Loop-mediated isothermal amplification for rapid detection of acute viral necrobiotic virus in scallop Chlamys farreri.
Loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of Acute viral necrobiotic virus (AVNV) in scallop Chlamys farreri was developed and evaluated. Four primers recognizing six targets on distinct AVNV DNA sequences were designed and the LAMP reaction was carried out in a water bath. Reaction temperature and time were optimized at 64 degrees C for 60 mins and LAMP products were detected using agarose gel electrophoresis and visual assessment. Confirmation of the expected LAMP products was performed with MboI restriction enzyme analysis. The detection limit of LAMP assay was as low as 1 fg AVNV DNA and accordingly, this assay was 100 times more sensitive than conventional PCR technique. A comparative evaluation of 20 samples using the LAMP and PCR assays revealed a complete accord in positivity or negativity for AVNV. These results indicate that the LAMP assay is simple, sensitive, specific, and has a great potential for detection of AVNV in the laboratory and field.
Ren
W C
WC
Department of Aquaculture, College of Fisheries, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, P.R. China.
Wang
C M
CM
Cai
Y Y
YY
D000818:Animals; D001483:Base Sequence; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D049897:Pectinidae / Q000821:virology*; D012680:Sensitivity and Specificity