五种主要上呼吸道病毒多重RT-PCR检测方法的建立

Establishment of Multiplex RT-PCR for Detecting Five Kinds of Main Respiratory Viruses

目的:建立甲型、乙型流感病毒、呼吸道合胞病毒A型、B型(RSV-A、RSV-B)和腺病毒(ADV)五种主要上呼吸道病毒的多重RT-PCR检测方法。方法:利用Primer premier5.0分别针对甲型流感病毒的M基因、乙型流感病毒的PB1基因、RSV-A和RSV-B的F基因及ADV的hexon基因设计五对特异性引物,对Mg2+、dNTP、引物浓度及退火温度等进行优化,建立同时检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV的多重RT-PCR方法,并验证该检测方法的灵敏性。结果:所建立的五种病毒的多重RT-PCR方法可以同时或者分别扩增甲型、乙型流感病毒、RSV-A、RSV-B及ADV的141bp、635bp、525bp、377b和283bp基因片段,敏感度分别达到770PFU/ml、800PFU/ml、680PFU/ml、970PFU/ml和850PFU/ml,且五种病毒间无交叉反应。结论:所建立的多重RT-PCR方法可以迅速准确地检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV,为五种病毒的检测提供了一种方便易行的方法。

Objective： To establish multiplex RT-PCR method for detecting five kinds of main respiratory viruses such as influenza A and B virus,respiratory syncytial virus（RSV） types A and B and adenovirus（ADV）.Methods： Five pairs of specific primers were designed with the Primer Premier 5.0 to detect M gene of influenza A virus,PB1 gene of influenza B virus,F gene of RSV-A、 RSV-B and hexon gene for ADV;the optimal reaction conditions of Mg2＋ concentration and annealing temperature were optimized to establish a stable and specific multiplex RT-PCR reaction for the rapid detection of four kinds of main respiratory viruses;then the specificity and sensitivity of the RT-PCR reaction were checked.Results： The multiplex RT-PCR reaction established could amplify 141bp,635bp,525bp,377bp and 283bp of influenza A and B virus,respiratory syncytial virus（RSV） types A and B and ADV;the sensitivity were 770PFU/ml,800PFU/ml,680PFU/ml,970PFU/ml and 850PFU/ml and there was no cross-reactivity between the four types.Conclusion： The multiplex RT-PCR reaction established could differential diagnosis of influenza A and B virus,RSV-A,RSV-B and ADV rapidly.It supplied an easy method for detecting five kinds of main respiratory viruses.