牛冠状病毒TaqMan荧光定量PCR检测方法的建立及应用

Establishment and Application of Taq Man Fluorescence Quantitative PCR Detection Method for Bovine Coronavirus

为建立牛冠状病毒TaqMan荧光定量PCR检测方法,根据GenBank收录的牛冠状病毒AKS-01株N基因序列(KU886219)保守区设计特异性引物和探针,构建重组质粒并进行反应条件优化、特异性试验、重复性试验以及敏感性试验,建立一种检测BCoV的TaqMan荧光定量PCR方法。结果显示,建立的牛冠状病毒TaqMan荧光定量PCR检测方法特异性、敏感性和重复性均良好。该方法BCoV重组质粒标准品在5.75×10^(7)~5.75×10^(3)copies/μL时与Ct值呈现良好线性关系,该方法对牛轮状病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒、牛副流感病毒3型均无交叉反应,特异性良好;该方法对BCoV重组质粒标准品最低检测限为5.75×10^(1)copies/μL;批内和批间重复性试验结果稳定,变异系数均小于2%。利用所建立的TaqMan荧光定量PCR方法对收集的132份样品进行检测,与常规PCR相比,两者符合率为96.21%,可为BCoV的临床检测和流行病学调查提供技术支持。

In order to establish a Taq Man quantitative PCR detection method for bovine coronavirus,specific primers and probes were designed according to the conserved region of AKS-01 strain included on GenBank,with recombinant plasmid construction and reaction condition optimization,specificity test,repeatability test and sensitivity test,and a Taq Man fluorescence quantitative PCR method for BCoV was established.The results showed that the established bovine coronavirus Taq Man fluorescence quantitative PCR detection method showed good specificity,sensitivity and reproducibility.When BCoV recombinant plasmid standard showed good linear relationship with Ct value at 5.75×10^(7) to 5.75×10^(3) copies/μL,this method showed no cross-reaction for bovine rotavirus,bovine infectious rhinotracheitis virus,bovine viral diarrhoea virus,and bovine parainfluenza virus type 3;this method for BCoV recombinant plasmid standard was 5.75×10^(1) copies/μL;the interbatch and interbatch reproducibility results were stable,and the coefficient of variation was both less than 2%.132 samples were detected by this Taq Man fluorescence quantitative PCR method,the coincidence rate was 96.21%with conventional PCR,which provided technical support for clinical detection and epidemiological investigation of BCoV.