{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T11:01:32Z","timestamp":1785322892409,"version":"3.55.0"},"reference-count":28,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Health management in selected finfish and shellfish & bio-prospecting from marine resources","award":["MBT\/HLT\/23"],"award-info":[{"award-number":["MBT\/HLT\/23"]}]},{"name":"Health Monitoring and Management strategies for Marine Finfish and Shellfish","award":["MBFNHD\/HM\/23"],"award-info":[{"award-number":["MBFNHD\/HM\/23"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Betanodavirus is a causative agent of viral nervous necrosis (VNN) and a major threat to marine and brackish-water aquaculture globally. This virus causes epizootic outbreaks with particularly high morbidity and mortality in larval and juvenile stages and causes significant economic losses in aquaculture. Here, we developed a rapid and highly sensitive single-tube Reverse Transcription\u2013Loop-Mediated Isothermal Amplification (RT-LAMP) assay for the detection of red-spotted grouper nervous necrosis virus (RGNNV) genotype infection in fish tissue samples. Six primers targeting eight conserved regions of the RNA2 coat protein gene of RGNNV were designed with conservation regions across RGNNV genotypes. RT-LAMP assay was completed within 60 min at 65 \u00b0C using a single-tube format that combined reverse transcription and isothermal amplification, and results were directly visualized by the addition of SYBR Green I dye, producing a colour change from orange (negative) to green (positive), observable with the naked eye or under UV illumination. The developed RT-LAMP assay was able to detect five copies of RGNNV from infected samples, which was 20-fold more sensitive than conventional reverse transcription-PCR. The assay demonstrated diagnostic sensitivity and specificity in two fish hosts (Asian seabass and cobia) and showed no cross reactivity with other fish viruses such as tilapia lake virus and cyprinid herpesvirus-2. The developed assay is simple, cost-effective, specific, and enables rapid detection of RGNNV in fish tissues. This single-tube RT-LAMP assay can be applied to screening broodstock facilities, fingerlings, aquaculture farms, quarantine facilities, and juveniles before stocking in ponds or cages, helping prevent disease outbreaks and the transmission of RGNNV in aquaculture systems.<\/jats:p>","DOI":"10.3390\/v18080827","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T16:11:56Z","timestamp":1785168716000},"page":"827","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single-Tube Reverse Transcription\u2013Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Red-Spotted Grouper Nervous Necrosis Virus in Fish Species"],"prefix":"10.3390","volume":"18","author":[{"given":"Mangottil Ayyappan","family":"Pradeep","sequence":"first","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3998-5420","authenticated-orcid":false,"given":"Cherammpillil Sukumaran","family":"Subin","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1958-2579","authenticated-orcid":false,"given":"Gokhlesh","family":"Kumar","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sulumane Ramachandra Krupesha","family":"Sharma","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadiyath Karayi","family":"Sanil","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9074-8212","authenticated-orcid":false,"given":"Thaliyil Veetil","family":"Arun Kumar","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikathil Raveendranathan","family":"Dhanutha","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-4203-3533","authenticated-orcid":false,"given":"Thevanattil Sairanksha Azhar","family":"Shahansha","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koyadan Kizhakkedath","family":"Vijayan","sequence":"additional","affiliation":[{"name":"Marine Biotechnology, Fish Nutrition and Health Division, Indian Council of Agricultural Research\u2014Central Marine Fisheries Research Institute, Kochi 682018, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"46755","DOI":"10.1038\/srep46755","article-title":"Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV\/SJNNV: An emerging threat for Mediterranean aquaculture","volume":"7","author":"Toffan","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Band\u00edn, I., and Souto, S. (2020). Betanodavirus and VER Disease: A 30-year Research Review. Pathogens, 9.","DOI":"10.3390\/pathogens9020106"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1046\/j.1365-2761.2002.00350.x","article-title":"Betanodavirus infections of teleost fish: A review","volume":"25","author":"Munday","year":"2002","journal-title":"J. Fish Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"103","DOI":"10.3354\/dao018103","article-title":"Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV)","volume":"18","author":"Nishizawa","year":"1994","journal-title":"Dis. Aquat. Org."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Biasini, L., Berto, P., Abbadi, M., Buratin, A., Toson, M., Marsella, A., Toffan, A., and Pascoli, F. (2022). Pathogenicity of Different Betanodavirus RGNNV\/SJNNV Reassortant Strains in European Sea Bass. Pathogens, 11.","DOI":"10.3390\/pathogens11040458"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1256","DOI":"10.1128\/JVI.78.3.1256-1262.2004","article-title":"Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus","volume":"78","author":"Iwamoto","year":"2004","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"79","DOI":"10.3354\/dao039079","article-title":"Characterization of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay","volume":"39","author":"Grotmol","year":"2000","journal-title":"Dis. Aquat. Organ."