title: Evaluation of rapid and sensitive reverse transcription loop-mediated isothermal amplification method for detecting infectious pancreatic necrosis virus in chum salmon (Oncorhynchus keta).
abstract: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detecting Infectious pancreatic necrosis virus (IPNV) in chum salmon (Oncorhynchus keta) in Korea. The RT-LAMP is a novel approach of nucleic acid gene amplification with high specificity, sensitivity, and rapidity under isothermal conditions. Based on the VP2/NS gene sequence of VR-299 and Jasper strains, a set of 6 IPNV-specific primers was designed to recognize 8 diverse sequences of the IPNV RNA. The assay was successfully optimized to detect IPNV at 65°C in 30 min. The detection limit was 0.075 tissue culture infectious dose infecting 50% of inoculated cultures per milliliter (TCID(50)/ml) from IPNV-infected rainbow trout gonad (RTG)-2 cells, whereas nested reverse transcription polymerase chain reaction (nRT-PCR) had a sensitivity of 7.5 TCID(50)/ml. Using RT-LAMP assay, field samples were analyzed and the results compared with those of nRT-PCR assay. Two hundred and sixty-six out of 659 (40.4%) samples were IPNV-positive by RT-LAMP, whereas 182 of 659 samples (27.6%) were IPNV-positive by nRT-PCR. The results indicate that RT-LAMP can be a useful tool for early field diagnosis of IPNV.
authors: Faculty of Marine Bioscience & Technology, Gangneung-Wonju National University, Gangneung 210-702, Korea.
authors: Rungkarn
authors: R
authors: Suebsing
authors: Myung-Joo
authors: MJ
authors: Oh
authors: Jeong-Ho
authors: JH
authors: Kim
mesh_terms: D000818:Animals; D001483:Base Sequence; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D018061:Infectious pancreatic necrosis virus / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary*; D017685:Oncorhynchus keta; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D012367:RNA, Viral