蜱传脑炎病毒(TBEV)SYBR Green Ⅱ荧光定量PCR检测方法的建立

Establishment of SYBR Green Ⅱ fluorescent quantitative PCR for detection of Tick-borne encephalitis virus(TBEV)

根据TBEV NS5基因序列设计特异性引物,构建标准品,优化荧光定量PCR的反条件和反应体系,将标准品按10^8~10^1拷贝/μL梯度稀释,建立标准曲线。同时分析其敏感性和特异性,并对155份牛血清样品进行检测验证。结果显示,建立了TBEV基于SYBR Green Ⅱ荧光定量PCR的检测方法。标准曲线在8个浓度梯度间呈现良好的线性关系,扩增效率为97%,组间和组内变异系数均低于2%且无非特异杂峰。敏感性检测显示,荧光定量检测下限为10^1拷贝/μL,灵敏度比半巢式PCR高10倍。该方法对LCMV、SFTSV及TBEV毒株进行检测,仅TBEV出现特异性扩增。临床样品检测结果显示,荧光定量PCR检出TBEV阳性率为14.2%,比半巢式PCR检测阳性率提高了5.2%,两者检测一致率为63.6%。结果表明,成功建立TBEV基于SYBR Green Ⅱ荧光定量PCR的检测方法,为蜱传病防控奠定基础。

Specific primers were designed according to the NS5gene sequence of TBEV,the TBEV standard was prepared,the reverse conditions and reaction system of fluorescence quantitative PCR were optimized,the standard product was diluted as 10^8-10^1 copies/μL to establish the standard curve.Its sensitivity and specificity will be analyzed,and 155bovine serum samples were tested and verified.SYBR Green Ⅱ fluorescence quantitative PCR for detecting TBEV was established.The standard curve showed a good linear relationship among the 8concentration gradients,with 97% amplification efficiency,inter-group and intra-group variation coefficient was less than 2%,and no nonspecific peaks.Sensitivity detection showed that the lower limit of fluorescence quantitative detection was 10^1copies/μL,and the sensitivity was 10times higher than that of semi-nested PCR.LCMV,SFTSV and TBEV strains were detected by this method,and only TBEV showed specific amplification.The results of clinical samples showed that the positive rate of TBEV detected by fluorescence quantitative PCR was 14.2%,which was 5.2%higher than that by semi-nested PCR,and the consistent rate of the two detection was 63.6%.SYBR Green Ⅱ fluorescence quantitative PCR detection of TBEV was successfully estabished,which lays a good foundation for the tick-borne disease prevention and control.