Establishment of a TaqMan real-time PCR assay for detecting the Cyprinid herpesvirus Ⅱ

A 1 446 bp coding region of Cyprinid herpesvirus Ⅱ(CyHV-2)DNA helicase gene was amplified by PCR and cloned into pMD19T vector for the construction of recombinant plasmid.After being identified and confirmed with PCR reaction,10-fold serial dilutions of recombinant plasmid were used as standard templates for TaqMan real-time PCR to quantify the virus genomic copy number and generate standard curve.Herein,a TaqMan real-time PCR of detecting CyHV-2 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 1×10⁷ copies/μL,the correlation coefficient(R²)was 0.999 1,and the slope value of standard curve was -3.412.The detection results showed that the specificity of this assay was high for CyHV-2 without cross reactions with DNA templates from KHV and GSIV.The diseased crucian carp from Sheyang and Baoying,Jiangsu Province,were detected with the established method and the results showed that the content of CyHV-2 were 6.89×10⁴ copies/μL and 3.02×10² copies/μL,respectively.The real-time PCR assay described here with high sensitivity and accuracy is considered to be a powerful tool for the rapid detection and quantification of CyHV-2 in fish.