猪源牛病毒性腹泻病毒与猪瘟病毒一步法双重RT-PCR鉴别检测方法的建立及应用

Establishment and Application of One-step Double RT-PCR Identification Method for Pig Source Bovine Viral Diarrhea Virus and Swine Fever Virus

为了建立一种快速鉴别检测猪源牛病毒性腹泻病毒(猪源BVDV)和猪瘟病毒(CSFV)的方法,根据GenBank中猪源BVDV和CSFV 5′UTR基因序列分别设计合成特异性检测引物,经一步法双重RT-PCR反应条件的优化,建立了鉴别检测猪源BVDV和CSFV的一步法双重PCR方法。该方法对猪源BVDV、CSFV、猪源BVDV/CSFV混合物可分别扩增出185、342、185 bp和342 bp的特异性条带,检测PRRSV、PRV、PPV、PCV-2、PEDV、JEV、TGEV均为阴性,对猪源BVDV、CSFV、BVDV/CSFV混合物的RNA检测灵敏度分别达0.95 ng、0.85 ng、8.0 ng,与猪源BVDV一步法RT-PCR检测方法、CSFV一步法RT-PCR检测方法的符合率均为100%。该方法在检测94份疑似CSFV感染病料样品的临床应用中,猪源BVDV阳性率为14.89%,CSFV阳性率为24.47%,猪源BVDV/CSFV混合感染率为5.32%。表明该一步法双重RT-PCR鉴别检测方法具有良好的特异性、敏感性、重复性、准确性和临床适用性,可用于临床病料样品中猪源BVDV和CSFV的快速鉴别检测,为基层兽医工作者开展猪源BVDV和CSFV的临床鉴别诊断和流行病学监测,提供了一种简单、有效的分子生物学鉴别检测方法。

In order to establish a rapid identification method for pig source bovine viral diarrhea virus(BVDV) and classical swine fever virus(CSFV),we designed and synthesized specific detection primers according to the sequences of pig source BVDV and CSFV 5′UTR genes in GenBank.By one-step double RT-PCR reaction conditions optimization,a one-step dual PCR method was established for the identification of pig source BVDV and CSFV.This method could amplify 185 bp,342 bp,185 bp and 342 bp specific bands for pig source BVDV,CSFV,and pig source BVDV/CSFV mixtures.There was no cross-reaction with PRRSV,PRV,PPV,PCV-2,PEDV,JEV and TGEV.The sensitivity of the pig source BVDV,CSFV,and BVDV/CSFV mixtures was 0.95 ng,0.85 ng,and 8.0 ng RNA.The rate of compliance with BVDV one-step RT-PCR detection method and CSFV one-step RT-PCR detection method was 100%.In the clinical application of 94 suspected CSFV infected samples,the positive rate of BVDV was 14.89%,the positive rate of CSFV was 24.47%,and the mixed infection rate of pig source BVDV/CSFV was 5.32%.It indicates that the one-step double RT-PCR identification method has good specificity,sensitivity,repeatability,accuracy and clinical applicability,and can be used for rapid identification and detection of pig source BVDV and CSFV in clinical samples.It provides a simple and effective molecular biological identification method for primary veterinary workers to carry out clinical differential diagnosis and epidemiological monitoring of pig source BVDV and CSFV.