同时检测犬瘟热病毒和犬细小病毒双重PCR方法的建立

Establishment of the duplex PCR for the detection of canine distemper virus and canine parvovirus

为建立可以同时检测犬瘟热病毒(CDV)和犬细小病毒(CPV)的双重PCR方法,本研究根据GenBank登录的CDV N蛋白序列和CPV NS基因保守序列,设计合成2对特异性引物。通过优化反应条件,对CDV阳性病毒株反转录后的cDNA模板和CPV的DNA模板进行双重PCR扩增,同时得到2条与试验设计相符的669 bp(CDV)和392 bp(CPV)特异性条带,建立了同时检测CDV和CPV的双重PCR方法。实验结果表明:在同一PCR反应体系中可以同时检测这2种病毒,而对犬腺病毒Ⅰ型、犬腺病毒Ⅱ型、狂犬病毒检测均为阴性;CDV和CPV的最低检出限分别为101.8TCID50和101.4TCID50。采用该方法对在黑龙江省不同地区所采集的30份犬病料样品进行检测,CDV阳性率为30%;CPV阳性率为23.33%,表明建立的PCR方法可以用于临床诊断。

To develop a method for simultaneous detection of canine distemper virus （CDV） and canine parvovirus （CPV）, a duplex PCR was established with 2 pairs of specific primers designed based on the conserved sequences in N gene of CDV and NS gene of CPV. Two special fi＇agments of 669 bp （CDV） and 392 bp （CPV） were amplified by the optimized duplex PCR, with detecting as low as 1018 TCIDs0 （CDV） and 10TM TCIDs0 （CPV）, respectively. The specific tests showed that no PCR products were detected for CAV-I, CAV-II and RV. Thirty clinical tissue samples of sick dogs from different areas in Heilongjiang province were detected by the duplex PCR and the detection rate was 30% （9/30） for CDV and 23.33% （7/30） for CPV, respectively, which indicated that the duplex PCR for detecting CDV and CPV was rapid, specific and sensitive, and could be used in clinic diagnoses.