Establishment of a TaqMan real-time PCR assay for detecting the giant salamander iridovirus

A 1 392 bp coding region of giant salamander iridovirus(GSIV)MCP protein was amplified by PCR and cloned into pMD19-T vector for the construction of recombinant plasmid pMD19-T-MCP. After being identified and confirmed by PCR reaction, 10-fold serial dilutions of plasmid pMD19-T-MCP were used as standard templates for TaqMan real-time PCR to generate standard curve for quantifying the virus genomic copy number. A good linear correlation was demonstrated in the standard curve for the real-time PCR assay. The coefficients of variance (CV) were 0.52% for intra-assay tests, which indicated good reliability. The detection results showed that the specificity of this assay was high for giant salamander iridovirus without cross-reactions with DNA templates from KHV, Aeromonas hydrophila, Citrobacter freundii and EPC cells. A minimum of 10 copies of GSIV DNA (1.1×10-3 pg/μL total DNA) could be detected, which indicated that the sensitivity of real time PCR is about 1000 times higher than that of the conventional PCR assay. The TaqMan real-time PCR assay established in this study is considered to be a powerful tool for the rapid detection and quantification of GSIV in giant salamander.