【摘要】：目的：柯薩奇病毒A組6型病毒（coxsackievirus A6, CVA6）是引起手足口病的常見病原體，且其引起的手足口病常伴有甲病變等癥狀。本實驗的目的在于建立一種適于廣泛推廣的檢測CVA6的快速高效、靈敏特異的僅需在單個試管中一步完成的逆轉(zhuǎn)錄環(huán)介導等溫擴增方法（reverse transcription loop-mediatedisothermal amplification，RT-LAMP），以滿足快速識別CVA6感染的需要。 方法：采集手足口病病人的糞便樣本進行核酸擴增并鑒定分型后，選取合適標本用做反應(yīng)模板。在GenBank上下載編碼CVA6衣殼蛋白VP1的基因序列，，利用在線軟件設(shè)計針對CVA6基因組VP1片段保守區(qū)的引物，在特異性引物和具有鏈置換活性的Bst聚合酶作用下進行RT-LAMP反應(yīng)，探索CVA6-RT-LAMP方法的最佳反應(yīng)條件,肉眼觀察擴增結(jié)果或取產(chǎn)物進行瓊脂糖凝膠電泳分析，驗證其特異性并與常規(guī)的逆轉(zhuǎn)錄聚合酶鏈反應(yīng)（reverse transcription polymerasechain reaction, RT-PCR）比較靈敏度。利用熒光PCR儀得到擴增產(chǎn)物的熔解曲線。 結(jié)果：優(yōu)化后的CVA6-RT-LAMP體系特異性強，僅能擴增出CVA6核酸，與其他型別的腸道病毒不發(fā)生非特異性擴增反應(yīng)；靈敏度高，約為CVA6-RT-PCR法的100倍；反應(yīng)快速，可在40分鐘內(nèi)達到反應(yīng)平臺期；熒光熔解曲線呈單峰狀，也說明反應(yīng)產(chǎn)物單一，特異性強。 結(jié)論：本實驗建立的CVA6-RT-LAMP技術(shù)具有快速高效，靈敏度高、特異性強、操作簡便、對儀器設(shè)備的要求低，結(jié)果判定方便等優(yōu)點，適于廣泛推廣做為CVA6鑒定的方法，尤其適于在缺乏先進設(shè)備的基礎(chǔ)單位推廣。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R512.5

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