四重荧光定量RT-PCR检测新型甲型禽流感病毒H7N9方法学的研究与应用

发布时间：2018-04-26 16:19

本文选题：多重荧光定量RT-PCR + 甲型禽流感　；参考：《浙江大学》2015年硕士论文

【摘要】：2013年2-3月,新型甲型禽流感H7N9病毒在我国上海和安徽两地率先发现,H7N9禽流感是由禽H7N9亚型引起的急性呼吸道传染病。患者一般表现为流感样症状,如发热,咳嗽,少痰,可伴有头痛、肌肉酸痛和全身不适。重症患者病情发展迅速,表现为重症肺炎,体温大多持续在39℃以上,出现呼吸困难,可伴有咯血痰；H7N9感染的大部分病例都是恶性的,而且迅速发展为重症肺炎和急性呼吸窘迫症(ARDS)而致死。目前H7N9病毒感染的实验室检查包括血常规、血生化、病原学及相关检测及胸部影像学检查等,病原学检测是H7N9病毒筛查、确认至关重要的环节。病毒培养是诊断病原学检测的“金标准”,但与PCR比较,敏感性及特异性较低,本研究通过对甲型禽流感和H7N9亚型保守区基因序列进行分析,分别设计高度特异性的引物与Taqman探针,建立了四重荧光定量RT-PCR,采用一步法可同时检测M基因、H7基因、N9基因以及内参RP基因。本研究分两部分：第一,四重荧光定量RT-PCR法检测H7N9病毒方法的建立；第二,该方法的对临床标本检测的实际应用。第一部分四重荧光定量RT-PCR检测新型甲型禽流感病毒H7N9方-法学的建立方法：1.从美国NCBI基因库下载涵盖国内外F1uA及甲型流感病毒H7N9亚型的多条基因序列,用DNAman软件对其进行同源性比较,确定以上病毒基因组的保守区,用Primer Express3.0软件在其保守区设计高度特异性的引物与TaqMan探针,并进行BLAST序列比对验证引物特异性。并对RT-PCR反应体系中,四套引物及探针的浓度进行优化。2.合成各基因标准品片段,将其连接到质粒载体PmdTM19-T Simple Vector上进行转化和培养。经鉴定后提取质粒DNA,利用NanoDrop ND-2000核酸检测仪测量质粒DNA的浓度,确定DNA的拷贝数作为敏感度的标准品定量母液。根据实验需要,将标准品定量母液稀释至所需最高浓度(107copies/mL),并连续10倍稀释至最低浓度(102copies/mL),平行进行荧光定量RT-PCR反应,验证其灵敏度,并与WHO推荐的方法进行比对。3.在本方法的反应体系中加入其它21种呼吸道病原微生物[季节性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,肠道病毒EV71、肠道病毒CoxA16,肺炎支原体,肺炎克雷伯菌,鲍曼不动杆菌,铜绿假单胞菌,金黄色葡萄球菌,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,比较反应体系的特异性。4.将确定好的各质粒DNA浓度,根据实验需要,稀释至所需要的浓度(106copies/mL),连续三次10倍稀释至(104copies/mL),用本方法分别检测5次,得到的CT值计算其标准差和变异系数,检测反应体系的重复性。结果：1.四重荧光定量RT-PCR反应体系的灵敏度：本方法在检测甲型禽流感病毒基质蛋白(M)区、H7、N9及RNaseP的合成片段时,各自灵敏度与WHO推荐的方法一致,均在102copies/mL时能扩增出条带。3.四重荧光定量RT-PCR反应体系的特异性：将上述21种呼吸道病原菌的RNA提取物作为模板分别加入多重荧光定量RT-PCR进行测定,除流感病毒出现很好的阳性结果外,其它所有病毒的检测结果均呈阴性。4.四重荧光定量RT-PCR反应体系的重复性：不同浓度核酸各自的检测Ct值标准差在0.11～0.37之间,变异系数(CV均低于1.62%,具有较好的重复性。结论：所建立的四重荧光定量RT-PCR法可检测甲型流感病毒,并区分H7N9亚型,其检测快速、准确,具有临床推广价值。第二部分四重荧光定量RT-PCR法检测H7N9病毒方法的临床应用方法：1.利用所建立的四重荧光定量RT-PCR方法对1896例疑似患者的痰液标本进行检测,对结果进行统计分析,并与上海之江生物有限公司生产的市售试剂盒的方法结果比较。2.连续20天收集35例H7N9确诊病例的咽拭子标本和痰液标本,用本方法进行检测,探讨标本对结果的影响。结果：1.对临床1896例疑似患者的痰液标本应用本方法进行检测,共筛查出甲型流感病毒235份,其中127份为甲型流感病毒H7N9感染,与之江生物有限公司的市售试剂盒检测结果的符合度达100%。2.痰液标本的阳性率明显大于咽拭子标本,(26.57%vs8.86%,x2=75.34,p0.001),且在痰液标本中检测到H7N9病毒的时间明显长于咽拭子标本(平均6.14天vs 2.42天)。结论：本方法建立的实时荧光定量PCR技术简便快捷、重复性好,对临床上疑似甲型流感病毒感染的患者可提供早期明确诊断,并可区分高致病性H7N9亚型,为临床治疗方案的制定提供参考依据,并且本方法从疑似患者的咽拭子、痰液标本中都可以检测到病毒,但痰液标本的检测结果更加稳定、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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

【共引文献】