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。
Objective： To develop a real - time RT - PCR assay to rapidly detect influenza A virus. Methods： Primers and Taq-Man - 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：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.
出处
《中国卫生检验杂志》
CAS
2007年第1期46-48,共3页
Chinese Journal of Health Laboratory Technology
