{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T01:16:10Z","timestamp":1789694170733,"version":"4.0.1"},"reference-count":68,"publisher":"PeerJ","license":[{"start":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T00:00:00Z","timestamp":1747094400000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:sec>\n               <jats:title>Background<\/jats:title>\n               <jats:p>Tilapia aquaculture faces significant threats posed by four prominent pathogens: tilapia lake virus (TiLV), infectious spleen and kidney necrosis virus (ISKNV), <jats:italic>Francisella orientalis<\/jats:italic>, and <jats:italic>Streptococcus agalactiae<\/jats:italic>. Currently, employed molecular diagnostic methods for these pathogens rely on multiple singleplex polymerase chain reactions (PCR), which are time-consuming and expensive.<\/jats:p>\n            <\/jats:sec>\n            <jats:sec>\n               <jats:title>Methods<\/jats:title>\n               <jats:p>In this study, we present an approach utilizing a multiplex PCR (mPCR) assay, coupled with rapid Nanopore sequencing, enabling the one-tube simultaneous detection and one-reaction Nanopore sequencing-based validation of four pathogens.<\/jats:p>\n            <\/jats:sec>\n            <jats:sec>\n               <jats:title>Results<\/jats:title>\n               <jats:p>Our one-tube multiplex assay exhibits a detection limit of 1,000 copies per reaction for TiLV, ISKNV, and <jats:italic>S. agalactiae<\/jats:italic>, while for <jats:italic>F. orientalis<\/jats:italic>, the detection limit is 10,000 copies per reaction. This sensitivity is sufficient for diagnosing infections and co-infections in clinical samples from sick fish, enabling rapid confirmation of the presence of pathogens. Integrating multiplex PCR and Nanopore sequencing provides an alternative approach platform for fast and precise diagnostics of major tilapia pathogens in clinically sick animals, adding to the available toolbox for disease diagnostics.<\/jats:p>\n            <\/jats:sec>","DOI":"10.7717\/peerj.19425","type":"journal-article","created":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T08:33:17Z","timestamp":1747125197000},"page":"e19425","source":"Crossref","is-referenced-by-count":5,"title":["Multiplex polymerase chain reaction (PCR) with Nanopore sequencing for sequence-based detection of four tilapia pathogens"],"prefix":"10.7717","volume":"13","author":[{"given":"J\u00e9r\u00f4me","family":"Delamare-Deboutteville","sequence":"first","affiliation":[{"name":"Aquatic Food Biosciences, WorldFish, Batu Maung, Penang, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Watcharachai","family":"Meemetta","sequence":"additional","affiliation":[{"name":"Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Khaettareeya","family":"Pimsannil","sequence":"additional","affiliation":[{"name":"Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Han Ming","family":"Gan","sequence":"additional","affiliation":[{"name":"Microbial Genomics, Patriot Biotech Sdn Bhd, Bandar Sunway, Selangor, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Khor","sequence":"additional","affiliation":[{"name":"Aquatic Food Biosciences, WorldFish, Batu Maung, Penang, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohan","family":"Chadag","sequence":"additional","affiliation":[{"name":"Aquatic Food Biosciences, WorldFish, Batu Maung, Penang, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ha Thanh","family":"Dong","sequence":"additional","affiliation":[{"name":"School of Environment, Resources and Development, Asian Institute of Technology, Pathum Thani,  Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saengchan","family":"Senapin","sequence":"additional","affiliation":[{"name":"Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand"},{"name":"National Science and Technology Development Agency (NSTDA), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathum Thani, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"4443","published-online":{"date-parts":[[2025,5,13]]},"reference":[{"key":"10.7717\/peerj.19425\/ref-1","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1111\/jfd.13164","article-title":"The nature and consequences of co-infections in tilapia: a review","volume":"43","author":"Abdel-Latif","year":"2020","journal-title":"Journal of Fish Diseases"},{"key":"10.7717\/peerj.19425\/ref-2","doi-asserted-by":"publisher","first-page":"1880","DOI":"10.1111\/are.14538","article-title":"Natural co-infection of cultured Nile tilapia Oreochromis niloticus with Aeromonas hydrophila and Gyrodactylus cichlidarum experiencing high mortality during summer","volume":"51","author":"Abdel-Latif","year":"2020","journal-title":"Aquaculture Research"},{"key":"10.7717\/peerj.19425\/ref-3","doi-asserted-by":"publisher","first-page":"965","DOI":"10.3390\/v15040965","article-title":"A multiplexed, tiled PCR Method for rapid whole-genome sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in tilapia","volume":"15","author":"Alathari","year":"2023","journal-title":"Viruses"},{"key":"10.7717\/peerj.19425\/ref-4","article-title":"Impacts of climate change on fisheries and aquaculture: synthesis of current knowledge, adaptation and mitigation options","author":"Barange","year":"2018","journal-title":"FAO fisheries and aquaculture technical paper. 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