犬瘟热病毒、犬细小病毒和犬冠状病毒三重PCR检测方法的建立

Establishment of a multiplex PCR for simultaneous detection of CDV,CPV and CCV

参考Gen Bank上登录的犬瘟热病毒（CDV）、犬细小病毒（CPV）和犬冠状病毒（CCV）的基因序列保守片段分别设计特异性引物,建立了可以同时检测CDV（684 bp）、CPV（213 bp）和CCV（417 bp）的三重PCR方法。特异性结果表明,所建立的体系只能扩增CDV、CPV、CCV,而不能扩增犬腺病毒Ⅱ型（CAV-Ⅱ）和犬副流感病毒（CPIV）;敏感性试验表明,该方法对CDV、CPV和CCV的最低核酸检测量分别为1.39×10^- 2、6.0×10^-2和6.7×10^-2 copies/L,其敏感性比普通PCR高100倍。采用建立的三重PCR方法对北京、山东地区收集的临床粪便样品进行检测结果表明,使用三重PCR体系的扩增结果与单项PCR扩增结果一致,符合率为100%。以上结果表明该方法快速、敏感、特异性强,对上述3种病毒能够进行快速鉴定诊断,为临床诊断提供了快速简便的方法。

According to the gene sequences in Gen Bank of canine distemper virus（ CDV）,canine parvovirus（ CPV） and canine coronavirus（ CCV）,three pairs of specific primers were designed for amplifying the three specific fragments of CDV（ 684 bp）,CPV（ 213 bp）and CCV（ 417 bp）,respectively. The established detection method was examined for specificity and sensitivity. The results for specificity showed that the system could amplify CDV,CPV and CCV only,but could not amplify canine adenovirus type II（ CAV-II） or canine parainfluenza virus（ CPIV）. The sensitivity test showed that the system was highly sensitive with the minimum test amount of nucleic acid for CDV,CPV and CCV was 1. 39×10^-2,6. 0×10^-2 and 6. 7×10^-2 copies/L,respectively. Then,eight clinical fecal samples from Beijing and Tianjin were detected by the multiplex PCR and the single PCR,resulting in a coincidence rate of 100%. The above results show that the method developed here is rapid,sensitive and specific,and could be used to effectively detect and differentiate CDV,CPV and CCV individual-or co-infection in clinical samples.