多重逆转录聚合酶链反应检测主要上呼吸道病毒
Main respiratory viruses detected by multiplex reverse transcription polymerase chain reaction
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摘要:目的 建立用多重逆转录聚合酶链反应(mRT-PCR)检测甲1型、甲3型、乙型流感病毒以及呼吸道合胞病毒(RSV)A型、B型等主要上呼吸道病毒的方法, 以快速检测上呼吸道感染主要病毒及对甲型、甲亚型、乙型流感病毒在上海地区人群感染、流行情况调查。方法 采用经MDCK细胞接种培养的甲3型、乙型流感病毒和Hep-2细胞接种培养的RSVA型、B型标准毒株病毒液, 以及甲1型、甲3型、乙型流感病毒和RSVA型、B型混合毒株病毒液, 使用5组引物, 分别为HA1-5a1和HAl-3a1(甲1型流感病毒)、HA3-5a1和HA3-3a1(甲3型流感病毒)、NP-5b1和NP-3b1(乙型流感病毒)、RSVAF和RSVAR(RSVA型)、RSVBF和RSVBR(RSVB型), 经mRT-PCR, 琼脂糖凝胶电泳, 于紫外灯下观察上述病毒特异核苷酸条带。结果 甲1型、甲3型、乙型流感病毒, RSVA型、B型分别在431, 210, 390, 334, 183bp处出现清晰的5条DNA条带。结论 应用5组引物, mRT-PCR可用于快速检测临床上呼吸道感染标本中的流感病毒和RSV, 并可对其进行分型, 对上呼吸道病毒感染性疾病的诊断和流感的监测方面是非常重要的。Abstract:Objective A multiplex reverse transcription polymerase chain reaction(mRT-PCR)method is capable of detecting and subtyping the main respiratory viruses, including influenza A(H1N1 and H3N2)and B viruses as well as respiratory syncytial virus(RSV)types A and B.mRT-PCR method is establishd in order to detect the main viruses in respiratory clinical samples and the outbreak and epidemic of influenza in Shanghai.Methods Influenza A(H1N1, and H3N2), influenza B and RSV types A and B cultured by MDCK shell viral are adopted to detect their DNA zone spectrum in ultraviolet after mRT-PCR and agarose gel electrophoresis.Results Corresponding five obvious DNA zone spectrums at 431 bp, 210 bp, 390 bp, 334 bp and 183 bp respectively in ultraviolet after mRT-PCR and agarose gel electrophoresis.Conclusions Using five pairs of primers, mRT-PCR could be used to detect influenza virus and to subtype the influenza A and RSV in clinical samples rapidly, which is very important for diagnosis and surveillance of upper respiratory tract infectious diseases.
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