犬瘟热与犬副流感病毒双重一步法RT-PCR检测方法的建立

Establishment of the duplex one-step reverse transcription PCR for detection of canine distemper virus and canine parainfluenza virus

为建立可以同时检测犬瘟热病毒(CDV)和犬副流感病毒(CPIV)的检测方法,依据GenBank中登录的CDV的H基因和CPIV的NP基因保守序列,设计合成扩增序列分别为593 bp (CDV H基因)、1 530 bp (CPIV NP基因)的2对引物,通过优化反应条件,建立一种能够同时检测CDV和CPIV的双重一步法RT-PCR检测方法。特异性和敏感性试验显示,该方法能特异地检测CDV和CPIV,其最低检测限分别为3.58×10~6拷贝/μL和1.74×10~6拷贝/μL。对51份临床疑似病料样品进行检测,同时分别采用商品化的CDV和CPIV抗原检测试剂盒进行检测,结果二者符合率为100%。该结果表明本实验所建立的双重一步法RT-PCR方法的灵敏度及特异性均较好,可用于临床相关疫病的诊断。

To develop a method for simultaneous detection of canine distemper virus （CDV） and canine parainfluenza virus （CPIV）, a duplex one-step reverse transcription PCR （RT-PCR） was established with two pairs of specific primers designed according to the H gene of CDV and NP gene of CP1V. Under the optimized conditions of the duplex one-step RT-PCR, two special fragments of CDV （593bp） and CPIV （1,530bp） were amplified from the genomic RNAs of CDV and CPIV. It was showed that the duplex one-step RT-PCR assay was specificity and sensitivity with a detection limit of 3.58 x l06copies/txL and 1.74 x l06copies/ixL. Fifty-one suspected clinical samples were detected by the duplex one-step RT-PCR assay and commercial CDV and CPIV antigen test kits, respectively. It was showed that the coincidence rate of the two detection methods was 100%. The results showed that the sensitivity and specificity of the duplex one-step RT-PCR method could be application in clinical diagnoses of the related diseases.