牛轮状病毒NSP5基因SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立与应用

Establishment and application of SYBR Green Ⅰ real-time RT-PCR for detecting NSP5 gene of bovine rotavirus

为建立检测牛轮状病毒(BRV)快速特异的荧光定量RT-PCR方法,本研究针对BRV NSP5基因设计了一对特异性引物经PCR扩增目的片段,并克隆于pCI载体作为重组质粒标准品(pCI-NSP5),经优化反应条件,建立了基于NSP5基因的BRV荧光定量RT-PCR方法,并对其进行了特异性、敏感性及重复性试验。结果显示,建立的SYBR GreenⅠ荧光定量RT-PCR方法最佳引物浓度为10μmol/L,模板为2μL,退火温度为58℃;特异性试验结果显示,该方法除对BRV的检测结果为阳性外,对牛病毒性腹泻病毒、牛冠状病毒、牛副流感3型病毒、牛呼吸道合胞体病毒的检测结果均为阴性;敏感性试验结果显示,该方法对重组质粒标准品的最低检测限为12拷贝/μL,对BRV的最低检测限为1×102TCID50/反应;批内和批间重复性试验的变异系数均小于2%。利用该方法检测30份BRV接种犊牛的粪便样品,结果 25份呈阳性,而利用VP7基因的常规RT-PCR和病毒分离方法检出的阳性样品分别为20份和25份,表明本研究建立的检测方法的敏感性高于VP7基因的常规RT-PCR方法,而与病毒分离方法的符合率为100%。以上结果表明,本研究建立的BRV NSP5基因荧光定量RT-PCR检测方法具有良好的特异性、敏感性和重复性,可以用于BRV的快速检测、病原流行病学调查以及疫苗免疫攻毒试验的病毒载量定量等研究。

To establish a rapid and specific fluorescent quantitative RT-PCR method for bovine rotavirus(BRV) detection, a pair of specific primers were designed for PCR amplification of the BRV NSP5 gene, and the amplified fragment was cloned into the PCI vector(pCI-NSP5) as the recombinant plasmid standard. After optimized reaction conditions, a fluorescent quantitative RTPCR method for detection of BRV based on the NSP5 gene was established, and the specificity, sensitivity and repeatability tests were performed. The results showed that the SYBR Green Ⅰ fluorescence quantitative RT-PCR method was established with the optimal primer concentration of 10 μmol/L, template of 2μL and annealing temperature of 58 ℃. The specific test results showed that the detections of bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza 3 virus and bovine respiratory syncytial virus by this assay were negative, while the detection of BRV was positive. The sensitivity test results showed that the minimum detectable amount of this method for the recombinant plasmid standard and BRV was 12 copies/μL and 1×102 TCID50, respectively.The variation coefficients of detections were less than 2% in both intra-assay and inter-assay. A total of 30 samples from the experimental diarrheic calves were collected for BRV detection;25 samples were positive for BRV in real-time RT-PCR method,while 20 and 25 BRV samples were positive for BRV in VP7 RT-PCR and virus isolation, respectively. The coincidence of this realtime RT-PCR was 100% with the virus isolation, and the detection rate was higher than the VP7 gene-based RT-PCR method. The results indicated that the NSP5 gene-based fluorescence quantitative RT-PCR method established in this study is specific, sensitive and reproducible for BRV detection. It can be used to rapidly diagnose BRV infection, pathogen epidemiological investigation and viral load analysis in vaccine challenge tests.