基于重组酶聚合酶扩增技术的柯萨奇A6型病毒的快速检测方法的建立

发布时间：2018-05-29 23:10

本文选题：柯萨奇病毒A6型 + 重组酶聚合酶扩增　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景和目的:手足口病(HFMD,hand foot mouth disease)是全世界,尤其是在亚太地区流行的病毒性传染病,主要感染5岁以下儿童,严重威胁儿童健康和生命。HFMD由多种病毒导致,主要包括肠道病毒71(EV71 enterovirus 71)型、柯萨奇病毒(CA,coxsakievirus)A16、A4、A6、A10、B3、B5 以及艾克病毒 30 型等。近年来,多项研究表明HFMD的致病病原体谱发生改变,CA6成为越来越重要的病原体,在全世界范围内已导致多起HFMD暴发。CA6可以引起重症HFMD病例,并可导致部分重症患儿死亡。CA6常引起非典型的HFMD,容易导致误诊。HFMD尚无有效治疗药物,针对EV71的CA16的单价以及双价疫苗已进入临床试验阶段。但针对多种病毒的多价疫苗进展缓慢。HFMD主要预防和控制措施是切断传播,对症治疗以减少并发症的出现。加强HFMD的全球流行病学和实验室病原学监测,预测新的暴发疫情将有助于HFMD的预防和控制,而病原体的快速检测则是HFMD有效监测和诊断的关键。重组酶聚合酶扩增技术(RPA,recombinase polymerase amplification)是英国Twist DX公司商业化的新型核酸扩增检测技术,该技术不依赖于昂贵的仪器,在37-42度的恒温条件下可完成对目的基因的扩增和检测,有多种探针可应用于产物检测,大大提高了检测的特异性。另外,其扩增产物也可以进行测序分析,增加了其可核对性。RPA技术可以在20min内实现对病原体的检测,是目前最快速的核酸扩增检测方法,已被广泛用于病原体的快速检测。所以,RPA技术非常适合于传染病暴发现场的快速检测。本研究旨在建立针对CA6的实时荧光逆转录一步法重组酶聚合酶(RT-RPA,reverse transcription remconbinase polymerase amplification)快速检测方法。方法:在Genbank下载CA6病毒的VP1蛋白的基因序列,并比对序列寻找保守区。针对保守区设计特异性的引物,进行引物筛选并设计相应的探针,随后建立针对CA6的实时荧光RT-RPA快速检测方法。将含有目的片段的cDNA连接至载体,并进行体外转录,计算核酸浓度并转换成拷贝数/μ1,随后进行10倍梯度稀释,每个梯度设置8个重复,分别用实时荧光RT-RPA检测和商业化CA6荧光定量PCR检测试剂盒检测,概率元分析计算其95%检测限。用RT-RPA检测对照病毒,分析其特异度。分别利用RT-RPA和商业化CA6荧光定量PCR检测试剂盒检测234份EV71和CA16阴性的HFMD病例的粪便样本,分析检测结果一致性,计算Kappa值。结果:RT-RPA检测方法的反应时间为20min。RT-RPA检测方法只与CA6发生反应,产生扩增曲线,没有与任何对照病毒发生反应,其特异度为100%。RT-RPA的95%检测限低至231拷贝/反应,与商业化荧光定量PCR无差别。临床样本的检测结果显示RT-RPA检测方法与商业化CA6荧光定量PCR检测试剂盒的kappa值为0.93,p0.001,证明两者的检测结果高度一致。结论:本研究建立的针对CA6的实时荧光RT-RPA具备良好的特异性和灵敏度,反应快速,将有助于CA6相关的HFMD的监测和诊断。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.1;R3416

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