本文選題：呼吸道合胞病毒 + 黏附蛋白��； 參考：《鄭州大學(xué)》2008年碩士論文

【摘要】：呼吸道合胞病毒(respiratory syncytial virus,RSV)簡(jiǎn)稱合胞病毒,是重要的嬰幼兒急性呼吸道感染病原體,可引起間質(zhì)性肺炎及毛細(xì)支氣管炎等疾病。RSV感染非常普遍,超過90%的兒童在2歲以前感染過RSV,而且都經(jīng)歷過1次或多次感染。在發(fā)展中國家,5歲以下兒童因RSV感染住院病人的死亡率為7%,發(fā)達(dá)國家為0.5%～2.0%。我國RSV感染研究缺乏全國資料,部分地區(qū)資料顯示,北京市兒童醫(yī)院研究顯示在1996～1999年期間,住院的病人中有呼吸道感染癥狀病例1183例,RSV陽性者標(biāo)本255例,占送檢標(biāo)本總數(shù)的21.5%;沈陽地區(qū)1999～2000年調(diào)查顯示小兒急性呼吸道感染病例的44.62%由RSV引起。 我省尚未開展RSV感染的病原學(xué)研究,僅僅開展了RSV-IgM抗體的檢測(cè)工作,也沒有對(duì)于嬰幼兒急性呼吸道感染病例的病原學(xué)流行趨勢(shì)的研究。為了了解河南省嬰幼兒急性呼吸道感染病例病原學(xué)分布狀況,探討呼吸道合胞病毒在我省的流行規(guī)律,積累呼吸道合胞病毒毒株,了解呼吸道合胞病毒黏附蛋白G基因的分子生物學(xué)特征,確定我省呼吸道合胞病毒分離株的型別,從而填補(bǔ)我省呼吸道合胞病毒病原學(xué)研究的空白,我們?cè)O(shè)立并開展了此項(xiàng)研究工作。另外,通過對(duì)呼吸道合胞病毒各種檢測(cè)、鑒定方法的對(duì)比,比較各種檢測(cè)方法檢測(cè)效果的差異,在尋找呼吸道合胞病毒快速檢測(cè)方法、建立應(yīng)急檢驗(yàn)平臺(tái)等突發(fā)公共衛(wèi)生事件的預(yù)防控制工作方面,提供可靠的實(shí)驗(yàn)室數(shù)據(jù)支持。 材料與方法 研究對(duì)象來源于2006年10月-2007年5月期間河南省人民醫(yī)院兒科門診和住院病例,以及河南省流感樣病例監(jiān)測(cè)系統(tǒng)中的病例,對(duì)診斷為急性支氣管炎、毛細(xì)支氣管炎、肺炎,符合篩選條件的病例確定為研究對(duì)象。對(duì)研究對(duì)象進(jìn)行調(diào)查并采集咽拭液標(biāo)本,選擇Hep-2、Vero細(xì)胞系,采用組織培養(yǎng)的方法在咽拭液標(biāo)本中分離培養(yǎng)呼吸道合胞病毒株,運(yùn)用相關(guān)試劑盒提取咽拭液標(biāo)本RNA模板,采用Ag-ELISA、real-time PCR等方法鑒定呼吸道合胞病毒。設(shè)計(jì)并合成針對(duì)RSV黏附蛋白G基因的上下游引物,對(duì)鑒定陽性病例運(yùn)用RT-PCR方法擴(kuò)增該基因,陽性擴(kuò)增產(chǎn)物運(yùn)用核苷酸測(cè)序方法獲得G基因全長核苷酸序列,將所獲得的序列與已知參考毒株G基因序列一同導(dǎo)入DNASTAR軟件,進(jìn)行同源性分析和系統(tǒng)進(jìn)化樹分析。運(yùn)用卡方檢驗(yàn)等相關(guān)統(tǒng)計(jì)學(xué)方法比較各檢測(cè)方法的敏感性差異,結(jié)合實(shí)際工作,找出快速實(shí)用的檢測(cè)方法。 結(jié)果 通過收集和篩選資料,共有126例病例符合本研究所規(guī)定的條件,對(duì)所有研究對(duì)象進(jìn)行調(diào)查和采集臨床標(biāo)本,共采集咽拭液標(biāo)本126份。經(jīng)過組織培養(yǎng)共有58份標(biāo)本出現(xiàn)CPE,其中5份為典型細(xì)胞融合病變,53份為其他細(xì)胞病變。經(jīng)過real-time PCR方法鑒定出12份為呼吸道合胞病毒,經(jīng)過Ag-ELISA鑒定出8份呼吸道合胞病毒,通過針對(duì)黏附蛋白G蛋白基因的引物的RT-PCR反應(yīng),共有6份標(biāo)本擴(kuò)增出了陽性條帶,對(duì)這6份病毒標(biāo)本的RT-PCR擴(kuò)增產(chǎn)物進(jìn)行了核苷酸序列測(cè)定,獲得這些病毒的G蛋白基因全長核苷酸序列,分別編號(hào)為henan2006/A36、henan2006/A39、henan2006/207、henan2006/305、henan2006/417、henan2006/589,其核苷酸序列長度均為922bp。將所得到的核苷酸序列導(dǎo)入DNASTAR軟件MegAlign模塊中,進(jìn)行核苷酸序列同源性分析,通過分離毒株序列兩兩比較,其同源性最大99%,最小88%,將所測(cè)核苷酸序列推導(dǎo)出氨基酸序列進(jìn)行同源性比較,同源性在90%～95%之間,其與RSVA型毒株序列同源性為87%～94%,與RSVB型毒株序列同源性33%～35%。從Pubmed基因數(shù)據(jù)庫中查找并下載多條RSV毒株G蛋白基因核苷酸參考序列,與樣本序列導(dǎo)入DNASTAR軟件MegAlign模塊中進(jìn)行比較,并構(gòu)建系統(tǒng)進(jìn)化樹,確定病毒型別。 通過比較不同的RSV鑒定方法,實(shí)驗(yàn)結(jié)果表明,共檢測(cè)12株呼吸道合胞病毒,其中組織培養(yǎng)分離出5株病毒,real-time PCR檢出12株病毒,Ag-ELISA檢出8株病毒,對(duì)上述數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理,檢出率經(jīng)配對(duì)卡方檢驗(yàn),real-time PCR和Ag-ELISA兩種方法差異無統(tǒng)計(jì)學(xué)意義(P＞0.10,χ~2=2.25),檢測(cè)敏感性沒有顯著性差異,都可以用來進(jìn)行快速檢測(cè)。 結(jié)論 1.通過檢測(cè)結(jié)果的比較,real-time PCR實(shí)驗(yàn)與Ag-ELISA實(shí)驗(yàn)對(duì)于呼吸道合胞病毒的檢測(cè),檢測(cè)敏感性差異無統(tǒng)計(jì)學(xué)意義。 2.在我省部分地區(qū)2006～2007年度嬰幼兒急性呼吸道感染病例中,RSV感染毒株以A型毒株為主。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373

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