病毒性脑炎脑膜炎症候群荧光定量RT-PCR Array检测方法的建立

发布时间：2018-04-15 05:32

本文选题：病毒性脑炎脑膜炎症候群 + 实时荧光定量RT-PCR　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：病毒性脑炎脑膜炎症候群是由于病毒感染引起的急性传染病,临床上以发热、头疼、呕吐并伴有不同程度意识障碍或脑膜刺激为主要特征。目前,能够引起病毒性脑炎脑膜炎的病毒种类较多,而常见的病毒主要有虫媒病毒、疱疹病毒和人肠道病毒等。由于此类疾病所产生的风险及造成的社会负担很重,因此对于它们的早期识别、诊断、控制和治疗就显得尤为重要。在本研究中,采用TaqMan实时荧光定量RT-PCRArray结合全自动工作站方法首先对2015年-2016年山西省、河北省以及湖南省所采集的临床病例脑脊液、血液、粪便标本进行了与病毒性脑炎脑膜炎症候群相关的16种病毒病原学快速检测。结果显示,608份标本中脑脊液总检出率为25.91%,血液总检出率为3.56%,粪便总检出率为6.25%。在脑脊液标本中,检出的主要病原为肠道病毒(占23.77%)、人疱疹病毒(占0.61%)、乙型脑炎病毒(占0.61%);在血液标本中,检出的主要病原为肠道病毒(占2.94%),粪便标本检出的主要病原为肠道病毒(占6.25%)。本文建立的结合全自动工作站的TaqMan实时荧光定量RT-PCRArray方法能够在2个小时内确定样本中是否存在常见的病毒感染或常见病毒的混合感染,具有快速、灵敏、高通量和标准化的优势,为快速鉴定和识别脑炎脑膜炎症候群常见病毒性病原体提供了高效的解决方案。在上述对病毒性脑炎脑膜炎症候群相关病毒的检测中发现,检出最多的为肠道病毒(HEV),为了进一步实现肠道病毒的高灵敏检测和分型,本文随后建立了一种一步法巢式RT-PCR(real-time nested RT-PCR,RTN RT-PCR)方法。通过下载 GenBank 已收录的 33 株肠道病毒全基因组序列,并以此为依据设计内外部引物,并完成实验方案的优化。实验结果表明,当外引物浓度为10nM,内引物浓度为200nM时,较少形成引物二聚体;外引物的退火温度在64℃时的扩增效果最佳,内引物的退火温度在52℃时扩增效果最佳;外引物扩增循环数为17时扩增效果最佳。一步法巢式RT-PCR对EV71、CVA]6、CVA10和CVA6四个病毒株进行检测均出现典型的特异性扩增曲线,无交叉反应,其灵敏度达到10-8稀释度,高于肠道病毒通用型荧光定量PCR的灵敏度。一步法巢式、两步法巢式RT-PCR和实时荧光定量RT-PCR方法同时检测140份脑脊液和粪便标本的HEVs,结果显示,一步法巢式RT-PCR阳性率为(22.14%),两步法巢式RT-PCR阳性率为(33.57%),实时荧光定量RT-PCR阳性率为(17.14%)。一步法巢式RT-PCR主要优势为操作简单、快速、灵敏度高于实时荧光定量PCR、特异性好,此外,一步法巢式RT-PCR可实现肠道病毒的分型(测定PCR产物)或利用溶解曲线判断有无肠道病毒的感染(无需电泳),适合在偏远和预算有限的地方疾控系统推广,虽然一步法巢式RT-PCR的灵敏度略低于两步法巢式RT-PCR,但避免了由于两步法巢式RT-PCR转移再扩增所导致的交叉污染风险。本方法的建立为高灵敏的检测HEVs提供了一种更适用于基层推广应用的可靠手段。
[Abstract]:Viral encephalitis syndrome is due to an acute infectious disease caused by virus infection, fever, headache, vomiting and with varying degrees of consciousness or meningeal irritation as the main feature. At present, many kinds of virus can cause viral encephalitis, and common virus mainly arbovirus, herpes virus and human enterovirus because of the risk of this disease. And the burden is very heavy, so for the early identification, their diagnosis, control and treatment is particularly important. In this study, using TaqMan real-time fluorescence quantitative RT-PCRArray combined with automatic station method based on 2015 -2016 in Shanxi Province, Hebei province and cerebrospinal fluid, clinical cases in Hunan province collected blood and stool specimens for viral encephalitis syndrome related to 16 kinds of virus pathogen rapid detection. The results show that 608 samples of cerebrospinal fluid, the total positive rate was 25.91%, the blood total detection rate was 3.56%. The total detection rate of 6.25%. in feces in cerebrospinal fluid, the main pathogen detection for enterovirus (23.77%), herpes simplex virus (0.61%), Japanese encephalitis virus (0.61%) in the blood samples; the main pathogen detection, for enterovirus (2.94%), the main pathogen detection of stool specimens for enterovirus (6.25%). The combination of automatic workstation TaqMan real-time fluorescence quantitative RT-PCRArray method in 2 hours to determine whether there is mixed infection, common viral infection or common virus samples with a rapid, sensitive, high-throughput and standardized advantages, provides an efficient solution for rapid identification of meningitis encephalitis syndrome common viral pathogens. In the viral encephalitis syndrome The discovery of the virus detection, detection of most of the enterovirus (HEV), in order to further realize the high sensitive detection of enteroviruses and type, this paper then established a one-step nested RT-PCR (real-time nested RT-PCR, RTN RT-PCR) method. 33 strains of enteric virus genome sequence by downloading the GenBank is included. And as a basis for the design of internal and external primers, and complete the optimization experiment. Experimental results show that when the concentration of 10nM in the outer primer, primer concentration is 200nM, less likely to form dimers with two primer amplification effect; outer primer annealing temperature at 64 DEG C when the optimum annealing temperature, inner primer at 52 DEG C was the best effect; the outer primer amplification cycle number of 17 was the best effect. The one-step nested RT-PCR for EV71, CVA]6, CVA10 and CVA6 four virus detection showed typical specificity amplification curve, no cross reaction, The sensitivity reached 10-8 dilution, the sensitivity was higher than that of enterovirus universal type fluorescent quantitative PCR. The results showed that one-step nested, simultaneous detection of 140 CSF samples and fecal samples of HEVs, two step nested RT-PCR and real-time fluorescence quantitative RT-PCR method, the positive rate of one-step nested RT-PCR for (22.14%), the positive rate of nested RT-PCR two steps for (33.57%), real time fluorescence quantitative RT-PCR positive rate (17.14%). The main advantage of one-step nested RT-PCR is simple, rapid and sensitive than real-time PCR, good specificity, in addition, typing one-step nested RT-PCR can realize the intestinal virus (determination of PCR products) or by the dissolution curves to determine whether or not enterovirus infection (without electrophoresis), suitable for promotion in the remote control system and a limited budget of local disease, although the sensitivity of one-step nested RT-PCR is slightly lower than the two step nested RT-PCR, but to avoid the two The cross contamination risk caused by footwork nested RT-PCR transfer and re amplification. The establishment of this method provides a reliable means for high-sensitivity detection of HEVs, which is more suitable for grass-roots popularization and application.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.3;R440

【参考文献】