Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of ostreid herpesvirus 1 DNA.
A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of ostreid herpesvirus 1 (OsHV-1) DNA. A set of four primers was designed, based on the sequence of the ATPase subunit of the OsHV-1 DNA-packaging terminase gene. The reaction temperature and time were optimized to 64°C and 60min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of OsHV-1, and no cross-reaction was observed with other DNA viruses, such as White spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV), Turbot reddish body iridovirus (TRBIV) and Lymphocystis disease virus (LCDV) found commonly in China. The lower detection limit of the LAMP assay was approximately 20 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. A comparative evaluation of 10 oyster samples using LAMP and PCR assays showed overall correlation in positive and negative results for OsHV-1. These results indicate that the LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of OsHV-1. The LAMP technique has capacity for use for the detection of OsHV-1 both in the laboratory and on farms.
Ren
Weicheng
W
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Science, 106 Nanjing Road, Qingdao, Shandong, China.
Renault
Tristan
T
Cai
Yuyong
Y
Wang
Chongming
C
D000818:Animals; D001483:Base Sequence; D049896:Crassostrea / Q000821:virology*; D017931:DNA Primers; D004267:DNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes