【摘要】 目的:建立一种特异、灵敏、快速检测甲型流感病毒核酸的荧光定量RT-PCR方法。方法:根据GenBank登录的流感毒株序列,应用生物软件在甲型流感病毒膜蛋白(MP)基因的保守区设计与筛选引物和MGB探针,对荧光定量RT-PCR反应体系与条件进行优化,验证方法的特异性和敏感性。并通过对疑似流感临床样本的检测,以评价该方法的实际应用价值。结果:该方法对甲型流感病毒的检测有高度的特异性、通用性,对乙型流感、麻疹、风疹、腮腺炎、RSV和腺病毒等其他呼吸道病毒均无交叉反应,检测的灵敏度达0.1TC ID50,可从疑似流感患者含漱液中直接检测流感病毒核酸,从病毒核酸提取至完成检测仅需2.5 h左右,且操作简便,重复性好。结论:本研究建立的MGB荧光定量RT-PCR的方法具有特异、敏感、快速的特点,适用于甲型流感疫情的应急快速检测。更多还原

【Abstract】 Objective:To develop a real-time RT-PCR assay to rapidly detect influenza A virus.Methods:Primers and TaqMan-MGB probes were designed and selected from highly conserved regions of matrix protein（MP） gene of influenza A virus.The assay was optimized in reactive system and condition to improve the specificity and sensitivity.In addition,the practical value of the assay was estimated by detecting clinical specimens.Results:[WTBZ]The assay was highly specific and sensitive in detection of influenza A virus,and none of the negative control samples showed false-positive reaction in duplication.The detective limit of the assay was 0.1TCID₅₀.It took only 2.5 hours from viral RNA extraction to PCR completion.Meanwhile,the assay was simple and of good reproducibility.Conclusion:This real-time RT-PCR assay is an excellent method which is suitable for quick and quantitative detection of influenza A virus in clinical lab.更多还原