流感病毒检测新技术的建立及初步应用研究

发布时间：2018-01-10 07:20

本文关键词：流感病毒检测新技术的建立及初步应用研究　出处：《中国疾病预防控制中心》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：建立和评价两种基于实验室的同时检测新甲型流感病毒和季节性流感病毒的多重RT-PCR方法；建立和评价一种基于现场的快速诊断新甲型H1N1流感病毒的等温扩增技术。方法：(一)利用GeXP system建立多重RT-PCR方法1.从NCBI网站的GenBank数据库中下载所有甲型流感病毒的Matrix(M)基因、所有甲型流感病毒H1N1亚型的血凝素(HA)基因、季节性H1N1流感病毒多的血凝素(HA)基因、季节性H3N2流感病毒的血凝素(HA)基因、2009年流行的新甲型H1N1流感病毒的血凝素(HA)基因的核苷酸序列,利用GeXP eXpress Profiler分别设计针对上述各基因的特异性引物。利用人的RNaseP基因做为内参判断标本来源和实施质量控制的依据；同时利用Kan(?)RNA做为内参判断扩增体系是否正常。2.建立8重RT-PCR方法同时检测新甲型流感病毒和季节性流感病毒,对其进行方法学评价(包括灵敏度、特异性等),并与WHO推荐的rRT-PCR鉴定结果进行比较。(二)利用CFX96real-time system建立4重rRT-PCR方法1.从NCBI网站的GenBank数据库中下载季节性H1N1流感病毒的血凝素(HA)基因、季节性H3N2流感病毒的血凝素(HA)基因、2009年流行的新甲型H1N1流感病毒的血凝素(HA)基因的核苷酸序列,利用Primer Premier5分别设计针对上述各基因的特异性引物和探针。利用人的RNaseP基因做为内参判断标本来源和实施质量控制的依据。2.建立4重rRT-PCR方法检测新甲型流感病毒和季节性流感病毒,对其进行方法学(包括特异性、灵敏度等)评价,并与WHO推荐的rRT-PCR方法的鉴定结果进行比较。(三)建立环介导的逆转录等温扩增技术1、选择人甲型H1N1流感HA基因(GenBank:FJ966974.1)设计相应的RT-LAMP引物。2、建立RT-LAMP特异性检测新甲型流感病毒的方法,对其进行方法学(包括特异性、灵敏度等)评价,WHO推荐的rRT-PCR方法的鉴定结果进行比较。结果：1.利用GeXP system建立的8重RT-PCR方法检测新甲型流感病毒的灵敏度达到10copies RNA。通过对不同来源的流感病毒的验证,结果表明每对引物均未见交叉反应,特异性较好。通过对29份新甲型流感疑似患者的咽拭子标本进行检测,GeXP多重RT-PCR的阳性率为97%,明显高于WHO推荐的rRT-PCR方法(76%)(P0.05)。2.利用CFX96建立的4重rRT-PCR方法检测新甲型流感病毒的灵敏度达到20copies RNA。通过对不同来源的流感病毒的验证,结果表明每对引物均未见交叉反应,特异性较好。通过对29份新甲型流感疑似患者的咽拭子标本进行检测,CFX96的多重rRT-PCR阳性率为93%,高于WHO推荐的rRT-PCR方法(76%)(P0.05)。3.利用RT-LAMP检测新甲型流感病毒的灵敏度达到60copies RNA。通过对不同来源的流感病毒的验证,结果表明此法特异性高。通过对29份新甲型流感疑似患者的咽拭子标本进行检测,RT-LAMP阳性率为90%,高于WHO推荐的rRT-PCR方法(76%)(P0.05)。结论：成功建立两种多重RT-PCR同时检测新甲型流感病毒和季节性流感病毒的新技术和一种基于现场的快速准确易推广的检测新甲型流感病毒的等温扩增技术,有助于提高我国流感监测网络实验室的检测能力。
[Abstract]:Objective: to establish and evaluate two laboratory based multiple RT-PCR methods for simultaneous detection of influenza A and seasonal influenza viruses, and establish and evaluate a field based rapid isothermal amplification technology for diagnosing influenza A (H1N1) virus.
Methods: (a) using GeXP system to establish the multiplex RT-PCR 1. Download all influenza A virus from the website of NCBI GenBank database in Matrix (M) gene, all H1N1 influenza virus hemagglutinin (HA) gene, seasonal H1N1 influenza virus hemagglutinin (HA) gene, H3N2 seasonal influenza virus the hemagglutinin (HA) gene, the 2009 pandemic H1N1 influenza A virus hemagglutinin (HA) gene nucleotide sequence, using GeXP eXpress Profiler specific primers were designed for each of the above genes. Identify the source of the specimen and the implementation of quality control for reference by the human RNaseP gene; at the same time using Kan (?) RNA as a reference to judge whether the normal.2. amplification system established 8 new RT-PCR for simultaneous detection of influenza A virus and seasonal influenza virus, on the methodological evaluation (including sensitivity, specificity, etc.), Compared with WHO and rRT-PCR identification results. (two) recommended by CFX96real-time system 4 rRT-PCR method 1. download H1N1 seasonal influenza virus from the website of NCBI GenBank database (HA) of the hemagglutinin gene, H3N2 seasonal influenza virus hemagglutinin (HA) gene, the 2009 pandemic H1N1 influenza A virus the hemagglutinin (HA) gene nucleotide sequence, using Primer Premier5 were designed according to the gene specific primers and probes. As a reference to judge the source of the specimen and the implementation of quality control based on.2. model is 4 rRT-PCR for the detection of new influenza virus and seasonal influenza virus RNaseP gene by the method of (including its specificity, sensitivity evaluation, etc.) the identification results are compared with rRT-PCR method and recommended by WHO. (three) the establishment of reverse transcription loop mediated isothermal amplification technology 1, choice The influenza A (H1N1) HA gene (GenBank:FJ966974.1) is designed with the corresponding RT-LAMP primer.2, and the method of RT-LAMP specific detection of influenza A virus is established. Its methodology (including specificity, sensitivity, etc.) is evaluated, and the results of rRT-PCR recommended by WHO are compared.
Results: the sensitivity of 1. by 8 RT-PCR method of GeXP system to establish new detection of influenza A virus to 10copies RNA. through verification of different sources of influenza virus, results show that each pair of primers showed no cross reactivity, good specificity. Through testing of 29 samples of the new swine flu suspected patients with throat swab specimens the positive rate of GeXP multiplex RT-PCR was 97%, significantly higher than that of rRT-PCR method recommended by WHO (76%) (P0.05).2. detection sensitivity of new influenza virus using 4 rRT-PCR method established by CFX96 reached 20copies RNA. through verification of different sources of influenza virus, results show that each pair of primers showed no cross reaction specificity. 29. Based on a new influenza A patients with suspected throat swab samples were detected, the positive rate of multiple rRT-PCR CFX96 was 93%, higher than the rRT-PCR method recommended by WHO (76%) (P0.05).3. by RT-LAMP The detection sensitivity of the new influenza virus reached 60copies RNA. through the verification of different sources of influenza virus, the results show that this method has high specificity. The 29 new suspected influenza patients with throat swab specimens were detected, the positive rate of RT-LAMP was 90%, higher than the rRT-PCR method recommended by WHO (76%) (P0.05).
Conclusion: the new technology successfully established two kinds of isothermal RT-PCR multiple simultaneous detection of new influenza virus and seasonal influenza virus and a live fast and accurate easy detection based on the new influenza virus amplification technology, help to improve the detection ability of laboratory influenza surveillance network in China.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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