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1111\/j.1365-2761.2004.00559.x","article-title":"Histopathological studies on viral nervous necrosis of sevenband grouper, Epinephelus septemfasciatus Thunberg, at the grow-out stage","volume":"27","author":"Tanaka","year":"2004","journal-title":"J. Fish Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1016\/j.aquaculture.2011.02.040","article-title":"Betanodavirus infection in golden pompano, Trachinotus blochii, fingerlings cultured in deep-sea cage culture facility in Langkawi, Malaysia","volume":"315","author":"Ransangan","year":"2011","journal-title":"Aquaculture"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"33","DOI":"10.3354\/dao047033","article-title":"Sea bream Sparus aurata, an asymptomatic contagious fish host for nodavirus","volume":"47","author":"Castric","year":"2001","journal-title":"Dis. Aquat. Organ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"113","DOI":"10.3354\/dao063113","article-title":"Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: First report from India","volume":"63","author":"Azad","year":"2005","journal-title":"Dis. Aquat. Organ."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1481","DOI":"10.1111\/are.12095","article-title":"Isolation and characterization of Indian betanodavirus strain from infected farm-reared Asian seabass Lates calcarifer (Bloch, 1790) juveniles","volume":"45","author":"John","year":"2014","journal-title":"Aquac. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1007\/s13337-014-0226-8","article-title":"Isolation and characterization of a nodavirus associated with mass mortality in Asian seabass (Lates calcarifer) from the west coast of India","volume":"25","author":"Banerjee","year":"2014","journal-title":"Virusdisease"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"740047","DOI":"10.1016\/j.aquaculture.2023.740047","article-title":"Co-infection of viral nervous necrosis (VNN) and bacterial infection outbreak in cage cultured cobia fingerlings","volume":"578","author":"Rameshkumar","year":"2024","journal-title":"Aquaculture"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1007\/s10499-018-0322-7","article-title":"Betanodavirus-associated mortality in Asian seabass (Lates calcarifer, Bloch) cultured in indoor tanks and sea cages","volume":"27","author":"Sharma","year":"2019","journal-title":"Aquacult. Int."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"85","DOI":"10.3354\/dao029085","article-title":"Vacuolating encephalopathy and retinopathy associated with a nodavirus-like agent: A probable cause of mass mortality of cultured larval and juvenile Atlantic halibut Hippoglossus hippoglossus","volume":"29","author":"Grotmol","year":"1997","journal-title":"Dis. Aquat. Org."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2067","DOI":"10.1099\/0022-1317-77-9-2067","article-title":"Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax","volume":"77","author":"Frerichs","year":"1996","journal-title":"J. Gen. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"E63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated-isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1038\/nprot.2008.57","article-title":"Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products","volume":"3","author":"Tomita","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1006\/bbrc.2001.5921","article-title":"Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation","volume":"289","author":"Mori","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1016\/j.jbbm.2006.08.008","article-title":"Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances","volume":"70","author":"Kaneko","year":"2007","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/j.jviromet.2009.03.022","article-title":"Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers","volume":"159","author":"Sung","year":"2009","journal-title":"J. Virol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.jviromet.2009.09.009","article-title":"Detection of redspotted grouper nervous necrosis virus by loop-mediated isothermal amplification","volume":"163","author":"Xu","year":"2010","journal-title":"J. Virol. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.4014\/jmb.1201.01005","article-title":"Development of a reverse transcription loop-mediated isothermal amplification assay for detecting nervous necrosis virus in olive flounder Paralichthys olivaceus","volume":"22","author":"Suebsing","year":"2012","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.apjtm.2016.01.035","article-title":"Detection of coat protein gene of nervous necrosis virus using loop-mediated isothermal amplification","volume":"9","author":"Hwang","year":"2016","journal-title":"Asian Pac. J. Trop. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1111\/jfd.12297","article-title":"Development of simple, rapid and sensitive detection assay for grouper nervous necrosis virus using real-time loop-mediated isothermal amplification","volume":"38","author":"Mekata","year":"2015","journal-title":"J. Fish Dis."},{"key":"ref_27","unstructured":"Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [2nd ed.]."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1128\/AEM.70.1.621-624.2004","article-title":"Sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method","volume":"70","author":"Savan","year":"2004","journal-title":"Appl. Env. Microbiol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/8\/827\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T10:20:07Z","timestamp":1785320407000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/8\/827"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,27]]},"references-count":28,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["v18080827"],"URL":"https:\/\/doi.org\/10.3390\/v18080827","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,27]]}}}