霍乱弧菌毒力相关基因和整合子分布特征的研究

发布时间：2018-01-12 17:47

本文关键词：霍乱弧菌毒力相关基因和整合子分布特征的研究　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 【目的】对天津市1964至2008年分离的霍乱弧菌进行毒力相关基因检测,并对主要的毒力基因进行序列分析,在分子水平上了解霍乱弧菌毒力基因的分布特点和变迁及其流行规律。了解天津市收集的1995年23株霍乱弧菌耐药性情况。了解3类整合子在菌株中的分布特征及整合子可变区携带的耐药基因型,探讨整合子在细菌耐药性产生中的作用。【方法】采用PCR方法对1964至2008年天津市保存的176株霍乱弧菌的CTXΦ基因元件中的ctxAB、zot基因,TLC因子中的cri基因进行检测。采用PCR方法对随机选取的不同年代的6株霍乱弧菌进行ctxA、ctxB、cri、tcpA和toxR毒力基因扩增,对PCR产物进行基因测序,利用DNAStar软件对序列进行拼接及生物信息学分析。采用WHO推荐的琼脂扩散(改良K-B)法,对天津市保存的1995年的23株霍乱弧菌进行药敏试验,按照美国国家临床实验室标准化委员会CLSI(2009)文件规定判断耐药、中介和敏感。采用PCR方法对Ⅰ、Ⅱ、Ⅲ类整合酶基因和整合子基因盒的携带情况进行检测。整合子的可变区扩增阳性的PCR产物经切胶纯化后连接到载体pMD 18-T simple,转化入E. coli TOP10中,阳性重组子经PCR鉴定后,进行测序分析。采用BLAST对序列进行比对,确定可变区所含耐药基因盒。参照PulseNet监测网络提供的霍乱弧菌PFGE标准操作程序,进行脉冲场凝胶电泳,分析菌株间的同源性。【结果】 176株霍乱弧菌中有131株携带CTXΦ遗传单元中的ctxAB、zot基因,阳性率分别为72.72%(128/176株),73.30%(129/176株)。在1964～1996年间分离的01群霍乱弧菌中,有69.00%(69/100株)携带cri基因,而1998～2008年间分离的霍乱弧菌中cri基因的携带率仅为1.33%(1/75株)。1株0139群霍乱弧菌ctxAB、zot、cri均阳性。6株霍乱弧菌与EVC国际标准株N16961的多序列比对结果表明：ctxA和tcpA基因间同源均为100%, ctxB、cri和toxR基因间同源性分别99.5%-100%,98.4%-100%和99.4-100%。Ⅰ、Ⅱ、Ⅲ类整合酶基因在霍乱弧菌中携带率差异较大：所检测的23株霍乱弧菌中有19株Ⅰ类整合酶基因阳性,其阳性率为82.61%(19/23株),而Ⅱ、Ⅲ类整合子未检出。所有Ⅰ类整合子均携带一个基因盒aadA1,编码腺苷转移酶,介导对链霉素(streptomycin)/壮观霉素(spectinomycin)的耐药性。携带aadA1基因盒的霍乱弧菌对链霉素耐药。脉冲场凝胶电泳结果分析1995年部分霍乱弧菌可能来自同一克隆。【结论】霍乱弧菌毒力相关基因检测能反映不同年代菌株间的分子特征,部分毒力基因的测序分析能反映毒力基因的特征,这对于霍乱的分子流行病学研究具有重要意义。临床上可以选用氨苄青霉素、四环素、甲氧苄啶/磺胺甲恶唑、氯霉素作为治疗霍乱的参考药物。Ⅰ类整合子在1995年分离保存的01群菌株中有较高的携带率(82.61%)；药敏结果表明耐药基因型和表型一致,整合子与霍乱弧菌的链霉素耐药性相关。脉冲场凝胶电泳结果表明1995年部分菌株可能是同一克隆来源。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.3

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