鼠脑心肌炎病毒TaqMan探针荧光定量RT-PCR检测方法的建立

Establishment of TaqMan Real-time RT-PCR Method to Detect Mice Encephalomyocarditis virus

目的建立快速诊断鼠脑心肌炎病毒感染的荧光定量RT-PCR方法。方法根据鼠脑心肌炎病毒(EMV)PEC9毒株全基因组序列(Gen Bank登录号:DQ288856.1),设计一对特异性引物和TaqMan探针,建立EMV的TaqMan探针荧光定量RT-PCR方法,进行条件优化,检测其特异性,敏感性,稳定性。结果建立的荧光定量PCR方法,标准曲线的线性关系良好,r2值可达0.99,敏感性最低检测限为1个拷贝数,高于普通PCR方法 1000倍,其特异性强,与其他常见感染的小鼠病毒株均无特异性扩增,批内和批间变异系数均小于2%。利用该方法对上海市120份小鼠心肌样品进行检测,未检测到阳性感染。结论本研究为鼠脑心肌炎病毒感染的分子检测,为制定国家标准提供良好的方法。

Objective To develop a real-time RT-PCR assay （rRT-PCR） for efficient detection of Murine Encephalomyocarditis virus （EMV）.Method According to the genomic sequences of EMV strain PEC9 （DQ288856.1） download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed.Then reaction parameters were optimized to develop a real-time RT-PCR assay （rRTPCR）.Result The method showed a good linear relationship of standard curve with a r^2 value of 0.99.The sensitivity of the real-time PCR was less than 1 copies/μL.1 000 times higher than ordinary PCR method,and its specificity is strong,common mice strains had no specific amplification,between batch and batch of variation coefficient is less than 2%.120 mice feces samples epidemic materials provided by the Shanghai institute of experimental animals were detected by the real-time PCR.All the 120 mice samples were negative.Conclusion The real-time RT-PCRmethod is good in specificity and sensitivity,which can make a powerful technical support for EMV epidemiological investigation and detection.