牛轮状病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及应用

Establishment and Application of SYBR GreenⅠReal-time PCR for Detection of Bovine Rotavirus

旨在建立一种SYBR GreenⅠ实时荧光定量PCR(RT-qPCR)检测牛轮状病毒(BRV)的方法,并采用针对BRV vp6基因序列保守区设计的引物,使用RT-qPCR方法进行目的基因扩增,优化反应条件,制备阳性标准品,建立标准曲线,确定重复性、敏感性和特异性,与常规PCR方法比较。结果显示,建立的RT-qPCR方法最佳引物浓度为5μmol/L,退火温度为58℃,40个循环,熔解温度为77℃±0.5℃,最小检出量8.13copies/μL。两种方法检出率分别为30.4%和19.6%,RT-qPCR具有较高敏感性。建立的RT-qPCR可为BRV感染早期诊断、分子流行病学调查及定量检测分析等提供技术保障。

The aim of this study was to establish a real-time quantitative PCR(RT-qPCR)for detection of bovine rotavirus(BRV)with SYBR GreenⅠ.The primers designed by the conservative region of BRV vp6 gene sequence were used to amplify the target gene by RT-PCR.The reaction conditions were optimized,the positive standard sample was prepared,the standard curve was established,and the repeatability,sensitivity and specificity were determined,and then compared it with the regular PCR.Results showed that the optimum primer concentration and annealing temperature of the RT-qPCR method were 5μmol/L and 58μmo/L,respectively.At 40cycles,the dissolution temperature was 77℃±0.5℃,and the minimum detectable amount 8.13copies/μL.The detection rates of the two methods were 30.4%and 19.6%,respectively.RT-qPCR was highly sensitive.Therefore,the established RT-qPCR can provide reliable technical support for early diagnosis,molecular epidemiological investigation and quantitative analysis of BRV infection.