犬腺病毒、犬细小病毒联合PCR方法的建立与应用

Establishment and Application of C AVs-CPV Multi-PCR

根据GenBank报道的犬腺病毒(CAV)和犬细小病毒(CPV)序列,设计两对联合PCR引物,其中一对为CAV-1和CAV-2通用引物,另一对为CPV引物。在建立单项PCR基础上,通过优化Mg2+离子浓度和循环参数等反应条件建立联合PCR,确定联合PCR条件为:96℃180s,96℃30s,56℃30s,72℃80s,30个循环。联合PCR结果显示:CAV-1扩增片段大小为497bp,CAV-2为1019bp,CPV为719bp;细胞和其它相关病毒对照均无扩增带。上述PCR产物经用限制性内切酶酶切和克隆测序,结果均与相应病毒的应有条带和序列相同。敏感性比较试验结果表明,联合PCR比用细胞培养分离病毒敏感。将联合PCR应用于15份CAV和CPV细胞培养物,5份CAV-1、1份CAV-2和3份CPV人工感染犬病料以及30份临床病料检测,并与电镜负染、HA/HI及病毒分离等方法的结果进行比较,结果显示,联合PCR的检出率和病毒分离结果一致,高于电镜负染和HA/HI试验。以上结果说明:CAV-1/CAV-2和CPV联合PCR不仅具有很好的特异性和敏感性,而且可以在短时间内(2 5h～3h)同时鉴定出上述三种病毒,因此具有良好的实验室诊断和临床应用价值。

Two pairs of primers,one pair specific to CAVs,another spec ific to CPV,were designed for a mu lti-PCR according to the sequences of CAVs and CPV reported in GenBan kBased on the data of single PCR for each,the concentration of magn esium and the parameters of PCR cy cles were optimized and a multi-PC R assay was establishedThe protoc ol of the multi-PCR were determine d to be 96℃ 180s;96℃ 30s,56℃ 30s a nd 72℃ 80s for 30 cyclesIt was di splayed that the 513bp,1023bp and 71 9bp fragments were amplified from CAV-1,CAV-2 and CPV,respectivelyN o nonspecific products could be amp lified from cell cultures and the other related virusesThe PCR prod uct specificity was further identif ied by restriction endonuclease analysis and s equencing and the results indicate d they were specific to the corres ponding virusesResults also showe d that the multi-PCR is more sensi tive than TCID50 assayFifteen CAV and CPV cell cultures,5 CAV-1, 1 C AV-2 and 3 CPV experimentally infe cted samples and 30 clinical speci mens were detected by multi-PCR,el ectron microscopy,HA/HI and virus isolationThe PCR results were in accordance with that of virus isol ation,and were advantageous over t hat of electron microscopic observa tion and hemagglutination assayAl l above indicated that the establi shed multi-PCR not only is specifi c and sensitive,but also can ident ify the three viruses at the same timeIt can be applied in diag nosis of these virus infections i n laboratory and in clinical pract ice