本文選題：多重熒光定量RT-PCR + 甲型禽流感��； 參考：《浙江大學》2015年碩士論文

【摘要】：2013年2-3月,新型甲型禽流感H7N9病毒在我國上海和安徽兩地率先發(fā)現(xiàn),H7N9禽流感是由禽H7N9亞型引起的急性呼吸道傳染病�；颊咭话惚憩F(xiàn)為流感樣癥狀,如發(fā)熱,咳嗽,少痰,可伴有頭痛、肌肉酸痛和全身不適。重癥患者病情發(fā)展迅速,表現(xiàn)為重癥肺炎,體溫大多持續(xù)在39℃以上,出現(xiàn)呼吸困難,可伴有咯血痰；H7N9感染的大部分病例都是惡性的,而且迅速發(fā)展為重癥肺炎和急性呼吸窘迫癥(ARDS)而致死。目前H7N9病毒感染的實驗室檢查包括血常規(guī)、血生化、病原學及相關(guān)檢測及胸部影像學檢查等,病原學檢測是H7N9病毒篩查、確認至關(guān)重要的環(huán)節(jié)。病毒培養(yǎng)是診斷病原學檢測的“金標準”,但與PCR比較,敏感性及特異性較低,本研究通過對甲型禽流感和H7N9亞型保守區(qū)基因序列進行分析,分別設(shè)計高度特異性的引物與Taqman探針,建立了四重熒光定量RT-PCR,采用一步法可同時檢測M基因、H7基因、N9基因以及內(nèi)參RP基因。本研究分兩部分：第一,四重熒光定量RT-PCR法檢測H7N9病毒方法的建立；第二,該方法的對臨床標本檢測的實際應(yīng)用。第一部分 四重熒光定量RT-PCR檢測新型甲型禽流感病毒H7N9方-法學的建立方法：1.從美國NCBI基因庫下載涵蓋國內(nèi)外F1uA及甲型流感病毒H7N9亞型的多條基因序列,用DNAman軟件對其進行同源性比較,確定以上病毒基因組的保守區(qū),用Primer Express3.0軟件在其保守區(qū)設(shè)計高度特異性的引物與TaqMan探針,并進行BLAST序列比對驗證引物特異性。并對RT-PCR反應(yīng)體系中,四套引物及探針的濃度進行優(yōu)化。2.合成各基因標準品片段,將其連接到質(zhì)粒載體PmdTM19-T Simple Vector上進行轉(zhuǎn)化和培養(yǎng)。經(jīng)鑒定后提取質(zhì)粒DNA,利用NanoDrop ND-2000核酸檢測儀測量質(zhì)粒DNA的濃度,確定DNA的拷貝數(shù)作為敏感度的標準品定量母液。根據(jù)實驗需要,將標準品定量母液稀釋至所需最高濃度(107copies/mL),并連續(xù)10倍稀釋至最低濃度(102copies/mL),平行進行熒光定量RT-PCR反應(yīng),驗證其靈敏度,并與WHO推薦的方法進行比對。3.在本方法的反應(yīng)體系中加入其它21種呼吸道病原微生物[季節(jié)性H1N1、H3N2、新甲型H1N1 (2009)病毒,乙型流感病毒,呼吸道合胞病毒A和B型,麻疹病毒,腸道病毒EV71、腸道病毒CoxA16,肺炎支原體,肺炎克雷伯菌,鮑曼不動桿菌,銅綠假單胞菌,金黃色葡萄球菌,白念珠菌,禽流感H5N1、H5N3、H9N2病毒,人副流感病毒Ⅰ～Ⅲ型]提取的模板,比較反應(yīng)體系的特異性。4.將確定好的各質(zhì)粒DNA濃度,根據(jù)實驗需要,稀釋至所需要的濃度(106copies/mL),連續(xù)三次10倍稀釋至(104copies/mL),用本方法分別檢測5次,得到的CT值計算其標準差和變異系數(shù),檢測反應(yīng)體系的重復(fù)性。結(jié)果：1.四重熒光定量RT-PCR反應(yīng)體系的靈敏度：本方法在檢測甲型禽流感病毒基質(zhì)蛋白(M)區(qū)、H7、N9及RNaseP的合成片段時,各自靈敏度與WHO推薦的方法一致,均在102copies/mL時能擴增出條帶。3.四重熒光定量RT-PCR反應(yīng)體系的特異性：將上述21種呼吸道病原菌的RNA提取物作為模板分別加入多重熒光定量RT-PCR進行測定,除流感病毒出現(xiàn)很好的陽性結(jié)果外,其它所有病毒的檢測結(jié)果均呈陰性。4.四重熒光定量RT-PCR反應(yīng)體系的重復(fù)性：不同濃度核酸各自的檢測Ct值標準差在0.11～0.37之間,變異系數(shù)(CV均低于1.62%,具有較好的重復(fù)性。結(jié)論：所建立的四重熒光定量RT-PCR法可檢測甲型流感病毒,并區(qū)分H7N9亞型,其檢測快速、準確,具有臨床推廣價值。第二部分 四重熒光定量RT-PCR法檢測H7N9病毒方法的臨床應(yīng)用方法：1.利用所建立的四重熒光定量RT-PCR方法對1896例疑似患者的痰液標本進行檢測,對結(jié)果進行統(tǒng)計分析,并與上海之江生物有限公司生產(chǎn)的市售試劑盒的方法結(jié)果比較。2.連續(xù)20天收集35例H7N9確診病例的咽拭子標本和痰液標本,用本方法進行檢測,探討標本對結(jié)果的影響。結(jié)果：1.對臨床1896例疑似患者的痰液標本應(yīng)用本方法進行檢測,共篩查出甲型流感病毒235份,其中127份為甲型流感病毒H7N9感染,與之江生物有限公司的市售試劑盒檢測結(jié)果的符合度達100%。2.痰液標本的陽性率明顯大于咽拭子標本,(26.57%vs8.86%,x2=75.34,p0.001),且在痰液標本中檢測到H7N9病毒的時間明顯長于咽拭子標本(平均6.14天vs 2.42天)。結(jié)論：本方法建立的實時熒光定量PCR技術(shù)簡便快捷、重復(fù)性好,對臨床上疑似甲型流感病毒感染的患者可提供早期明確診斷,并可區(qū)分高致病性H7N9亞型,為臨床治療方案的制定提供參考依據(jù),并且本方法從疑似患者的咽拭子、痰液標本中都可以檢測到病毒,但痰液標本的檢測結(jié)果更加穩(wěn)定、可靠。
[Abstract]:In 2-3 months of 2013, the new type of avian influenza H7N9 virus was first found in Shanghai and Anhui in China. H7N9 avian influenza is an acute respiratory infection caused by avian H7N9 subtype. The patients are usually characterized by influenza like symptoms such as fever, cough, and phlegm, with headache, muscle acid pain and general discomfort. Severe pneumonia, most of the body temperature above 39 degrees centigrade, can be accompanied by dyspnea accompanied by hemoptysis; most cases of H7N9 infection are malignant and rapidly develop to severe pneumonia and acute