犬瘟热病毒实时荧光RT-RPA检测方法的建立及应用

Establishment and Application of Real-time Fluorescent RT-RPA for Detection of Canine Distemper Virus

为建立犬瘟热病毒(CDV)临床快速检测方法,针对CDV保守N基因依据重组酶聚合酶等温扩增(RPA)引物设计原理进行RPA引物探针设计,通过反应条件优化、敏感性试验、特异性试验和临床样品检测,建立了CDV实时荧光RT-RPA检测方法。结果显示,反应体系中M-MLV反转录酶最适浓度为4 U/μL,所建CDV实时荧光RT-RPA检测方法特异性较好,敏感性最低可检测到5.68×102 copies/μL,高于国标RT-PCR(GB/T 27532-2011)检测方法。建立的CDV实时荧光RT-RPA检测方法具有较高的敏感性和特异性、操作简便、20 min内判定结果,可用于临床犬瘟热快速诊断,对犬瘟热的防控具有重要意义。

In order to establish a rapid clinical detection method for canine distemper virus(CDV),RPA primers and probe were designed for the conserved N gene of canine distemper virus based on the recombinase polymerase amplification(RPA)primer design principle.RT-RPA assay was established by optimization of reaction conditions,sensitivity test,specificity test and clinical sample test.The results showed that the optimum concentration of M-MLV reverse transcriptase was 4 U/μL,the RT-RPA assay for CDV had a better specificity and a lowest sensitivity of 5.68×102 copies/μL,higher than the national standard RT-PCR(GB/T 27532-2011)assay.The method which has high sensitivity and specificity is easy to operate and get the results within 20min,and can be effectively applied to the rapid detection of clinical canine distemper,which would be significant to the prevention and control of canine distemper.