CDV、CPV、CAV-2及CPIV多重PCR的建立及应用

Establishment and Rudimentary Application of the Multiplex PCR(mPCR)to the Detection of CDV、CPV、CAV-2 and CPIV

为建立一种快速检测犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬腺病毒Ⅱ型(CAV-2)和犬副流感病毒(CPIV)的多重PCR方法,参照Gen Bank中的相关病毒基因序列,分别设计了4对引物,用于特异性扩增CDV H基因、CPV VP2基因、CAV-2 E3基因和CPIV NP基因上的目的片段。通过优化反应条件,建立了同时扩增CDV(410 bp)、CPV(253 bp)、CVA-2(655 bp)和CPIV(868 bp)的多重PCR方法。利用建立的多重PCR方法,对CDV、CPV、CAV-2、CPIV、CDV+CPV+CAV-2+CPIV和犬冠状病毒(CCV)的DNA或c DNA模板进行扩增,发现该方法的特异性良好。利用建立的多重PCR方法与单一PCR方法进行敏感性比较,发现两者敏感性相差10倍,证明多重PCR方法敏感性良好。利用该方法对从北京市采集的30份犬病料样品进行检测,发现CDV阳性率为63.33%(19/30)、CPV阳性率为33.33%(10/30)、CAV-2阳性率为6.66%(2/30)、CPIV阳性率为0(0/30)。检测结果证明建立的多重PCR方法可用于临床诊断。

In order to establish a multiple PCR method for rapid detection of CDV,CPV,CAV-2 and CPIV,according to the gene sequences in Gen Bank,four pairs of specific primers were designed for amplifying the four specific fragments of CDV H,CPV VP2,CAV-2 E3 and CPIV NP. By optimization of annealing temperature,a multiplex PCR assay of CDV H（410 bp）,CPV VP2（253 bp）,CAV-2 E3（655 bp）and CPIV NP（868 bp）was established for simultaneous detection of the four viruses. The DNA or c DNA template of CDV,CPV,CAV-2,CPIV,CDV＋CPV＋CAV-2＋ CPIV and CCV were detected by multiplex PCR,and the results showed that the specificity was good. The multiplex PCR method and the single PCR method were compared with the sensitivity. The results of difference was ten times,and the sensitivity of the multiplex PCR was good. Thirty clinical samples of affected dogs from Beijing City were detected by the multiplex PCR,and the positive rate of CDV,CPV,CAV-2 and CPIV were 63.33%（19/30）,33.33%（10/30）,6.66%（2/30）and 0（0/30）,respectively. The results indicated that the established PCR method could be used for clinical diagnosis.