本文關(guān)鍵詞：霍亂弧菌毒力相關(guān)基因和整合子分布特征的研究　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 霍亂弧菌 毒力基因 整合子 聚合酶鏈反應(yīng) 脈沖場凝膠電泳

【摘要】： 【目的】 對天津市1964至2008年分離的霍亂弧菌進(jìn)行毒力相關(guān)基因檢測,并對主要的毒力基因進(jìn)行序列分析,在分子水平上了解霍亂弧菌毒力基因的分布特點(diǎn)和變遷及其流行規(guī)律。了解天津市收集的1995年23株霍亂弧菌耐藥性情況。了解3類整合子在菌株中的分布特征及整合子可變區(qū)攜帶的耐藥基因型,探討整合子在細(xì)菌耐藥性產(chǎn)生中的作用。 【方法】 采用PCR方法對1964至2008年天津市保存的176株霍亂弧菌的CTXΦ基因元件中的ctxAB、zot基因,TLC因子中的cri基因進(jìn)行檢測。采用PCR方法對隨機(jī)選取的不同年代的6株霍亂弧菌進(jìn)行ctxA、ctxB、cri、tcpA和toxR毒力基因擴(kuò)增,對PCR產(chǎn)物進(jìn)行基因測序,利用DNAStar軟件對序列進(jìn)行拼接及生物信息學(xué)分析。采用WHO推薦的瓊脂擴(kuò)散(改良K-B)法,對天津市保存的1995年的23株霍亂弧菌進(jìn)行藥敏試驗(yàn),按照美國國家臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化委員會CLSI(2009)文件規(guī)定判斷耐藥、中介和敏感。采用PCR方法對Ⅰ、Ⅱ、Ⅲ類整合酶基因和整合子基因盒的攜帶情況進(jìn)行檢測。整合子的可變區(qū)擴(kuò)增陽性的PCR產(chǎn)物經(jīng)切膠純化后連接到載體pMD 18-T simple,轉(zhuǎn)化入E. coli TOP10中,陽性重組子經(jīng)PCR鑒定后,進(jìn)行測序分析。采用BLAST對序列進(jìn)行比對,確定可變區(qū)所含耐藥基因盒。參照PulseNet監(jiān)測網(wǎng)絡(luò)提供的霍亂弧菌PFGE標(biāo)準(zhǔn)操作程序,進(jìn)行脈沖場凝膠電泳,分析菌株間的同源性。 【結(jié)果】 176株霍亂弧菌中有131株攜帶CTXΦ遺傳單元中的ctxAB、zot基因,陽性率分別為72.72%(128/176株),73.30%(129/176株)。在1964～1996年間分離的01群霍亂弧菌中,有69.00%(69/100株)攜帶cri基因,而1998～2008年間分離的霍亂弧菌中cri基因的攜帶率僅為1.33%(1/75株)。1株0139群霍亂弧菌ctxAB、zot、cri均陽性。6株霍亂弧菌與EVC國際標(biāo)準(zhǔn)株N16961的多序列比對結(jié)果表明：ctxA和tcpA基因間同源均為100%, ctxB、cri和toxR基因間同源性分別99.5%-100%,98.4%-100%和99.4-100%。Ⅰ、Ⅱ、Ⅲ類整合酶基因在霍亂弧菌中攜帶率差異較大：所檢測的23株霍亂弧菌中有19株Ⅰ類整合酶基因陽性,其陽性率為82.61%(19/23株),而Ⅱ、Ⅲ類整合子未檢出。所有Ⅰ類整合子均攜帶一個(gè)基因盒aadA1,編碼腺苷轉(zhuǎn)移酶,介導(dǎo)對鏈霉素(streptomycin)/壯觀霉素(spectinomycin)的耐藥性。攜帶aadA1基因盒的霍亂弧菌對鏈霉素耐藥。脈沖場凝膠電泳結(jié)果分析1995年部分霍亂弧菌可能來自同一克隆。 【結(jié)論】 霍亂弧菌毒力相關(guān)基因檢測能反映不同年代菌株間的分子特征,部分毒力基因的測序分析能反映毒力基因的特征,這對于霍亂的分子流行病學(xué)研究具有重要意義。臨床上可以選用氨芐青霉素、四環(huán)素、甲氧芐啶/磺胺甲惡唑、氯霉素作為治療霍亂的參考藥物。Ⅰ類整合子在1995年分離保存的01群菌株中有較高的攜帶率(82.61%)；藥敏結(jié)果表明耐藥基因型和表型一致,整合子與霍亂弧菌的鏈霉素耐藥性相關(guān)。脈沖場凝膠電泳結(jié)果表明1995年部分菌株可能是同一克隆來源。
[Abstract]:[Objective]
In the city of Tianjin from 1964 to 2008 for detection of Vibrio cholerae virulence related genes, and the main virulence gene sequence analysis, to understand the distribution characteristics and change of virulence genes of Vibrio cholerae and its epidemiology at the molecular level. In Tianjin, collected in 1995 23 strains of Vibrio cholerae. Drug resistant genotypes carrying 3 kinds of understanding the distribution of integrons and characteristics of integron in strains in the variable region, to investigate the integron in bacterial resistance in vitro.