respiratory distress syndrome (ARDS). The laboratory examination of H7N9 virus infection includes blood routine, blood biochemistry, etiology and related detection and chest. H7N9 virus screening is a vital link. Virus culture is the "gold standard" for diagnosis of pathogenic detection, but compared with PCR, the sensitivity and specificity are low. This study designed a highly specific gene sequence by analyzing the sequence of the conservative region of avian influenza A and H7N9 subtype. The four heavy fluorescence quantitative RT-PCR was established with the Taqman probe. One step method could be used to detect the M gene, H7 gene, N9 gene and the internal parameter RP gene simultaneously. This study was divided into two parts: the establishment of the first, fourth heavy fluorescence quantitative RT-PCR method for the detection of the H7N9 virus method; second, the practical application of this method to the detection of clinical specimens. The first part was four heavy fluoret. Optical quantitative RT-PCR is used to detect the establishment of a new avian influenza A virus (H7N9) recipe for a new avian influenza A virus: 1. downloading multiple gene sequences of F1uA and the H7N9 subtype of influenza A virus from the NCBI gene bank of the United States, using DNAman software to compare their homology to the conserved region of the above virus gene group, and using Primer Express3.0 software in it A highly specific primer and TaqMan probe were designed in the conservative area, and the specificity of the primers was verified by BLAST sequence alignment. In the RT-PCR reaction system, the concentration of four primers and probes was optimized for.2. synthesis of each gene standard fragment, which was connected to the plasmid vector PmdTM19-T Simple Vector to be transformed and cultured. Plasmid DNA was taken with the NanoDrop ND-2000 nucleic acid detector to measure the concentration of plasmid DNA and determine the copy number of DNA as a standard quantitative mother liquid for sensitivity. According to the experimental needs, the standard quantitative mother liquid was diluted to the maximum required concentration (107copies/mL), and 10 times diluted to the lowest concentration (102copies/mL) continuously, and the fluorescence quantitative RT-P was carried out in parallel. CR reaction, verifying its sensitivity, and comparing with the methods recommended by WHO to add 21 other respiratory pathogenic microorganisms in the reaction system of this method [seasonal H1N1, H3N2, new type a H1N1 (2009) virus, influenza B virus, respiratory syncytial virus A and B, hemp virus, enterovirus EV71, enterovirus CoxA16, Mycoplasma pneumoniae, Klebsiella pneumoniae, Acinetobacter Bauman, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, avian influenza H5N1, H5N3, H9N2 virus, human parainfluenza virus type I - III) extracted template, the specific.4. of the reaction system will determine the good DNA concentration of each plasmid, diluted to the required concentration (106copies/mL) according to the needs of the experiment. Three