PCR快速检测伪狂犬病病毒野毒感染

Establishment and Application of PCR to Detect Wild-Type Pseudorabies Virus

根据已发表的伪狂犬病病毒(PrV)gE、gI基因的序列,设计并合成了一对引物,以PrV容A株细胞培养毒为模板,筛选最佳反应条件和试剂工作浓度,建立了区分PrV野毒株和疫苗弱毒的鉴别PCR方法.该方法能从PrV容A株(RA)、上海株(SH)、鲁A株(LA)中扩增出一条848bp的片段,但Bartha-K61株没有扩增出该片段.测序结果表明PCR扩增产物和方法的特异性.对正常细胞与其它6种引起猪病毒性疫病相关病毒进行检测,结果均为阴性,没有出现交叉反应.对PrV容A株细胞毒提取物DNA进行检测,其最低检出量为5pg.PCR对感染野毒的发病猪不同组织器官检测发现,淋巴结检出率最高,依次为脾、脑(海马角)、肺、肾、肝等.对2003～2004年期间江苏、浙江、安徽、福建、上海等省市的37个大中型猪场送检的172份病料进行PCR检测病料阳性率为20.34%(35/172),猪场阳性率为40.54%(15/37).实验结果表明所建立的PCR技术可用于伪狂犬病野毒感染的快速鉴定和流行病学调查.

In order to effectively differentiate between gene-deleted vaccine and wild-type isolates of Pseudorabies virus(PrV), a PCR method, based on sequences of gE and gI of the PrV genome, was established after selection of the optimal reaction conditions. By applying this technique, the 848bp gene fragment was amplified from PrV RA strain, SH strain and LA strain, respectively. The negative results were achieved from Bartha-K61, RK-13, VSV, HCV, PRRSV, JEV, PPV and PCV2. Sequencing of the amplified products showed that the PCR method was specific. The sensitivity of PCR reached to 5pg DNA of PrV RA strain . We applied the PCR method to detect 172 tissue samples from 37 pig farms in Jiangsu, An’hui, Zhejiang, Fujian and Shanghai during 2003-2004, wild-type isolates of PrV were found in 35 tissue samples (20.34%) and 15 pig farms(40.54%). PrV distributed widely in naturally infected pig’s tissues including brain, liver, spleen, kidney, lung and lymphnodes. The PrV positive detection rate in lymphnodes was the highest in all tissues examined and reached 83.33%. The established PCR method provided a more sensitive, specific and reliable method to rapidly detect wild-type PrV and epizootic study of PrV.