Establishment and application of real-time fluorescence quantitative PCR detection method of Yak-derived Bovine enterovirus
In order to rapidly and accurately detect the Bovine enterovirus(BEV)from yaks,this study designed specific primer for real-time fluorescent quantitative PCR(RT-PCR)to detect yak BEV based on the 3D gene sequence of Qinghai yak BEV-NH2 strain(entry number PP746571)in GenBank.A RT-PCR method for yak BEV detection was established by constructing standard plasmids and generating standard curves.The specificity,sensitivity and repeatability tests were performed on the method.This method and conventional RT-PCR were used to detect BEV in 286 diarrheal yak fecal samples collected from 6 breeding bases in Haibei,Qinghai Province,and the coincidence rate of the two methods was calculated.The results showed that the melting curve of the established yak BEV RT-PCR amplification product was single,with a Tm value ranging from 85.6～85.8℃,and no primer dimerization and non-specific amplification was observed.The standard curve exhibited a good linear relationship within the plasmid concentration range of 3×102 to 3×108 copies/μL,with Ct values ranging from 10.88 to 29.63,a regression equation of y=-3.16x+38.01,and R2=0.996 4.The method specifically detected yak BEV NH2 strain,but were not detected Bovine viral diarrhea virus(BVDV)MY01 strain,Bovine rotavirus(BRV)MY20 strain,and Bovine coronavirus(BcoV)QL21 strain.The minimum detection limit of yak BEV was 3×102 copies/μL.At template concentrations of 3×102 copies/μL、3×103 copies/μL and 3×107 copies/μL,the variation coefficients of Ct were 1.28%,1.74%and 0.15%,all below 1.80%.Among 286 fecal samples,182 BEV-positive samples were detected by the established RT-PCR method,with a positive rate of 63.64%,while 114 BEV-positive samples were detected by conventional RT-PCR,with a positive rate of 39.86%.The coincidence rate of the two methods was 76.22%.The results indicated that the RT-PCR method developed in this study was highly specific,sensitive and repeatable,making it suitable for detecting BEV in clinical diarrhea samples from yak.
万方数据
维普