times of 10 times dilution to (104copies/mL), 5 times were detected by this method. The obtained CT value calculated its standard deviation and coefficient of variation, and detected the repeatability of the reaction system. Results: the sensitivity of 1. four heavy fluorescence quantitative RT-PCR reaction system: this method was used to detect the synthetic fragments of the matrix protein (M) region, H7, N9 and RNaseP in the detection of avian influenza A virus Each sensitivity is consistent with the method recommended by WHO, which can amplify the specificity of the.3. four heavy fluorescence quantitative RT-PCR reaction system at 102copies/mL: the RNA extracts of the 21 pathogens of the respiratory tract were added as the template to the multiple fluorescent quantitative RT-PCR, respectively, in addition to the good positive results of the influenza virus. The results of all the virus detection showed the repeatability of the negative.4. four heavy fluorescence quantitative RT-PCR reaction system: the Ct values of different concentrations of nucleic acids were between 0.11 and 0.37, and the coefficient of variation (CV was lower than 1.62%, with good repeatability. Conclusion: the four heavy fluorescein RT-PCR method can be used to detect influenza A virus, and To distinguish H7N9 subtype, its detection is fast, accurate and has clinical value. Second part of the four heavy fluorescence quantitative RT-PCR method for the detection of H7N9 virus method: 1. using the established four heavy fluorescence quantitative RT-PCR method to detect the sputum specimens of 1896 cases of suspected patients, statistical analysis of the results, and the Shanghai River The method of marketing reagent boxes produced by biological Co., Ltd. was compared with.2. for 20 days to collect 35 cases of pharynx swab specimens and sputum specimens of H7N9 confirmed cases. The results were examined by this method. Results: 1. of the sputum specimens from 1896 clinically suspected patients should be detected by this method, and a total of the type a flow was screened. 235 samples of the virus were infected by influenza A virus H7N9, and the positive rate of 100%.2. sputum specimens was significantly higher than that of swab specimens, (26.57%vs8.86%, x2=75.34, p0.001), and the time of detection of H7N9 virus in the sputum mark was significantly longer than that of swab specimens. (an average of 6.14 days, vs and 2.42 days). Conclusion: the real-time fluorescence quantitative PCR technique established by this method is simple, quick and reproducible. It can provide early and clear diagnosis for patients with suspected influenza A virus infection, and can distinguish high pathogenic H7N9 subtypes, which provides a reference for the formulation of clinical treatment scheme, and this method is from suspected patients. The virus can be detected in throat swabs and sputum samples, but sputum samples are more stable and reliable.

【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.5

【共引文獻】

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