本文選題：病毒性腦炎腦膜炎癥候群 + 實時熒光定量RT-PCR　； 參考：《中國疾病預防控制中心》2017年碩士論文

【摘要】：病毒性腦炎腦膜炎癥候群是由于病毒感染引起的急性傳染病,臨床上以發(fā)熱、頭疼、嘔吐并伴有不同程度意識障礙或腦膜刺激為主要特征。目前,能夠引起病毒性腦炎腦膜炎的病毒種類較多,而常見的病毒主要有蟲媒病毒、皰疹病毒和人腸道病毒等。由于此類疾病所產(chǎn)生的風險及造成的社會負擔很重,因此對于它們的早期識別、診斷、控制和治療就顯得尤為重要。在本研究中,采用TaqMan實時熒光定量RT-PCRArray結(jié)合全自動工作站方法首先對2015年-2016年山西省、河北省以及湖南省所采集的臨床病例腦脊液、血液、糞便標本進行了與病毒性腦炎腦膜炎癥候群相關(guān)的16種病毒病原學快速檢測。結(jié)果顯示,608份標本中腦脊液總檢出率為25.91%,血液總檢出率為3.56%,糞便總檢出率為6.25%。在腦脊液標本中,檢出的主要病原為腸道病毒(占23.77%)、人皰疹病毒(占0.61%)、乙型腦炎病毒(占0.61%);在血液標本中,檢出的主要病原為腸道病毒(占2.94%),糞便標本檢出的主要病原為腸道病毒(占6.25%)。本文建立的結(jié)合全自動工作站的TaqMan實時熒光定量RT-PCRArray方法能夠在2個小時內(nèi)確定樣本中是否存在常見的病毒感染或常見病毒的混合感染,具有快速、靈敏、高通量和標準化的優(yōu)勢,為快速鑒定和識別腦炎腦膜炎癥候群常見病毒性病原體提供了高效的解決方案。在上述對病毒性腦炎腦膜炎癥候群相關(guān)病毒的檢測中發(fā)現(xiàn),檢出最多的為腸道病毒(HEV),為了進一步實現(xiàn)腸道病毒的高靈敏檢測和分型,本文隨后建立了一種一步法巢式RT-PCR(real-time nested RT-PCR,RTN RT-PCR)方法。通過下載 GenBank 已收錄的 33 株腸道病毒全基因組序列,并以此為依據(jù)設計內(nèi)外部引物,并完成實驗方案的優(yōu)化。實驗結(jié)果表明,當外引物濃度為10nM,內(nèi)引物濃度為200nM時,較少形成引物二聚體;外引物的退火溫度在64℃時的擴增效果最佳,內(nèi)引物的退火溫度在52℃時擴增效果最佳;外引物擴增循環(huán)數(shù)為17時擴增效果最佳。一步法巢式RT-PCR對EV71、CVA]6、CVA10和CVA6四個病毒株進行檢測均出現(xiàn)典型的特異性擴增曲線,無交叉反應,其靈敏度達到10-8稀釋度,高于腸道病毒通用型熒光定量PCR的靈敏度。一步法巢式、兩步法巢式RT-PCR和實時熒光定量RT-PCR方法同時檢測140份腦脊液和糞便標本的HEVs,結(jié)果顯示,一步法巢式RT-PCR陽性率為(22.14%),兩步法巢式RT-PCR陽性率為(33.57%),實時熒光定量RT-PCR陽性率為(17.14%)。一步法巢式RT-PCR主要優(yōu)勢為操作簡單、快速、靈敏度高于實時熒光定量PCR、特異性好,此外,一步法巢式RT-PCR可實現(xiàn)腸道病毒的分型(測定PCR產(chǎn)物)或利用溶解曲線判斷有無腸道病毒的感染(無需電泳),適合在偏遠和預算有限的地方疾控系統(tǒng)推廣,雖然一步法巢式RT-PCR的靈敏度略低于兩步法巢式RT-PCR,但避免了由于兩步法巢式RT-PCR轉(zhuǎn)移再擴增所導致的交叉污染風險。本方法的建立為高靈敏的檢測HEVs提供了一種更適用于基層推廣應用的可靠手段。
[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.

【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.3;R440

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