猪伪狂犬病毒PCR检测方法的建立及应用

Establishment and Application of PCR for Detection of Pseudorabies Virus

根据猪伪狂犬病毒(PRV)gE基因序列保守区段,设计一对特异性引物,通过优化反应条件,建立了可区分猪伪狂犬病毒野毒株与基因缺失疫苗株的PCR检测方法,并对该方法的敏感性、特异性和重复性进行了验证。结果显示,该PCR方法可扩增出388 bp的目的片段;对模板的最低检测量为1.1 pg;与猪圆环病毒Ⅱ型、猪细小病毒、猪支原体、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪乙型脑炎病毒、猪流行性腹泻病毒无交叉反应,具有高特异性。采用建立的PCR方法对2014年以来全国不同地区81个猪场421份疑似病料进行检测,发现PRV猪场平均阳性率为35.80%,样品平均阳性率为25.42%。该方法灵敏度高、特异性强、重复性好,可用于PRV的临床诊断和流行病学调查。

Based on the conserved region of gE gene, a pair of primers were designed, and a PCR assay which can distinguish wild strain and vaccine strains with gene deletion was established by improving condition. Meanwhile, the sensibility, specificity and repeatability of the assays were verified. The results demonstrated that the established PCR assay could amplify 388 bp target fragment, and could detect 1.1 pg DNA and no cross with porcine circovirus type 2, porcine parvovirus, Mycoplasma hyopneumoniae, classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine Japanese encephalitis virus, porcine epidemic diarrhea virus. Using the established PCR method, 421 suspected materials from 81 pig farms since 2014 were detected, the positive detection rate of pig farms and the materials were 35.80% and 25.42%, respectively. The PCR assay was sensitive, specific and accurate that could be used for massive samples detection and diagnosis of PRV infection.