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目的通过分析GenBank中丙型流行性感冒(流感)病毒的序列,建立丙型流感病毒实时荧光定量-反转录聚合酶链反应(rRT-PCR)检测方法,并制备丙型流感病毒的RNA标准品。方法下载GenBank中丙型流感病毒的序列,使用Beacon Designer 8软件在保守区域设计特异性引物和TaqMan荧光探针。人工合成病毒HE、NP、MP和NS基因序列,克隆到pBluescriptⅡSK(+)质粒载体,进一步通过体外转录获得RNA模板,并验证检测方法的检测极限值和特异性。结果建立了针对丙型流感病毒HE、NP、MP和NS基因的快速、可靠的实时荧光定量RT-PCR检测方法,同时制备了RNA标准品。结论本文建立的丙型流感病毒实时荧光定量RT-PCR检测方法,可以在日常监测和临床鉴定中快速、准确地检测丙型流感病毒。
Objective To establish a real-time fluorescence quantitative-reverse transcription-polymerase chain reaction (rRT-PCR) assay for influenza C virus and to prepare RNA standard of influenza C virus by analyzing the sequence of influenza C (influenza) virus in GenBank. Product Methods The sequence of influenza virus type C in GenBank was downloaded and specific primers and TaqMan fluorescent probes were designed in the conserved region using Beacon Designer 8 software. Synthetic HE, NP, MP and NS gene sequences were synthesized and cloned into pBluescriptⅡSK (+) plasmid vector. The RNA template was further obtained by in vitro transcription, and the detection limit and specificity of the method were verified. Results A rapid and reliable real-time fluorescence quantitative RT-PCR assay for HE, NP, MP and NS genes of influenza virus C was established and RNA standards were also prepared. Conclusion The real-time fluorescence quantitative RT-PCR assay of influenza C virus established in this paper can rapidly and accurately detect influenza C virus in routine monitoring and clinical evaluation.