Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers.
The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 degrees C for 60min. The detection limit of RT-LAMP is 10(-6) NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers.
Sung
Chia-Hsuan
CH
Animal Health Inspection Division, Bureau of Animal and Plant Health Inspection and Quarantine, 3F, No. 51, Sec. 2, Chungcing S. Road, Taipei City 100, Taiwan.
Lu
Jenn-Kan
JK
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D005399:Fishes; D030381:Nodaviridae / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012327:RNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers