五重荧光定量RT-PCR检测甲型流感病毒方法的建立和应用及2010～2013年杭州地区人H3N2病毒流行病学分析

发布时间：2018-07-11 11:06

本文选题：多重荧光定量RT-PCR + 甲型流感H3N2　；参考：《浙江大学》2015年硕士论文

【摘要】：目的：建立一种快速、敏感、特异的五重荧光定量RT-PCR方法检测甲型流感的不同亚型,并对2010～2013年杭州地区甲型流感H3N2进行分子流行病学调查,通过选择压力分析为疫苗研制提供依据。方法：1.设计甲型禽流感病毒基质蛋白(M)区、H3、H5、H7及内参基因(RNaseP,RP)的引物及Taqman探针,并对RT-PCR反应体系中,五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),然后用本方法和WHO推荐的方法进行灵敏度比较。3.在本方法的反应体系中加入其它18种呼吸道病原微生物[乙型流感病毒,禽流感H5N3、H5N1、H9N2病毒,人副流感病毒Ⅰ～Ⅲ型,呼吸道合胞病毒A和B型,肺炎支原体,铜绿假单胞菌,肺炎克雷伯菌,鲍曼不动杆菌,金黄色葡萄球菌,白念珠菌]提取的模板,检测反应体系的特异性。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),用本方法分别检测三次,检测反应体系的(批内)重复性,并与WHO推荐方法的结果进行比较。5.用本方法对262例疑似患者与正常入的痰液标本进行检测,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。6.2010年1月至2013年12月浙江大学附属第一医院流感监测网络相关数据,各年度流感样病例分别为：2406例、995例、766例和1156例。分别采用研究试剂所有病例的咽拭子均进行H3N2流感病毒检测,并经过血凝抑制实验和流感病毒核酸监测证实。7.病原学检测：样本先进行流感病毒核酸检测,采用五重RT-PCR检测试剂盒。按照不同年份、性别以及年龄段对所有结果进行统计分析。8.序列收集：所有H3N2的HA (hemagglutinin)与NA (neuraminidase)基因完整序列均在NCBI上搜索查找获取,搜索关键词分别为:"China", "Hangzhou", "Hong Kong"加"H3N2 HA gene complete cds","H3N2 NA gene complete cds",香港地区H3N2基因序列并入中国整个国家序列中。9.HA与NA基因序列筛选与比对：依据病毒的分离地、时间、宿主为前提条件进行整合,条件相同序列只取一条。H3N2基因序列按照地区分为中国HA(133)、杭州HA(69)和杭州NA(69)三组。分组的序列分别用软件Clustal X(v1.8)进行比对、分析、剪切对齐。杭州地区的序列比对完成后,直接运用Mega(v6.0)软件,采用邻位相接法进行系统进化树分析。用FigTree软件对图形进行编辑。10.分子钟与人口感染动力学分析：利用jModeltest软件找出序列最适碱基替换模型,按照手册中要求参数设置(http://code.google.com/p/jmodeltest2).再使用Beast软件中贝叶斯MCMC (Bayesian Markov chain Monte Carlo)法对数据集进行碱基替换速率、最近共同祖先(Time of most recent common ancestor, TMRCA)和Skyline plot分析,依据操作手册中要求的参数(http://beast.bio.ed.ac.uk/Tutorials/)进行设置,我们建立一个严格分子钟的指数生长模型,通过Tracer软件对所得结果进行分析,并规定有效样本大小(Effective sampling Size, ESS)200,最终计算分子钟(碱基替换速率)与时间人口感染动力学关系图,用Beast软件包中TreeAnnotator程序,计算获最大可信树(MCCT, Maximum clade credibility tree),用FigTree对所得到的树进行编辑,获得H3N2病毒的HA基因在时间标尺与地理位置上的进化图。11.甲型流感病毒H3N2选择压力分析：选择压力(dN-dS)可以反映病毒在收到外界环境进行选择时的进化情况,其中,dN (non-synonymous substitutions)代表非同义突变率,dS (synonymous substitutions)表示同义突变率。选择压力计算时以密码子发生非同义突变率dN与同义突变率dS的差值来确定选择的方向性,中性选择(dN=dS,dN-dS=O),正向选择(dN-dS0),净化选择(负向选择,dN-dS0).整组基因选择压力计算使用在线的Datamonkey webserve计算,导向进化树使用Neighbor Joining方法,选用在线检测的推荐模型作为序列的进化模型,因其序列数50,算法采用singlelikelihood ancestor counting (SLAC),使用Datamonkey在线网站提供的序列选择压力分析程序进行选择压力分析。12.与选择压力(正向选择)相关的蛋白质表达：在PDB数据库中找出与H3N2基因密码子相似程度较高(30%)的蛋白质结构,以此蛋白质结构进行同源建模,比较各选择压力位点下的蛋白质结构的变化。分别在蛋白质空间三维结构图中进行标记。结果：1.引物与探针的设计与浓度优化：从美国NCBI基因库下载涵盖国内外FluA及H3、H5、H7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。引物与探针浓度采用矩阵法进行,以获得的Ct值较小而荧光强度最大的浓度为反应体系最合适浓度。2.五重荧光定量RT-PCR反应体系的灵敏度：五重荧光定量RT-PCR反应在检测甲型禽流感病毒基质蛋白(M)区、H3、 H5、 H7及RNase P的合成片段时,各自灵敏度与WHO推荐的方法一致,均达到了在102copies/mL时能扩增出条带。3.五重荧光定量RT-PCR反应体系的特异性：将上述18种呼吸道病原菌的核酸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。特异性达到了100%。4.五重荧光定量RT-PCR反应体系的重复性(批内)：不同浓度核酸各自的检测Ct值标准差在0.193～0.451之间,变异系数(CV)最高达1.999%,本方法具有较好的批内重复性。5.五重荧光定量RT-PCR反应体系的检测符合率：使用本方法与已有试剂对临床262例疑似患者和正常人的痰液标本应用多重荧光定量RT-PCR方法进行检测,共检查出甲型流感病毒52例,其中H3N2型11例,H7N9型4例,H5N1未检测到阳性病例,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%。6.2010～2013年杭州地区H3N2感染率分析,不同年度间H3N2感染率差异具有统计学意义(χ2=14.004,P0.05),不同性别和年龄组间差异无统计学意义(χ2=3.552,χ2=2.691,P0.05)。杭州地区2010年与2012,2013年甲型流感H3N2的HA、NA序列进化来自广东省甲型流感H3N2进化的不同时间点的两个分支。7.我国流行甲型流感病毒H3N2的HA基因碱基替换速率为1.6584×10-3(95%可信区间：1.5325×10-3～1.7988×10-3), TMRCA:1945年(95%可信区间：1940～1952年),人口感染规模评价出现了两次高峰且感染率总趋势呈现上升状态。8.通过SLA法计算可得到阳性选择位点4个,14,153,209,278。阴性位点有238个。结论：1.多重荧光定量RT-PCR技术是利用TaqMan技术在普通PCR原有的一对特异性引物基础上增加了一条特异性的荧光双标记探针。