[method]
With CTX gene elements of 176 strains of Vibrio cholerae by using PCR method to save from 1964 to 2008 in Tianjin city in ctxAB, zot gene and cri gene were detected in the TLC factor. Using PCR method 6 strains of Vibrio cholerae in different age were randomly selected for ctxA, ctxB, CRI, tcpA and toxR virulence gene amplification and gene sequencing of PCR products, sequence splicing and bioinformatics analysis using DNAStar software. Recommended by WHO agar diffusion method (modified K-B), 23 strains of Vibrio cholera in Tianjin city in 1995 to preserve the drug sensitive test was conducted in accordance with the United States, the National Committee for clinical laboratory standards (CLSI 2009) judgment documents drug resistance, intermediate and sensitive. By the method of PCR I, II, III were detected with the integrase gene and integron gene cassette. The variable region of integron positive PCR amplification products by gel purified linked to carrier pMD 18-T simple, E. coli was transformed into TOP10. The positive recombinant was identified by PCR and sequenced. BLAST was used to determine the variable region of the sequence alignment, containing the resistance gene cassette. According to the operation procedure of Vibrio cholerae PFGE standard PulseNet monitoring network, by pulsed field gel electrophoresis analysis of homology between the strains.
[results]
176 strains of Vibrio cholerae in 131 strains carrying CTX with genetic element ctxAB, zot gene, the positive rate was 72.72% (128/176 strain), 73.30% (129/176 strain) in 1964 to 1996 years. The separation of the 01 Vibrio cholerae, 69% (strain 69/100) carrying CRI gene, and 1998 to 2008 years isolation of Vibrio cholerae CRI gene carrying rate was only 1.33% (1/75 strain).1 0139 strains of Vibrio cholerae ctxAB, zot, CRI multiple sequence alignment results were positive.6 strains of Vibrio cholerae with international standard EVC strain N16961 showed that ctxA and tcpA homologous genes were 100%, ctxB, CRI and toxR genes homology of 99.5%-100%, 98.4%-100% and 99.4-100%. I, II, III class integrase gene carrying rate differences in Vibrio cholerae: 23 strains of Vibrio cholerae detected in 19 strains of integrase gene positive, the positive rate was 82.61% (19/23 strain), and II, III integron was not detected. All I Integron were carrying a box of aadA1 gene, encoding adenosine transferase mediated (streptomycin) to streptomycin / spectinomycin (spectinomycin). The drug resistance of Vibrio cholerae box carrying aadA1 gene resistant to streptomycin. Pulsed field gel electrophoresis analysis of Vibrio cholera in 1995 may be from the same clone.
[Conclusion]
Detection of virulence genes of Vibrio cholerae in different years can reflect the molecular characteristics of strains, sequencing of partial virulence gene analysis can reflect the characteristics of virulence genes, which is of great significance for molecular epidemiology of cholera. Clinical can choose four ampicillin, tetracycline, trimethoprim / sulfamethoxazole, chloramphenicol as a reference drug for the treatment of cholera. Integron isolated in 1995 carrying a higher rate of preservation of the 01 strains in (82.61%); drug sensitivity results showed consistent resistance genotype and phenotype, and Vibrio cholerae integration chain mildew resistance related factors. Pulsed field gel electrophoresis results showed that some strains may 1995 come from the same clone.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378.3

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