甲型副伤寒沙门菌PFGE和MLVA分型方案的建立与优化

发布时间：2018-03-19 20:07

本文选题：甲型副伤寒沙门菌　切入点：脉冲场凝胶电泳(PFGE)　出处：《中国疾病预防控制中心》2012年博士论文　论文类型：学位论文

【摘要】：甲型副伤寒沙门菌(Salmonellaentericaserovar Paratyphi A,S.Paratyphi A)是引起甲型副伤寒(Paratyphoid fever)的病原菌。在过去几十年,尤其在二十世纪九十年代中期开始,甲型副伤寒的暴发在许多国家报道增多,尤其是东南亚的一些国家,甚至超过伤寒沙门菌的感染,已成为新的公共卫生问题。脉冲场凝胶电泳(Pulsed-field gel electrophoresis,PFGE)由于其重复性好、分辨率高,已经用于多种致病细菌的分子分型、流行病学调查和微生物学研究。然而,我们发现目前基于PulseNet国际网络实验室用于分析沙门菌的PFGE的一天标准化方案对我国分离自不同的年份和省份的甲型副伤寒沙门菌进行分析,仅仅获得有限的带型。那么这种标准化PFGE方案在分析甲型副伤寒沙门菌时,是分型方法不适宜该菌基因组分析而导致区分能力有限还是这些跨越如此多年和省份的流行菌株就是保守性克隆,还需要进行分析。为探索可能更适合于甲型副伤寒沙门菌分离株的PFGE分析程序,本研究中我们基于PulseNet国际网络实验室用于分析沙门菌的PFGE 一天标准化方案,选择分离自我国不同年份和省份的33株甲型副伤寒沙门菌,通过比较不同核酸内切酶和电泳参数,对PFGE方案进行比较优化以用于分型甲型副伤寒沙门菌。然后用新的优化方案,对分离自不同年代(1998-2010)和12个省份的菌株进行回顾性分析。结果显示SpeI,XbaI,XhoI and BlnI四种酶都适合于分析甲型副伤寒沙门菌,通过对33株菌的相似度和分辨率的分类和比较分析,发现SpeI-PFGE比XbaI-PFGE和XhoI-PFGE产生更多的带型和更高分辨率,所以SpeI被考虑是首选酶,XbaI-PFGE的分辨率比XhoI-PFGE稍低,但是XbaI-PFGE所获得的带型数目比XhoI-PFGE多,因此我们认为XbaI作为次选酶,而XhoI作为第三种酶。BlnI-PFGE与上面三种酶相比,产生最少的带型和最低的分辨率,没有在进一步的研究中采用。此外,在对每种酶使用的三种参数进行比较分析,发现使用XbaI酶切的PFGE电泳参数(1.5-29s,20h)、使用SpeI的电泳参数(1-20s,20h)、使用XhoI的电泳参数(2.2-29s,20h)分辨率在各酶中最高,分别为17种、23种和16种。用新的电泳参数对106株甲型副伤寒沙门菌的分析结果显示中国的甲型副伤寒流行中存在高度克隆化的菌株引起全国范围的扩散,但随着年份变迁和流行地区扩大,也积累了散在的变异。多位点可变数目串联重复序列分析(Multiple Locus Variable Numbers Tandem Repeat Analysis,MLVA)是近年发展起来的以PCR技术为基础的分子分型方法。已进行的评价发现该方法分辨率高、重复性好、操作过程不复杂,已经用于多种致病菌包括沙门菌多种血清型的流行病学分析。本研究中我们探索建立用于分析甲型副伤寒沙门菌的MLVA方法并对其在流行病学中的应用价值进行评价。应用TRF和TRD软件扫描甲型副伤寒沙门菌测序株ATCC9150的全基因组,共获得196个串联重复位点(Tandem Repeat,TR),对获得的TR设计引物,选择33株在分离时间与地点尽可能分散、PFGE带型上有差异的甲型副伤寒沙门菌菌株作为评价样本(panel),从所有TR中筛选具有多态性的位点,利用这些位点建立可用于甲型副伤寒沙门菌的MLVA实验方法。再对我国12个地区1998年-2010年分离的209株甲型副伤寒菌株进行MLVA和PFGE分析,将这两种分型方法的实验结果进行比较分析,评价MLVA用于甲型副伤寒沙门菌分型的价值。在33株分离株的检测中,仅发现7个VNTR位点拷贝数变化。基于这7个VNTRs位点,对209株甲型副伤寒沙门菌分离株进行MLVA分析,与PFGE(本研究优化的SpeI酶切和电泳参数)实验结果比较,发现其分型能力明显弱于PFGE,MLVA区分出了 21种型别而PFGE区分出了 65种型别。也提示在甲型副伤寒沙门菌中,尤其在一个区域流行扩散中,基因组VNTR不容易发生拷贝数的变化。我们认为MLVA不适于对一个区域(国家或地区)流行的甲型副伤寒菌株的分子分型监测和流行病学调查,但在遗传进化分析中的克隆群确定上能够发挥作用。MLVA操作比PFGE简便,时间短,实验过程容易标准化,一些PFGE相同的甲型副伤寒沙门菌菌株可以用MLVA进一步分型,与PFGE联合应用可以提供较PFGE更高的分辨力,因此可以作为一种辅助的分子分型方法,用于在甲型副伤寒的流行病学调查中需要对PFGE分型结果进行深入分析时的补充。
[Abstract]:Salmonella paratyphi A (Salmonellaentericaserovar Paratyphi A, S.Paratyphi A) is caused by Salmonella paratyphi A (Paratyphoid fever) of the pathogen. In the past few decades, especially beginning in the mid 1990s, the outbreak of paratyphoid A in many countries reported increased, especially in some Southeast Asian countries, even more than the Salmonella typhi infection has become the new public health problem. Pulsed field gel electrophoresis (Pulsed-field gel electrophoresis, PFGE) because of its good repeatability, high resolution, a variety of pathogenic bacteria have been used for molecular typing, epidemiology and microbiology studies. However, we found that the current PulseNet international network laboratory based on analysis for Salmonella PFGE day standard scheme Analysis on China from different provinces and years of Salmonella paratyphi A, only limited The belt type. Then