牛冠状病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及初步应用

Development and preliminary application of a SYBR GreenⅠReal-time PCR assay for detection of bovine coronavirus

针对牛冠状病毒(BCoV)N基因保守序列设计合成了1对特异性引物,以体外转录法制备的cDNA标准品为模板,建立了检测BCoV的SYBR GreenⅠ实时荧光定量PCR方法。在1个反应体系中模板最低检测限为7.8 copies/μL,比普通RT-PCR更加灵敏;与牛主要消化道和呼吸道病毒病病原均无交叉反应;批内、批间重复变异系数均低于1.5%;应用定量PCR检测了39份疑似临床病料,阳性检出率为51.28%(20/39),普通RT-PCR为41.03%(16/39)。结果表明,该方法特异性强、稳定性好、准确率高,本方法将为BCoV的临床诊断和流行病学调查提供技术保障。

In order to detect bovine coronavirus（BCoV）,we developed a SYBR GreenⅠreal-time PCR assay. According to the sequence of bovine coronavirus N gene,a pair of primers was designed. The cDNA was prepared by in vitro transcription and used as standard positive template.Then,the SYBR GreenⅠreal-time fluorescence quantitative RT-PCR assay was established to detect BCoV.The developed SYBR GreenⅠreal-time fluorescence quantitative RT-PCR was much more sensitive than the conventional RT-PCR and could detect the template as low as 7.8 copies/μL. There were no cross reactions with the other digestive and respiratory pathogens from cattle. The Inter-and intra-assays were conducted using the standard plasmid and the coefficients of variation were less than 1.5%.The 39 suspected samples were detected both by SYBR GreenⅠreal-time RT-PCR,and gel-based RT-PCR,and the positive detection rate were 51.28% and 41.03%,respectively. The SYBR GreenⅠreal-time PCR assay was much more specific,sensitive,rapid and accurate and could be used as an effective tool for clinical diagnosis,epidemiological investigation and quantification of bovine coronavirus.