the writing of the manuscript and approved the submitted version of themanuscript.\u003c/p\u003e\n\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\n\n\n"},{"header":"References","content":"\u003cp\u003e[1]Study on the stability and specificity of the kit for rapid detection of \u003cem\u003ePlasmodium falciparum \u003c/em\u003eand \u003cem\u003ePlasmodium vivax\u003c/em\u003e by Colloidal gold method［J］.Straits Science,2019,147（3）:3-6\u003c/p\u003e\n\u003cp\u003e[2]Wang ZH,Wei CY,Wang H.Research development on non-coding RNA of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e ［J］. 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18: 98.doi:\u0026nbsp;\u003ca href=\"https://dx.doi.org/10.1186/s12936-019-2722-1\"\u003e10.1186/s12936-019-2722-1\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e[25]\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Tegegne%20B%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=28103883\"\u003eTegegne B\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Getie%20S%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=28103883\"\u003eGetie S\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Lemma%20W%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=28103883\"\u003eLemma W\u003c/a\u003e，et al.Performance of loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria among malaria suspected pregnant women in Northwest Ethiopia［J］.\u003ca href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5244525/\"\u003eMalar J\u003c/a\u003e. 2017; 16: 34.doi:\u0026nbsp;\u003ca href=\"https://dx.doi.org/10.1186/s12936-017-1692-4\"\u003e10.1186/s12936-017-1692-4\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e[26]\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Sattabongkot%20J%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=30208895\"\u003eSattabongkot J\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Suansomjit%20C%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=30208895\"\u003eSuansomjit C\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Nguitragool%20W%5bAuthor%5d\u0026amp;cauthor=true\u0026amp;cauthor_uid=30208895\"\u003eNguitragool W\u003c/a\u003e,et al.Prevalence of asymptomatic\u0026nbsp;Plasmodium\u0026nbsp;infections with sub-microscopic parasite densities in the northwestern border of Thailand: a potential threat to malaria elimination［J］.\u003ca href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134695/\"\u003eMalar J\u003c/a\u003e. 2018; 17: 329.doi:\u0026nbsp;\u003ca href=\"https://dx.doi.org/10.1186/s12936-018-2476-1\"\u003e10.1186/s12936-018-2476\u003c/a\u003e-1\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Malaria, LAMP, Nest-PCR, Visualization","lastPublishedDoi":"10.21203/rs.2.17953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.17953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground:\u0026nbsp;To detect the DNA of the malaria using Loop-mediated isothermal amplification (LAMP) and evaluate the effect. \u003c/p\u003e\u003cp\u003eMethod;\u0026nbsp;According to the 18s rRNA sequence of the malaria, the LAMP primer of Plasmodium\u0026nbsp;falciparum and non-Plasmodium\u0026nbsp;falciparum\u0026nbsp;were designed using Primer Explorer V5 software. The method of Visual LAMP detecting malaria was formation. The sensitivity, specificity and amplification efficiency were tested, compared with the Nest-PCR. \u003c/p\u003e\u003cp\u003eResult:\u0026nbsp;The filter papers of blood from 47 patients\u0026nbsp;with Plasmodium falciparum\u0026nbsp;and 49 with non-Plasmodium falciparum\u0026nbsp;were detected using Visual LAMP. The patients with Plasmodium falciparum\u0026nbsp;were all positive. In the patients with non-Plasmodium falciparum\u0026nbsp;, false negative rate was 2.1%. In the 10 patients with Leishmaniasis, 8 with Schistosomiasis japonicum, 38 healthy human, none was positive. The results using visual LAMP were consistent with Nest-PCR (Kappa value = 0.956). All of the Tt in Visual LAMP are less than 60 min, especially the Tt of Plasmodium falciparum\u0026nbsp;is 18.2 min. When adding calcium pyridoxine indicator during the Visual LAMP detection, the progress of the reaction to different substes of malaria delayed of about 9-23 min. \u003c/p\u003e\u003cp\u003eConclusion:\u0026nbsp;LAMP is efficient and visible, deserved the prospect of application in the on-site and primary medical institutions.\u003c/p\u003e","manuscriptTitle":"Evaluation of Visual LAMP detection of Plasmodium falciparum and non-Plasmodium falciparum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-12-03 20:54:36","doi":"10.21203/rs.2.17953/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93183d2f-221b-4f7b-bccc-4abfe7ace467","owner":[],"postedDate":"December 3rd, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39144,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-12-03 20:54:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-8582","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}