title: Development of a reverse transcription loop-mediated isothermal amplification assay for detecting nervous necrosis virus in olive flounder Paralichthys olivaceus.
abstract: In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at 65 degrees C. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at 2.58 × 10(-2) TCID50/ml, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at 2.58 × 10(2) TCID50/ml and 2.58 TCID50/ml, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/ 102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.
authors.affiliation: Department of Marine Bioscience, Gangnung-Wonju National University, Gangnung 210-702, Korea.
authors.forename: Rungkarn
authors.initials: R
authors.lastname: Suebsing
authors.forename: Myung-Joo
authors.initials: MJ
authors.lastname: Oh
authors.forename: Jeong-Ho
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000818:Animals; D019411:Clinical Laboratory Techniques / Q000191:economics / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D005412:Flatfishes / Q000821:virology*; D030381:Nodaviridae / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D015995:Prevalence; D012327:RNA Virus Infections / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D056910:Republic of Korea / Q000453:epidemiology; D012680:Sensitivity and Specificity; D013997:Time Factors; D014730:Veterinary Medicine / Q000191:economics / Q000379:methods*; D014773:Virology / Q000191:economics / Q000379:methods
chemical_list: D017931:DNA Primers