Abstract:A sensitive and specific SYBR GreenⅠreal-time PCR assay for the detection of red-sea bream iridovirus (RSIV) was established.The real time PCR primers were designed according to the conserved region of major capsid protein (MCP) gene by using Primer Express 3.0 software.The RSIV MCP gene was inserted into pMD18-T vector to construct the recombinant plasmid.The resulted plasmid was serially diluted and used as the standards.The relationship between plasmid copy number(X) and Ct value was described as a standard curve: Ct=－3.184 lgX+40.270(with an R2 value of 0.996 9).The detection limit of the assay was 220×102 virus copies per reaction.The assay showed specificity and could not be amplified with RSIV and epizootic haematopoietic necrosis virus (EHNV),lymphocystis disease virus (LCDV),frog virus 3 (FV 3),or soft-shelled turtle iridovirus (STIV).The Tm of the specific product was obtained as 82.5 ℃ through the melting curve analysis.This assay was applied in detecting whether the sea fish samples (stone flounder,turbot,and weever) of 84 batches were infected by RSIV.It was found that the samples of 5 batches presented positive signals,and the virus was quantified by using the obtained standard curve.The results indicate that the real-time PCR assay is specific,sensitive,fast,and accurate and it has great potential in detecting RSIV and studying the pathogenic mechanism of RSIV.
