牛病毒性腹泻病毒RT-PCR检测方法的建立及应用

Establishment and application of RT-PCR for detection of bovine viral diarrhea virus

根据GenBank中登录的牛病毒性腹泻病毒(BVDV)5′UTR基因序列,设计合成了1对特异性引物,建立了检测BVDV218bp片段的RT-PCR方法。通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法从BVDV标准毒株OregonC24V中扩增出了218bp的特异性片段,而对猪瘟病毒、蓝舌病病毒、牛传染性鼻气管炎病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、MDBK正常细胞的扩增结果均为阴性。经对标准毒株的细胞毒进行检测,其敏感度达10-1TCID50。不同人员用该方法进行检测,结果均一致,表明其重复性好。应用该方法对70份临床疑似发病猪样品进行检测,结果检出11份阳性,阳性检出率约为15.7%。在对5份细胞培养用犊牛血清的检测中,亦检出2份阳性。表明,建立的RT-PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的临床检测及流行病学监测。

A reverse transcription PCR（RT-PCR） for detection of bovine viral diarrhea virus（BVDV） was established using a pair of specific primers based on the 5＇UTR gene of BVDV published in GenBank. This method could specifically amplify a 218 bp fragment from BVDV Oregon C_（24） V strain, but not from classical swine fever virus, blue tongue virus, infectious bovine rhinotracheitis virus, porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus and normal MDBK cells. In a test of 70 samples suspected with swine disease, the detection rate was about 15.7 %. Out of 5 caff sera for cell culture,2 were positive with BVDV. The established RT-PCR was specific,sensitive, efficient and rapid for the detection, monitoring and epidemiological investigation of BVDV infection.