Development and comparative evaluation of real-time PCR and real-time RPA assays for detection of tilapia lake virus |
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| Affiliation: | 1. Aquaculture Vaccine Platform, Department of Microbiology, Faculty of Science, King Mongkut''s University of Technology Thonburi (KMUTT), Bangkok 10140, Thailand;2. Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok 10400, Thailand;3. Department of Fisheries & Aquaculture, University of Agriculture Makurdi, P.M.B 2373 Makurdi, Nigeria;4. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand;1. Department of Microbiology, Faculty of Science, King Mongkut''s University of Technology Thonburi (KMUTT), Bangkok 10140, Thailand;2. Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok 10400, Thailand;3. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand;4. Wildlife, Exotic and Aquatic Pathology-Special Task Force for Activating Research, Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand;5. Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand;1. Department of Pharmacology, Hebei University of Chinese Medicine, No.326 South Xinshi Road, Shijiazhuang, Hebei Province 050200, People''s Republic of China;2. College of Life Sciences, Hebei Normal University, No. 20, Road E. 2nd Ring South, Yuhua District, Shijiazhuang, Hebei 050024, People''s Republic of China;3. College of Life Sciences and Food Engineering, Hebei University of Engineering, No. 178 Zhonghua South Street, Handan, Hebei 056038, People''s Republic of China;4. Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, No. 318 Hepingxilu Road, Shijiazhuang, Hebei Province 050051, People''s Republic of China;5. Hebei Academy of Inspection and Quarantine Science and Technology, No. 318 Hepingxilu Road, Shijiazhuang, Hebei Province 050051, People''s Republic of China;6. The Eighth Hospital of Shjiazhuang, No. 620, Xinhua Road, Shijiazhuang, Hebei Province 050081, People''s Republic of China |
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| Abstract: | Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Early and accurate diagnosis is an important priority for TiLV disease control. In order to evaluate the methodology in the molecular diagnosis of TiLV, we compared newly developed quantitative real-time PCR (qPCR) and real-time recombinase polymerase amplification (real-time RPA) assays regarding their sensitivities, specificities and detection effect on clinical samples. Real-time RPA amplified the target pathogen in less than 30 min at 39 °C with a detection limit of 620 copies, while qPCR required about 60 min with a detection limit of 62 copies. Both assays were specific for TiLV and there were no cross-reactions observed with other common fish pathogens. The assays were validated using 35 tissue samples from clinically infected and 60 from artificially infected animals. The sensitivities for the real-time RPA and qPCR assays were 93.33 and 100%, respectively, and the specificity was 100% for both. Both methods have their advantages and can play their roles in different situations. The qPCR is more suitable for quantitative analysis and accurate detection of TiLV in a diagnostic laboratory, whereas real-time RPA is more suitable for the diagnosis of clinical diseases and preliminary screening for TiLV infection in poorly equipped laboratories as well as in fish farms. |
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| Keywords: | Tilapia lake virus Recombinase polymerase amplification Real-time PCR Detection |
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