快速检测犬瘟热病毒的实时荧光RPA方法的建立

Establishment of real-time recombinase polymerase amplification assay for rapid detection of canine distemper virus

针对犬瘟热病毒(canine distemper virus,CDV)基因保守序列,设计特异性引物和荧光探针,并进行了敏感性和特异性试验,建立了CDV实时重组聚合酶等温扩增(real-time recombinase polymerase amplification,real-timeRPA)检测方法。结果表明,该实时荧光RPA方法在39℃恒温反应25min能特异性扩增目的基因,与其他犬病毒以及CDV同属的麻疹病毒均未出现交叉反应;该检测方法具有与RT-PCR一致的敏感性;通过对CDV阳性犬的组织样品、体表样品和不同时期分离鉴定的临床样本的检测,证实建立的实时荧光RPA方法具有良好的稳定性和可靠性。与现有的核酸检测方法相比,该实时荧光RPA检测方法在核酸上样25min内即可判读结果,可满足CDV快速检疫的需要。

Using pairs of special primers and probes for conserved sequence of canine distemper virus (CDV),the real-time recombinase polymerase amplification assay (RPA)was developed after tests of sensitivity and specificity.The results showed that the new method could detect target gene specifically at 39℃ within 25 min and there was no cross-reaction with other canine virus and measles virus.The new method had high sensitivity,which was in accordance with RT-PCR.The excellent stability of the new method was confirmed by detection of CDV infected positive and negative samples and clinical samples seperated at different time.Compared with other method,the real-time RPAmethod could finish detection within 25 min after nucleic acid isolated and meet the requirement of rapid detection of CDV.