利用该探针能与病原核酸特异性结合,而且结合部位位于引物结合区域,探针的5'端和3'端分别标记不同的荧光素,在PCR扩增过程中,利用检测系统中荧光量的变化,通过检测仪检测并记录荧光信号的变化判定检测结果,我们利用此原理建立了多重荧光RT-PCR法检测不同的流感亚型。2.检测方法的评价：通过标准毒株与临床样本的检测比较,发现我们建立的多重荧光RT-PCR特异性和灵敏度均达到了WHO推荐方法的特异性和灵敏度水平,而且我们的方法更快速,简便,并只需一次反应可同时检测甲型禽流感病毒和H3、H5、H7不同甲型禽流感不同亚型。3.临床标本验证：通过分别使用我们试剂与之江在售试剂,同时检测临床病人与正常人标本后,我们得出两者符合率达到了100%。4.通过对该方法的多角度评价,可知该方法具有快速、稳定、灵敏度和特异性高、重复性好的特点,对于临床上检测流感病毒及其不同亚型具有一定的意义。5.我们推测2012年后的H3N2病毒株可能是其他省份病毒迁徙所致；2013年病毒株HA与NA基因序列,均处于2012年病毒株树分支的下级,我们推断2013年的病毒株为2012年病毒株复苏后感染再传播,2012年H3N2病毒株感染后,使部分感染人群产生获得免疫,对于此病毒株再次感染具有一定的保护作用,致使2013年杭州地区H3N2病毒流感样人群中感染率下降。6.H3N2基因碱基置换速率为1.6525×10-3(95%可信区间：1.4796×10-3～1.8287×10-3),要明显低于H1N1(7.06×10-3,95%可信区间：5.4×10-3-8.8×10-3)、H5N1(8.87×10-3,95%可信区间：7.0×10-3～10.72×10-3)等高致病性禽流感的碱基置换速率,这也是H3N2在我国流行的感染病毒株,多以低变异株为主的主要原因。由碱基置换速率我们推测H3N2病毒H3基因的产生时间：1945年(95%可信区间：1940～1952年),由于H3N2病毒存在一组未在人类感染的病毒进化分支,因此剔除未感染人病毒组,计算人感染H3N2的H3基因产生时间为：1960年(95%可信区间：1957～1962年)与实际H3N2流行发生时间相符。同时H3N2病毒在我国感染的规模与时间的分布出现了两次高峰且感染率总趋势呈现上升状态。7.可将HA1基因的153(137)位于A抗原区,209(193)位于B抗原区,278(262)位于E抗原区,作为研制H3N2流感疫苗的依据,在H3N2流行中应重点观察该病毒在这些区域的变异情况
[Abstract]:Objective: to establish a fast, sensitive and specific five heavy fluorescence quantitative RT-PCR method to detect the different subtypes of influenza A, and to investigate the molecular epidemiology of influenza A influenza A (H3N2) in Hangzhou for 2010~2013 years, and to provide the basis for the development of the vaccine by selecting pressure analysis. Method: 1. to design the matrix protein (M) region of avian influenza A virus (H3). H5, H7 and RNaseP, RP (RNaseP, RP) primers and Taqman probes, and the concentration of five sets of primers and probes in the RT-PCR reaction system were 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. The plasmid DNA was extracted and detected by NanoDrop nucleic acid. The concentration of plasmid DNA was measured by the instrument, and the copy number of DNA was determined as the standard quantitative maternal 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), and then the sensitivity was compared with this method and the method recommended by WHO to.3. in this method. In the reaction system, 18 other respiratory pathogenic microorganisms (influenza B virus, avian influenza H5N3, H5N1, H9N2 virus, human parainfluenza virus I to III, respiratory syncytial virus A and B, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter Bauman, Staphylococcus aureus, Candida albicans) were extracted. The specific.4. of the reaction system will determine the good plasmid DNA concentration, dilute it to the required concentration (106copies/mL) according to the experimental needs, dilute it to (104copies/mL) three times continuously, and detect three times by this method, and detect the repeatability of the reaction system (in batch), and compare with the results of the WHO recommendation method,.5. with this method to 262. Cases of suspected patients and normal sputum specimens were tested, and compared with the method of marketing reagents produced by Shanghai Zhijiang biological Co., Ltd., the data of influenza surveillance network in the First Affiliated Hospital of Zhejiang University from January to December 2013.6.2010 were compared. The annual influenza like cases were 2406 cases, 995 cases, 766 cases and 1156 cases respectively. Do not use the swabs of all the cases of the reagents to detect the H3N2 influenza virus, and through the hemagglutination inhibition test and the influenza virus nucleic acid monitoring to confirm the.7. pathogenic test: the sample first carried out the detection of influenza virus nucleic acid, and the five RT-PCR detection kit was used. All the results were counted according to the different years, sex and age. .8. sequence collection: all H3N2 HA (hemagglutinin) and NA (neuraminidase) gene complete sequences are searched and obtained on NCBI, and the search key words are "China", "Hangzhou", "Hong Kong" and "Hong Kong". The sequence screening and comparison of.9.HA and NA gene sequences in the column: Based on the isolation, time and host of the virus, the same sequence only takes one.H3N2 sequence according to the region of China HA (133), Hangzhou HA (69) and Hangzhou NA (69). The sequence of the group is compared with the software Clustal X (v1.8), analysis, shear pairs, respectively. After the sequence alignment of Hangzhou area is completed, the Mega (V6.0) software is used directly to analyze the phylogenetic tree with the neighbor connection method. The graph is used to edit the.10. molecular clock and the dynamics analysis of the population infection: using the jModeltest software to find the optimal base replacement model of the sequence, set the parameters in the manual (HTTP). //code.google.com/p/jmodeltest2). Then use the Bayesian MCMC (Bayesian Markov chain Monte Carlo) method in the Beast software to carry out the base substitution rate for the data set. Als/) set up, we set up an exponential growth model of a strict molecular clock, analyze the results through Tracer software, and specify the size of the effective sample (Effective sampling Size, ESS) 200, and finally calculate the relationship diagram of the molecular clock (base substitution rate) and the temporal human mouth infection, and use the TreeAnnotator process in the Beast software package. In order, the MCCT, Maximum clade credibility tree was obtained, and the tree was edited with FigTree to obtain the evolution diagram of the HA gene of the H3N2 virus in the time scale and geographic location. The pressure analysis of the H3N2 selection of influenza A virus (.11.) of the influenza A virus was analyzed. The selection of pressure (dN-dS) could reflect the selection of the virus in the environment of receiving the environment. DN (non-synonymous substitutions) represents the non synonymous mutation rate, and dS (synonymous substitutions) represents the synonymous mutation rate. The selection pressure is calculated with the difference between the non synonymous mutation rate dN and the synonymous mutation rate dS in the selection pressure calculation, and the neutral selection (dN=dS, dN-dS=O), positive selection (dN-dS0). The purification selection (negative selection, dN-dS0). The whole group of gene selection pressure calculation uses the online Datamonkey webserve calculation, directing the evolutionary tree using the Neighbor Joining method, selecting the recommended model for on-line detection as the evolutionary model of the sequence, because the sequence number is 50, the algorithm uses singlelikelihood ancestor counting (SLAC), and uses Datamonkey. The sequence selection pressure analysis program provided by the online web site selects the protein expression associated with the selection pressure (positive selection) of the pressure analysis.12.: in the PDB database, the protein structure with a higher similarity to the H3N2 gene codon (30%) is