应用RT-LAMP技术检测肠道病毒Coxsackievirus A6的研究

发布时间：2019-06-20 09:51

【摘要】：目的：柯萨奇病毒A组6型病毒（coxsackievirus A6, CVA6）是引起手足口病的常见病原体，且其引起的手足口病常伴有甲病变等症状。本实验的目的在于建立一种适于广泛推广的检测CVA6的快速高效、灵敏特异的仅需在单个试管中一步完成的逆转录环介导等温扩增方法（reverse transcription loop-mediatedisothermal amplification，RT-LAMP），以满足快速识别CVA6感染的需要。方法：采集手足口病病人的粪便样本进行核酸扩增并鉴定分型后，选取合适标本用做反应模板。在GenBank上下载编码CVA6衣壳蛋白VP1的基因序列，，利用在线软件设计针对CVA6基因组VP1片段保守区的引物，在特异性引物和具有链置换活性的Bst聚合酶作用下进行RT-LAMP反应，探索CVA6-RT-LAMP方法的最佳反应条件,肉眼观察扩增结果或取产物进行琼脂糖凝胶电泳分析，验证其特异性并与常规的逆转录聚合酶链反应（reverse transcription polymerasechain reaction, RT-PCR）比较灵敏度。利用荧光PCR仪得到扩增产物的熔解曲线。结果：优化后的CVA6-RT-LAMP体系特异性强，仅能扩增出CVA6核酸，与其他型别的肠道病毒不发生非特异性扩增反应；灵敏度高，约为CVA6-RT-PCR法的100倍；反应快速，可在40分钟内达到反应平台期；荧光熔解曲线呈单峰状，也说明反应产物单一，特异性强。结论：本实验建立的CVA6-RT-LAMP技术具有快速高效，灵敏度高、特异性强、操作简便、对仪器设备的要求低，结果判定方便等优点，适于广泛推广做为CVA6鉴定的方法，尤其适于在缺乏先进设备的基础单位推广。
[Abstract]:Objective: coxsackievirus A6 (CVA6) is a common pathogen of hand, foot and mouth disease (HFMD) caused by Coxsackievirus group A, and it is often accompanied by nail disease and other symptoms. The purpose of this experiment is to establish a rapid, efficient, sensitive and specific reverse transcriptional loop-mediated isotherm amplification method for CVA6 detection, which only needs to be completed in one step in a single test tube, in order to meet the needs of rapid identification of CVA6 infection. Methods: the fecal samples of patients with HFMD were collected for nucleic acid amplification and genotyping, and the suitable samples were selected as reaction templates. The gene sequence encoding CVA6 capsid protein VP1 was downloaded on GenBank. Primers for the conserved region of CVA6 genomic VP1 fragment were designed by online software. RT-LAMP reaction was carried out under the action of specific primers and Bst polymerase with chain replacement activity. the optimum reaction conditions of CVA6-RT-LAMP method were explored, and the amplification results were observed by naked eye or the products were analyzed by agarose gel electrophoresis. The specificity was verified and the sensitivity was compared with that of conventional reverse transcription polymerase chain reaction (reverse transcription polymerasechain reaction, RT-PCR). The melting curve of the amplified product was obtained by fluorescence PCR. Results: the optimized CVA6-RT-LAMP system was specific and could only amplify CVA6 nucleic acid, and there was no nonspecific amplification reaction with other types of enterovirus, the sensitivity was about 100 times of that of CVA6-RT-PCR method, the reaction was rapid and could reach the platform stage within 40 minutes, and the fluorescence melting curve was unimodal, which also showed that the reaction product was single and the specificity was strong. Conclusion: the CVA6-RT-LAMP technique established in this experiment has the advantages of rapid and high efficiency, high sensitivity, strong specificity, simple operation, low requirements for instruments and equipment, and convenient determination of results. It is suitable for wide application as a method of CVA6 identification, especially in basic units lacking advanced equipment.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.5

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