本文選題：柯薩奇病毒A6型 + 重組酶聚合酶擴增　； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：背景和目的:手足口病(HFMD,hand foot mouth disease)是全世界,尤其是在亞太地區(qū)流行的病毒性傳染病,主要感染5歲以下兒童,嚴重威脅兒童健康和生命。HFMD由多種病毒導致,主要包括腸道病毒71(EV71 enterovirus 71)型、柯薩奇病毒(CA,coxsakievirus)A16、A4、A6、A10、B3、B5 以及艾克病毒 30 型等。近年來,多項研究表明HFMD的致病病原體譜發(fā)生改變,CA6成為越來越重要的病原體,在全世界范圍內(nèi)已導致多起HFMD暴發(fā)。CA6可以引起重癥HFMD病例,并可導致部分重癥患兒死亡。CA6常引起非典型的HFMD,容易導致誤診。HFMD尚無有效治療藥物,針對EV71的CA16的單價以及雙價疫苗已進入臨床試驗階段。但針對多種病毒的多價疫苗進展緩慢。HFMD主要預防和控制措施是切斷傳播,對癥治療以減少并發(fā)癥的出現(xiàn)。加強HFMD的全球流行病學和實驗室病原學監(jiān)測,預測新的暴發(fā)疫情將有助于HFMD的預防和控制,而病原體的快速檢測則是HFMD有效監(jiān)測和診斷的關(guān)鍵。重組酶聚合酶擴增技術(shù)(RPA,recombinase polymerase amplification)是英國Twist DX公司商業(yè)化的新型核酸擴增檢測技術(shù),該技術(shù)不依賴于昂貴的儀器,在37-42度的恒溫條件下可完成對目的基因的擴增和檢測,有多種探針可應(yīng)用于產(chǎn)物檢測,大大提高了檢測的特異性。另外,其擴增產(chǎn)物也可以進行測序分析,增加了其可核對性。RPA技術(shù)可以在20min內(nèi)實現(xiàn)對病原體的檢測,是目前最快速的核酸擴增檢測方法,已被廣泛用于病原體的快速檢測。所以,RPA技術(shù)非常適合于傳染病暴發(fā)現(xiàn)場的快速檢測。本研究旨在建立針對CA6的實時熒光逆轉(zhuǎn)錄一步法重組酶聚合酶(RT-RPA,reverse transcription remconbinase polymerase amplification)快速檢測方法。方法:在Genbank下載CA6病毒的VP1蛋白的基因序列,并比對序列尋找保守區(qū)。針對保守區(qū)設(shè)計特異性的引物,進行引物篩選并設(shè)計相應(yīng)的探針,隨后建立針對CA6的實時熒光RT-RPA快速檢測方法。將含有目的片段的cDNA連接至載體,并進行體外轉(zhuǎn)錄,計算核酸濃度并轉(zhuǎn)換成拷貝數(shù)/μ1,隨后進行10倍梯度稀釋,每個梯度設(shè)置8個重復,分別用實時熒光RT-RPA檢測和商業(yè)化CA6熒光定量PCR檢測試劑盒檢測,概率元分析計算其95%檢測限。用RT-RPA檢測對照病毒,分析其特異度。分別利用RT-RPA和商業(yè)化CA6熒光定量PCR檢測試劑盒檢測234份EV71和CA16陰性的HFMD病例的糞便樣本,分析檢測結(jié)果一致性,計算Kappa值。結(jié)果:RT-RPA檢測方法的反應(yīng)時間為20min。RT-RPA檢測方法只與CA6發(fā)生反應(yīng),產(chǎn)生擴增曲線,沒有與任何對照病毒發(fā)生反應(yīng),其特異度為100%。RT-RPA的95%檢測限低至231拷貝/反應(yīng),與商業(yè)化熒光定量PCR無差別。臨床樣本的檢測結(jié)果顯示RT-RPA檢測方法與商業(yè)化CA6熒光定量PCR檢測試劑盒的kappa值為0.93,p0.001,證明兩者的檢測結(jié)果高度一致。結(jié)論:本研究建立的針對CA6的實時熒光RT-RPA具備良好的特異性和靈敏度,反應(yīng)快速,將有助于CA6相關(guān)的HFMD的監(jiān)測和診斷。
[Abstract]:Background and objective: HFMD hand foot mouth disease) is a viral infectious disease that is prevalent all over the world, especially in the Asia-Pacific region. It mainly infects children under 5 years of age, and is a serious threat to children's health and life. HFMD is caused by many viruses. It mainly includes enterovirus 71(EV71 enterovirus 71), coxsackievirus A16A4, A10A10, B3B5 and Ike virus 30, etc. In recent years, many studies have shown that the pathogenicity of HFMD has changed. CA6 has become a more and more important pathogen, and has led to a number of HFMD outbreaks. CA6 can cause severe HFMD cases worldwide. CA6 can often cause atypical HFMDs, which may lead to misdiagnosis. There is no effective therapeutic drug. The monovalent and bivalent vaccine against CA16 of EV71 has entered the stage of clinical trial. But the progress of multivalent vaccine against many viruses is slow. The main prevention and control measures of HFMD are cut off transmission and symptomatic treatment to reduce the occurrence of complications. To strengthen the global epidemiological and laboratory etiological surveillance of HFMD and predict the new outbreak situation will be helpful to the prevention and control of HFMD, and the rapid detection of