河南省呼吸道合胞病毒分子生物学研究

发布时间：2018-05-23 12:56

本文选题：呼吸道合胞病毒 + 黏附蛋白　；参考：《郑州大学》2008年硕士论文

【摘要】：呼吸道合胞病毒(respiratory syncytial virus,RSV)简称合胞病毒,是重要的婴幼儿急性呼吸道感染病原体,可引起间质性肺炎及毛细支气管炎等疾病。RSV感染非常普遍,超过90%的儿童在2岁以前感染过RSV,而且都经历过1次或多次感染。在发展中国家,5岁以下儿童因RSV感染住院病人的死亡率为7%,发达国家为0.5%～2.0%。我国RSV感染研究缺乏全国资料,部分地区资料显示,北京市儿童医院研究显示在1996～1999年期间,住院的病人中有呼吸道感染症状病例1183例,RSV阳性者标本255例,占送检标本总数的21.5%;沈阳地区1999～2000年调查显示小儿急性呼吸道感染病例的44.62%由RSV引起。我省尚未开展RSV感染的病原学研究,仅仅开展了RSV-IgM抗体的检测工作,也没有对于婴幼儿急性呼吸道感染病例的病原学流行趋势的研究。为了了解河南省婴幼儿急性呼吸道感染病例病原学分布状况,探讨呼吸道合胞病毒在我省的流行规律,积累呼吸道合胞病毒毒株,了解呼吸道合胞病毒黏附蛋白G基因的分子生物学特征,确定我省呼吸道合胞病毒分离株的型别,从而填补我省呼吸道合胞病毒病原学研究的空白,我们设立并开展了此项研究工作。另外,通过对呼吸道合胞病毒各种检测、鉴定方法的对比,比较各种检测方法检测效果的差异,在寻找呼吸道合胞病毒快速检测方法、建立应急检验平台等突发公共卫生事件的预防控制工作方面,提供可靠的实验室数据支持。材料与方法研究对象来源于2006年10月-2007年5月期间河南省人民医院儿科门诊和住院病例,以及河南省流感样病例监测系统中的病例,对诊断为急性支气管炎、毛细支气管炎、肺炎,符合筛选条件的病例确定为研究对象。对研究对象进行调查并采集咽拭液标本,选择Hep-2、Vero细胞系,采用组织培养的方法在咽拭液标本中分离培养呼吸道合胞病毒株,运用相关试剂盒提取咽拭液标本RNA模板,采用Ag-ELISA、real-time PCR等方法鉴定呼吸道合胞病毒。设计并合成针对RSV黏附蛋白G基因的上下游引物,对鉴定阳性病例运用RT-PCR方法扩增该基因,阳性扩增产物运用核苷酸测序方法获得G基因全长核苷酸序列,将所获得的序列与已知参考毒株G基因序列一同导入DNASTAR软件,进行同源性分析和系统进化树分析。运用卡方检验等相关统计学方法比较各检测方法的敏感性差异,结合实际工作,找出快速实用的检测方法。结果通过收集和筛选资料,共有126例病例符合本研究所规定的条件,对所有研究对象进行调查和采集临床标本,共采集咽拭液标本126份。经过组织培养共有58份标本出现CPE,其中5份为典型细胞融合病变,53份为其他细胞病变。经过real-time PCR方法鉴定出12份为呼吸道合胞病毒,经过Ag-ELISA鉴定出8份呼吸道合胞病毒,通过针对黏附蛋白G蛋白基因的引物的RT-PCR反应,共有6份标本扩增出了阳性条带,对这6份病毒标本的RT-PCR扩增产物进行了核苷酸序列测定,获得这些病毒的G蛋白基因全长核苷酸序列,分别编号为henan2006/A36、henan2006/A39、henan2006/207、henan2006/305、henan2006/417、henan2006/589,其核苷酸序列长度均为922bp。将所得到的核苷酸序列导入DNASTAR软件MegAlign模块中,进行核苷酸序列同源性分析,通过分离毒株序列两两比较,其同源性最大99%,最小88%,将所测核苷酸序列推导出氨基酸序列进行同源性比较,同源性在90%～95%之间,其与RSVA型毒株序列同源性为87%～94%,与RSVB型毒株序列同源性33%～35%。从Pubmed基因数据库中查找并下载多条RSV毒株G蛋白基因核苷酸参考序列,与样本序列导入DNASTAR软件MegAlign模块中进行比较,并构建系统进化树,确定病毒型别。通过比较不同的RSV鉴定方法,实验结果表明,共检测12株呼吸道合胞病毒,其中组织培养分离出5株病毒,real-time PCR检出12株病毒,Ag-ELISA检出8株病毒,对上述数据进行统计学处理,检出率经配对卡方检验,real-time PCR和Ag-ELISA两种方法差异无统计学意义(P＞0.10,χ~2=2.25),检测敏感性没有显著性差异,都可以用来进行快速检测。结论 1.通过检测结果的比较,real-time PCR实验与Ag-ELISA实验对于呼吸道合胞病毒的检测,检测敏感性差异无统计学意义。 2.在我省部分地区2006～2007年度婴幼儿急性呼吸道感染病例中,RSV感染毒株以A型毒株为主。
[Abstract]:Respiratory syncytial virus (RSV), abbreviated as syncytial virus, is an important pathogen of acute respiratory infection in infants and infants. It can cause interstitial pneumonia and bronchiolitis and other diseases of.RSV. More than 90% of children have infected RSV before 2 years of age, and have experienced 1 or more times of infection. The death rate of hospitalized patients under 5 years old for RSV infection was 7%. The research on RSV infection in China was from 0.5% to 2.0%. in developed countries. Some data showed that there were 1183 cases of respiratory infection symptoms in hospitalized patients and 255 cases of RSV positive patients during the period of 1996~1999 years. The total number of specimens tested was 21.5%; 1999~2000 years of investigation in the Shenyang area showed that 44.62% of children with acute respiratory tract infections were caused by RSV.
The pathogenic study of RSV infection has not been carried out in our province, only the detection of RSV-IgM antibody has been carried out, and there is no study on the epidemic trend of the etiology of acute respiratory infection in infants and young children. In order to understand the distribution of the etiology of acute respiratory infection in infants and young children in Henan, the epidemic of respiratory syncytial virus in our province is discussed. Regularities, accumulating the respiratory syncytial virus strains, understanding the molecular biological characteristics of the respiratory syncytial virus adhesion protein G gene and determining the type of the respiratory syncytial virus isolates in our province, so as to fill the gap in the study of the etiology of respiratory syncytial virus in our province. Comparison of various detection and identification methods of cytosolic virus, compare the differences of detection results of various detection methods, and provide reliable laboratory data support in the search for the rapid detection methods of respiratory syncytial virus, the establishment of emergency inspection platform and other public health emergency prevention and control work.
