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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。
Objective: To develop a multiplex RT-PCR method for the detection of five major respiratory viruses of Influenza A and B, RSV A, B (RSV-A, RSV-B) and adenovirus (ADV). Methods: Primer premier 5.0 was used to design five pairs of specific primers for M gene of influenza A virus, PB1 gene of influenza B virus, F gene of RSV-A and RSV-B and hexon gene of ADV, dNTP, primer concentration and annealing temperature were optimized to establish a multiplex RT-PCR method for simultaneous detection of influenza A and B, RSV-A, RSV-B and ADV, and to verify the sensitivity of the assay. Results: The multiplex RT-PCR method of five viruses could amplify 141bp, 635bp, 525bp, 377b and 283bp gene fragments of influenza A and B, RSV-A, RSV-B and ADV simultaneously or separately, Sensitivity reached 770PFU / ml, 800PFU / ml, 680PFU / ml, 970PFU / ml and 850PFU / ml, respectively, and no cross-reaction between the five viruses. Conclusion: The established multiplex RT-PCR method can detect influenza A and B, RSV-A, RSV-B and ADV rapidly and accurately, providing a convenient and convenient method for the detection of five viruses.