found, and the protein structure is used for homologous modeling, and the eggs under the selection of pressure loci are compared. The changes in white matter structure are marked in the three-dimensional structure map of protein. Results: design and concentration optimization of 1. primers and probes: download the multiple gene sequences of FluA and H3, H5, H7 subtypes at home and abroad from the American NCBI gene pool, and compare the homology with DNAman software to determine the conserved area of the above virus genome. Primer Express3.0 software was used to design highly specific primers and TaqMan probes in the conserved area, and the specificity of primers was verified by BLAST sequence alignment. The primer and probe concentration was carried out by matrix method to obtain the lowest Ct value and the maximum concentration of fluorescence intensity was.2. five heavy fluorescence quantitative RT-PCR reactant. Sensitivity: the sensitivity of five heavy fluorescence quantitative RT-PCR reaction in detecting the matrix protein (M) region of avian influenza A (M), H3, H5, H7 and RNase P, each sensitivity is consistent with the recommended method of WHO, all of which are able to amplify the specificity of the band.3. five heavy fluorescein quantitative RT-PCR reaction system at 102copies/mL: the above 18 kinds of calls are called. The nucleic acid extracts of the pathogen of the pathogeny were tested with multiple fluorescent quantitative RT-PCR as the template. Except for the positive results of the influenza virus, all the other viruses were negative. The specificity reached the repeatability of the 100%.4. five heavy fluorescence quantitative RT-PCR reaction system (in batch): different concentrations of nucleic acids respectively The standard deviation of Ct values is between 0.193 and 0.451, and the coefficient of variation (CV) is up to 1.999%. This method has a good detection coincidence rate of the internal repetitive.5. five heavy fluorescence quantitative RT-PCR reaction system: using this method and the existing reagents, the multiple fluorescence quantitative RT-PCR method is applied to the sputum specimens of 262 clinically suspected patients and normal people. A total of 52 cases of influenza A virus were examined, including 11 cases of H3N2, 4 cases of type H7N9, and no positive cases detected by H5N1. The coincidence of the test results with the marketing reagent box of Zhijiang biological Co., Ltd. reached 100%.6.2010 ~ 2013 in Hangzhou area, and the difference of H3N2 infection rate between different years was statistically significant (x 2=14.004, P) 0.05) there was no statistical difference between different sex and age groups (x 2=3.552, X 2=2.691, P0.05). The HA of influenza a H3N2 in 2010 and 20122013 years in Hangzhou region, NA sequence evolution came from two branches of the different time points of the evolution of influenza A influenza A in Guangdong Province,.7., the rate of the HA gene base substitution of influenza A influenza A virus in China was 1.658 4 x 10-3 (95% confidence interval: 1.5325 x 10-3 ~ 1.7988 x 10-3), TMRCA:1945 years (95% confidence interval: 1940~1952 years), population infection scale evaluation appeared in two peaks and the total trend of infection rate showed an increasing state..8. can be calculated by SLA method by SLA method and 14153209278. negative loci have 238. The optical quantitative RT-PCR technique