Establishment of a Taq Man real-time PCR assay for detecting the grass carp reovirus

A 1 250 bp coding region of grass carp reovirus(GCRV)VP6 protein was amplified by RT-PCR and cloned into pEGFP-N1 vector for the construction of recombinant plasmid pEGFP-N1-VP6.After being identified and confirmed by PCR reaction with specific primer paires,10-fold serial dilutions of plasmid pEGFP-N1-VP6 were used as standard templates for Taq Man real-time PCR to quantify the virus genomic copy number and generate standard curve.Herein,a Taq Man real-time PCR of detecting GCRV was developed.It had a good linear relationship between the initial templates and C_t values with a detection range from 1×10¹ copies/μL to 1×10⁶ copies/μL,the correlation coefficient(R²)was 0.998 09 and the slope value of standard curve was -3.373.The assay for specificity of the method established revealed that the TaqMan real-time PCR had a specific detection of GCRV,but had no detection signals to spring viremia carp virus(SVCV)and infectious hematopoietic necrosis virus(IHNV).The Taq Man real-time RT-PCR assay described here with high sensitivity and accuracy is considered to be a powerful tool for the rapid detection and quantification of GCRV both in vivo and in vitro.