Materials and methods
The subjects were selected from the outpatient department of Pediatrics and the hospitalized cases in Henan Province People's Hospital during the May -2007 period, and the cases in the influenza like case monitoring system in Henan province. The subjects were determined to be diagnosed as acute bronchitis, bronchiolitis, pneumonia, and screening conditions. The subjects were investigated and collected. The pharynx swab specimens were selected for Hep-2, Vero cell lines, and the respiratory syncytial virus strains were isolated and cultured in the pharynx swab specimens by tissue culture. The RNA template of pharynx swab was extracted with the related kit, and the respiratory syncytial virus was identified by Ag-ELISA and real-time PCR. The upstream and downstream of the RSV adherent protein G gene was designed and synthesized. Primers were used to amplify the gene by RT-PCR method, and the positive amplification product obtained the full nucleotide sequence of the G gene by nucleotide sequencing. The sequence was introduced into the DNASTAR software with the G gene sequence of the known reference strain, and the homology analysis and phylogenetic tree analysis were carried out. The sensitivity of each method is compared with the method of calculation. Combined with practical work, a fast and practical detection method is found.
Result
By collecting and screening data, a total of 126 cases were conformed to the conditions stipulated in this study. All the subjects were investigated and collected, and 126 specimens of pharynx swab specimens were collected. Through tissue culture, 58 specimens appeared CPE, of which 5 were typical cell fusion diseases and 53 were other cytopathic lesions. After real-time PCR prescription 12 respiratory syncytial viruses were identified and 8 respiratory syncytial viruses were identified by Ag-ELISA. A total of 6 specimens were amplified by RT-PCR response to the primers of the G protein gene of the adhesion protein. The nucleotide sequence of the RT-PCR products of the 6 virus specimens was determined and the G protein group of these viruses was obtained. The nucleotide sequence was numbered henan2006/A36, henan2006/A39, henan2006/207, henan2006/305, henan2006/417, henan2006/589 respectively. The nucleotide sequence of the nucleotide sequence was 922bp., and the nucleotide sequence was introduced into the MegAlign module of DNASTAR software, and the nucleotide sequence homology of the nucleoside sequence was analyzed by separating the 22 ratio of the sequence of the strain. The homology of the nucleotide sequence was compared with the sequence of amino acids from 90% to 95%. The homology of the RSVA strain was 87% to 94%, and the sequence homology of the RSVB strain was 33% to 35%. from the Pubmed gene database to find and download a number of RSV gene nucleosides from the RSV strain G gene. The acid reference sequence was compared with the sample sequence in the DNASTAR software MegAlign module, and the phylogenetic tree was constructed to identify the virus type.
By comparing different RSV identification methods, the experimental results showed that 12 strains of respiratory syncytial virus were detected, of which 5 viruses were isolated from tissue culture, 12 viruses were detected by real-time PCR, 8 viruses were detected by Ag-ELISA, and the above data were statistically processed. The detection rate was tested by paired chi square, and the two methods of real-time PCR and Ag-ELISA were not different. Statistical significance (P > 0.10, ~2=2.25) showed no significant difference in sensitivity, and could be used for rapid detection.
conclusion
1. by comparing the detection results, there was no significant difference in sensitivity between real-time PCR test and Ag-ELISA test for detection of respiratory syncytial virus.
2. in 2006~2007 of the infants and young children with acute respiratory tract infections in some areas of our province, RSV infection is mainly A strain.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

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