动物冠状病毒通用PCR方法的建立及基因序列分析

Establishment of universal PCR and sequence analyse of polymerase genes of animal coronaviruses

参考GenBank中公布的冠状病毒相关序列,根据动物冠状病毒聚合酶基因的保守区段设计一对通用引物。用这对引物对犬冠状病毒、猫冠状病毒等8种动物冠状病毒的cDNA模板进行通用PCR扩增和通用PCR反应条件的优化,结果均得到与试验设计相符的449 bp条带;特异性试验结果表明犬冠状病毒、猫冠状病毒等均扩增出449 bp目的条带,而阴性对照没有。敏感性试验结果表明,其敏感程度与常规PCR方法相同。将扩增的聚合酶基因与冠状病毒的参考毒株进行同源性比对,同源率在56.69%～99.55%之间。进化树分析结果与文献中对冠状病毒根据血清学与基因学角度分类报道相一致。

A pair of universal primers were designed according to conservative regions of RNA polymemse genes of animal coronavirues published at GenBank, and used to amplify DNA fragment from cDNA of canine comnavirus （CCV）, feline coronavirus and other 6 coronaviruses. The PCR conditions were optimized and a specific 449 bp gene segment was amplified from CCV and other coronaviruses. Sequence of PCR fragments of RNA polymerase genes were compared with those of reference strains, which showed nucleotide homology of 56.69 % to 99.55 %. The results also showed a phylogenetic tree that was identical to the published results.