牛病毒性腹泻病毒实时定量RT-PCR检测技术的建立及初步应用

Establishment and Initial Application of a Real-time Fluorescent Quantitative PCR Method for Detection of Bovine Viral Diarrhea Virus

以牛病毒性腹泻病毒（BVDV）的保守基因5＇-非编码区为参考,设计、优化一对特异的荧光定量PCR引物,应用Smart CycleⅡ分析系统,结合Eva Green荧光染料结合原理,建立一种快速、定量检测牛病毒性腹泻病毒的荧光定量PCR技术。该方法线形范围为101~106copies/μL,相关系数为R2=0.997,扩增效率为E=0.78。灵敏性比常规PCR高102倍。该方法具有良好的特异性,检测变异系数低于1%。应用本实验建立的方法检测25份不同地区采集的临床症状疑似BVDV感染的牛粪样品,阳性检出率为72%（18/25）,与病毒分离培养和电检观察相比,该方法具有快速、灵敏、特异、重复性好和能定量检测等优点,为实验室快速、准确检测BVDV奠定了基础。

The aim of this study was to develop a Eva-Green real-time quantitative PCR which can detect Bovine viral diarrhea virus（BVDV） quickly and flexibly.According to genome sequences within 5＇-UTR of BVDV published in GenBank,a pair of primers was designed.The assay had a dependent coefficient R2=0.997,amplified efficiency E=0.78 and detection sensibility is 100 times than conventional PCR,with variation coefficient less than 1.0%.In this study,twenty five samples from bovine were detected by this method.Eighteen samples were positive and the ratio was 72%.Compared with isolation and electron microscope observation,this method was more rapid,sensitive,specific and has good reproducibility.Besides,it also can detect in quantization.These results demonstrated that the real-time quantitative PCR was a reliable and precise tool for detection of BVDV in the lab.