基于HCR技术对流感病毒H1N1新型可视化检测方法的建立

发布时间：2018-03-22 19:10

本文选题：流感病毒H1N1　切入点：杂交链式反应　出处：《黑龙江八一农垦大学》2017年硕士论文　论文类型：学位论文

【摘要】：极具感染性的流感病毒,尤其是甲型流感病毒,会引发全球范围内流感大规模爆发,造成巨大的经济损失,长期威胁人类与动物健康安全。因此快速、准确地对流感病毒检测是防控流感疫情的关键环节。为了实现对流感病毒快速、方便的检测,我们建立了三种流感病毒H1N1的可视化检测方法。本研究基于杂交链式反应(Hybridization Chain Reaction,HCR)核酸扩增技术结合胶体金核酸试纸条信号输出方式,实现对流感病毒H1N1简便、快速的可视化检测。具体如下:1利用HCR信号放大技术结合胶体金核酸试纸条,建立对流感病毒H1N1可视化检测方法。针对H1N1的特异性核酸片段设计了标记6-羧基荧光素(FAM)的抓取探针CP与检测探针DP,当有H1N1 RNA存在时,会形成标记生物素(Biotin)的HCR产物与标记FAM-CP的核酸复合产物,结合试纸条加样检测,会被检测线上的anti-FAM抓住,并通过链霉亲和素(SA)与生物素(Biotin)之间的特异性亲和力,将包被链霉亲和素(SA)的红色胶体金粒子(Au NPs)固定到检测线上,显示出红色条带,实现对H1N1的可视化检测,可达到2.96 p M的检测限。2分子信标(Molecular beacons,MBs)核酸探针技术与HCR信号放大技术结合,实现信号双重放大。当存在检测靶标H1N1 RNA时,H1N1 RNA会与MBs结合,使MBs构象发生改变,进而引发两段辅助探针Helper A、Helper B与MBs杂交,形成新的DNA双链,将靶标H1N1替换下来,H1N1 RNA循环结合其他MBs,实现信号一重放大。暴露出的MBs茎部设计有与检测探针DP的互补序列,能够与标记Biotin的HCR产物有效结合,进而实现信号的再次放大。结合试纸条传感器实现流感病毒H1N1的可视化检测,可达到21.9 f M的检测限。3将双链特异性核酸酶(DSN)与HCR信号放大技术相结合,设计了同时标记生物素(Biotin)与6-羧基荧光素(FAM)的抓取探针CP,当存在H1N1 RNA时,CP与H1N1 RNA形成杂交双链,由于DSN酶的特性,酶切DNA-RNA杂合双链中的互补DNA,RNA可以循环利用,结合其他CP,实现信号一重放大。由CP释放出的DNA短链与标记Biotin的H1、H2引发HCR反应,实现信号双重放大。结合试纸条传感器实现流感病毒H1N1的可视化检测,可达到3.12 f M的检测限。综上所述,本研究建立的三种对流感病毒H1N1的可视化检测方法,能够实现方便、快速的靶标检测,节省检测时间与成本,对流感病毒的基层大规模筛检与疫情防控具有应用价值。
[Abstract]:Highly infectious influenza viruses, especially influenza A viruses, can cause large-scale outbreaks of influenza worldwide, causing huge economic losses and long-term threats to human and animal health and safety. Accurate detection of influenza viruses is a key link in the prevention and control of influenza outbreaks. In order to achieve rapid and convenient detection of influenza viruses, We have established three visual detection methods for influenza virus H1N1. This study is based on hybrid chain reaction (hybridization Chain reaction) nucleic acid amplification technique combined with the method of colloidal gold nucleic acid test strip signal output to realize a simple and convenient method for influenza virus H1N1. Rapid visual detection. Specific as follows: 1 using HCR signal amplification technology combined with colloidal gold nucleic acid test paper, A visual detection method for influenza virus H1N1 was established. The grab probe CP and the detection probe DPDP RNA were designed for the specific nucleic acid fragment of H1N1. When there was H1N1 RNA, the grab probe CP and the detection probe were designed. The HCR product labeled biotinin and the nucleic acid compound product labeled FAM-CP, combined with the test strip to add sample detection, will be caught by the anti-FAM on the detection line, and the specific affinity between the HCR product and biotinin can be obtained by using streptavidin) and biotinin. The red colloidal gold particle (au NPs) coated with streptavidin (SAA) was immobilized on the detection line, and the red band was displayed to realize the visual detection of H1N1. The detection limit of 2.96 pm can be achieved by combining the molecular beaconsm nucleic acid probe technique with the HCR signal amplification technique to realize the double amplification of the signal. When the detection target H1N1 RNA exists, the H 1 N 1 RNA will combine with the MBs, and the conformation of MBs will change. Then the two-segment auxiliary probe Helper Agna Helper B was hybridized with MBs to form a new double strand of DNA. The target H1N1 was replaced by the H1N1 RNA cycle and combined with other MBs to realize signal amplification. The exposed stem of MBs was designed with complementary sequence to detect the probe DP. It can effectively combine with the HCR products labeled with Biotin, and then realize the signal re-amplification. In combination with the test strip sensor, the visual detection of influenza virus H1N1 can be realized. The detection limit of 21.9 FM can be reached by combining double-strand specific nuclease (DSNs) with HCR signal amplification technique. A grab probe, CP-labeled biotinin) and 6-carboxyfluorescein (FAM), was designed. When there was H1N1 RNA, CP and H1N1 RNA formed hybrid double strands. Because of the characteristic of DSN enzyme, the complementary DNA-RNA RNA in the DNA-RNA heterozygous double strand can be recycled and combined with other CPs to amplify the signal. The short DNA strand released by CP induces HCR reaction with the labeled Biotin H _ (1) O _ (2). Realize double amplification of signal. Realize the visual detection of influenza virus H1N1 with test strip sensor, can reach the detection limit of 3.12 FM. To sum up, the three visual detection methods of influenza virus H1N1 established in this study can be realized conveniently. Rapid target detection saves detection time and cost and is valuable for mass screening of influenza virus and prevention and control of influenza virus.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440;R511.7

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