is a specific fluorescent double labeled probe based on TaqMan technology on the basis of a pair of specific primers in common PCR. Using the probe, the probe can combine with the specific nucleic acid of the pathogen, and the binding site is located in the primer binding region, and the 5'and 3' terminals of the probe are labeled with different fluorescein, and the amplification is amplified in PCR. In the process, we use the detector to detect and record the change of fluorescence signal by detecting and recording the changes of fluorescence signal in the detection system. By using this principle, we establish a multiple fluorescence RT-PCR method for detecting different influenza subtypes.2. detection methods. The specificity and sensitivity of the heavy fluorescence RT-PCR have reached the specificity and sensitivity of the WHO recommendation method, and our method is more rapid and simple, and only one response can be used to detect the avian influenza A virus and H3, H5, H7 different subtypes of avian influenza a different subtype.3.. After selling the reagents and testing the clinical and normal human specimens, we found that the coincidence rate reached 100%.4. through the multi angle evaluation of the method. It is known that the method has the characteristics of rapid, stable, high sensitivity, high specificity and good repeatability. It has a certain significance for the detection of influenza virus and its different subtypes in clinical.5.. It is presumed that the H3N2 virus strain after 2012 may be the result of the virus migration in other provinces, and the sequence of HA and NA in 2013 is in the lower grade of the branch of the virus tree in 2012. We infer that the virus strain of 2013 is transmitted again after the 2012 virus strain is resuscitated, and after the infection of the H3N2 virus in 2012, some infected people will be immune. It has a certain protective effect on the reinfection of the virus strain, resulting in the decrease of.6.H3N2 gene base replacement rate of 1.6525 H3N2 10-3 (95% confidence interval: 1.4796 x 10-3 to 1.8287 x 10-3) in 2013, which is significantly lower than that of H1N1 (7.06 x 10-3,95% confidence interval: 5.4 x 10-3-8.8 * 10-3), H5N1 (8.87). X 10-3,95% confidence interval: 7 * 10-3 ~ 10.72 * 10-3) base replacement rate of high pathogenic avian influenza, which is the main reason that H3N2 is prevalent in our country, mainly with low variation strain. By the base replacement rate, we speculate the generation time of the H3N2 virus H3 gene: 1945 (95% confidence interval: 1940~1952 years), because The H3N2 virus has a group of uninfected virus evolution branches. Therefore, the H3 gene generation time of the human infected H3N2 is: 1960 (95% confidence interval: 1957~1962 years) is in accordance with the actual incidence of the actual H3N2 epidemic. Meanwhile, the distribution of the H3N2 virus in the scale and time of the infection in our country is two times higher The general trend of peak and infection rate showed a rising state of.7., which could locate 153 (137) of HA1 gene in A antigen region, 209 (193) located in B antigen region, 278 (262) in E antigen region, as the basis for developing H3N2 influenza vaccine. In H3N2 epidemic, the variation of the virus in these regions should be observed.
【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

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