基于重组酶聚合酶扩增技术的犬冠状病毒快速检测方法的建立

Development of the Recombinase Polymerase Amplification Assay for Rapid Detection of Canine Coronavirus

为建立一种快速、简便检测犬冠状病毒(CCoV)的方法,本试验基于犬冠状病毒M基因的保守区域设计特异性引物,通过优化反应条件,建立了CCoV的重组酶聚合酶扩增检测方法(RT-RPA)。试验结果表明,所建立的RT-RPA方法在35℃恒温反应15 min即可检测出100个拷贝的体外转录RNA,且与犬的其他常见病毒,如犬瘟热病毒、犬细小病毒、犬副流感病毒等,无非交叉反应,表明所建立的CCoV RT-RPA具有良好的特异性及较高的敏感性。使用RT-RPA对25份临床样品的检测结果显示,CCoV的检出率为48%(12/25),该结果与荧光定量PCR的检测结果完全一致。表明本试验所建立的CCoV RT-RPA检测方法操作简单、反应快速灵敏,适用于CCoV的快速检测。

To establish a rapid and simple detection method for canine coronavirus(CCoV),the recombinase polymerase amplification assay was developed and optimized with the specific primers designed according to the conserve sequence of M gene of CCoV.The results showed that the whole procedure of RT-RPA can dectect 100 copies of in vitro transcription RNA in 15 min at 35℃.The new developed assay was highly specific and no cross-reaction was observed with canine distemper virus,and canine parvovirus and canine parainfluenza virus.The results showed that the method was highly sensitive.25 clinical samples were detected using both RT-RPA and real time PCR,and these two methods shared 100%coincidence with 48%(12/25)positive rate.In this study,a simple and sensitive RT-RPA assay is developed and evaluated,which is useful for rapid detection of CCoV.