pathogens is the key to the effective monitoring and diagnosis of HFMD. Recombinant enzyme polymerase amplification (RPA) is a new type of nucleic acid amplification technique commercialized by Twist DX Company in UK. This technique does not depend on expensive instruments and can be used to amplify and detect the target gene under constant temperature of 37-42 degrees. A variety of probes can be used for product detection, which greatly improves the specificity of detection. In addition, its amplification products can also be sequenced and analyzed, adding its verifiability. RPA technology can detect pathogens in 20min. It is the most rapid method of nucleic acid amplification and has been widely used in the rapid detection of pathogens. Therefore, RPA technology is very suitable for rapid detection of infectious disease outbreaks. The aim of this study was to establish a one step real-time fluorescent reverse transcriptase polymerase reverse transcription remconbinase polymerase amplification) assay for CA6. Methods: the gene sequence of VP1 protein of CA6 virus was downloaded from Genbank, and the conserved region was found. The specific primers were designed for the conserved region, the primers were screened and the corresponding probes were designed, and then the rapid detection method of real-time fluorescent RT-RPA for CA6 was established. The cDNA containing the target fragment was ligated to the vector and transcribed in vitro. The nucleic acid concentration was calculated and converted into copy number / 渭 1, then diluted by 10 times gradient, each gradient was set up by 8 repeats. The detection limit of 95% was calculated by using real-time fluorescence RT-RPA detection kit and commercial CA6 fluorescence quantitative PCR detection kit. The control virus was detected by RT-RPA and its specificity was analyzed. The fecal samples of 234 EV71 and CA16 negative HFMD patients were detected by RT-RPA and commercial CA6 fluorescence quantitative PCR assay kit. The results were consistent and the Kappa values were calculated. Results the reaction time of the 20min.RT-RPA detection method was that the reaction time of the 20min.RT-RPA method only reacted with CA6, and the amplification curve was generated. The specificity of the reaction time was as low as 231copy / reaction limit of the 95% detection limit of 100%.RT-RPA, but not with any control virus. There was no difference with commercial fluorescent quantitative PCR. The detection results of clinical samples showed that the kappa value of RT-RPA detection method and commercial CA6 fluorescent quantitative PCR kit was 0.93p0.001, which proved that the detection results were highly consistent. Conclusion: the real-time fluorescent RT-RPA for CA6 has good specificity, sensitivity and rapid response, which will be helpful to the monitoring and diagnosis of CA6 related HFMD.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R725.1;R3416

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本文編號：1952827