Development of a Fluorescence Quantitative RT-PCR Method for Detection of a Novel Variant of Infectious Bursal Disease Virus
The Novel variant Infectious bursal disease virus(nVarIBDV)has brought greater challenges to the prevention and control of bursal bursal disease in the poultry industry in China.At present,there is a lack of rapid and sensitive detection methods for nVarIBDV.In order to solve this problem,based on the published VP2 gene sequence of nVarIBD,we designed a pair of primers and a specific Taq Man probe,and established a real-time Taq Man real-time RT-PCR detection method for nVarIBD.This method can determine whether the sample is a new variant of IBDV by specifically amplifying the nVarIBD VP2 gene.The sensitivity,repeatability and specificity of the primers and probes were tested by constructing the recombinant plasmid pMD18-T-VP2,and finally the detection method was used to verify the clinical samples.The sensitivity of real-time RT-PCR was 1.32×101copies/μL,which was 100 times higher than that of conventional RT-PCR.The coefficient of variation(CV)of intra-assay and inter-assay was less than 0.5%.The results showed that the method had good repeatability and stability.There was no cross reaction between the method and the common avian virus nucleic acid,indicating that the detection method had good specificity.The nVarIBD real-time RT-PCR assay established in this study has high specificity,sensitivity and repeatability,and can be used for the detection of new IBDV variants in clinical samples.
万方数据
维普
