检测CSFV、PRRSV和JEV感染的多重RT-PCR方法的建立及初步应用

为了建立1种能够应用于临床样本可以同时检测CSFV、PRRSV、JEV 3种RNA病毒感染的多重RT-PCR方法。根据GenBank收录的CSFV、PRRSV、JEV的基因组全序列,选择3种病毒的特异性保守区序列设计了3对特异性引物,优化多重RT-PCR反应条件,建立了能够同时检测3种病毒混合感染的多重RT-PCR诊断方法。特异性分析表明,应用该方法分别从CSFV、PRRSV、JEV毒株以及3种病毒的混合物中扩增出大小分别为777、451、605bp的3条特异性条带,其他对照组检测均为阴性;敏感性分析表明,该方法最低检测量分别为14.2、10.0、8.0pg的CSFV、PRRSV、JEV的RNA;初步应用性试验表明,52份疑似病料中PRRSV、CSFV和JEV的阳性率分别为46.1%、21.1%和1.92%;CSFV和PRRSV混合感染阳性率为21.1%。CSFV、PRRSV和JEV混合感染阳性率为3.84%。本试验建立的多重RT-PCR检测方法具有敏感性高、特异性强、重复性好的特点,可以有效检测CSFV、PRRSV和JEV的混合感染。

Foundation and initial application of multiplex RT-PCR method for detecting CSFV,PRRSV and JEV infection

A multiplex RT-PCR assay was developed and evaluated for the clinical detection of CSFV,PRRSV and JEV infection of swine.Three pairs of primers were designed and synthesized according to the highly conserved regions of CSFV,PRRSV and JEV genome sequence in GenBank.The multiplex RT-PCR reaction condition was optimized and the multiplex RT-PCR method for detecting CSFV,PRRSV and JEV infection was established.The specificity test showed that a fragment of 777,451 and 605 bp was amplified from genomic RNA of CSFV,PRRSV and JEV,respectively.Three fragments were amplified from the mixed RNA sample of CSFV,PRRSV and JEV simultaneously,and no amplification was achieved from other negative control groups.The sensitivity test showed that the RT-PCR could detect 14.2 pg of CSFV RNA,10.0 pg of PRRSV RNA and 8.0 pg of JEV RNA.The initial amplication test showed that among 52 clinical samples,PRRSV-positive percentage was 46.1%,CSFV-positive percentage was 21.1%,JEV-positive percentage was 1.92%,co-infection of CSFV and PRRSV percentage was 21.1%,co-infection of CSFV,PRRSV and JEV percentage was 3.84%.The result indicated that RT-PCR with method had specificity,sensitivity,repetitive characteristics can be used for detection of CSFV,PRRSV and JEV co-infection.