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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。

英文摘要:

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.

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