this standard PFGE solution in the analysis of Salmonella paratyphi A, is a type of method is not suitable for the analysis of the genome of bacteria due to limited ability to distinguish these or across so many years and provinces of the epidemic strains is conservative cloning, need analysis. In order to explore may be more suitable for Salmonella paratyphi A isolates PFGE analysis program, in this study we used PulseNet international network laboratory based on analysis of Salmonella PFGE day standard scheme, choose from China in different years and provinces of the 33 strains of Salmonella paratyphi A, by comparing the endonuclease and electrophoresis parameters, comparing optimized for genotyping of Salmonella paratyphi. The PFGE scheme is then used to optimize the new scheme, isolated from different ages (1998-2010) and 12 provinces were analyzed retrospectively. The results showed that Sp EI, XbaI, XhoI and BlnI four enzymes are suitable for the analysis of Salmonella paratyphi A, through the classification of the 33 strains of the similarity and the resolution and comparative analysis, found that SpeI-PFGE produced with more and more high resolution than XbaI-PFGE and XhoI-PFGE, so SpeI is considered to be the first selected enzyme, XbaI-PFGE slightly lower resolution than the XhoI-PFGE, but with the number of XbaI-PFGE type was more than XhoI-PFGE, so we think XbaI as an alternative enzyme, and XhoI as third kinds of enzyme.BlnI-PFGE and above three compared to the enzyme band type at least and the lowest resolution, not used in further study. In addition, in each of three parameters the use of enzymes for comparative analysis, found that the PFGE electrophoresis parameters using the XbaI enzyme (1.5-29s, 20h), electrophoresis parameters using SpeI (1-20s, 20h), electrophoresis parameters using XhoI (2.2-29s, 20h) the highest resolution in each enzyme, respectively. As of 17, 23 and 16. With the new electrophoresis parameters analysis of 106 strains of Salmonella paratyphi A results show that diffusion are highly Chinese cloning of paratyphoid epidemic strains in the country caused, but with the expansion of the year changes and epidemic areas, but also the accumulation of multilocus sporadic mutations. Variable number tandem repeat analysis (Multiple Locus Variable Numbers Tandem Repeat Analysis, MLVA) is based on PCR technology developed in recent years the molecular typing method. It is found that the method of evaluation has high resolution, good repeatability, the operation process is not complicated, has been used for epidemiological analysis of various pathogenic bacteria including Salmonella serotype variety. In this study, we explore the establishment of MLVA method for the analysis of Salmonella paratyphi A and evaluate its application value in epidemiology. The application of TRF and TRD software A genome-wide scan of Salmonella paratyphi A strains of ATCC9150 sequencing, we obtained 196 tandem repeat loci (Tandem, Repeat, TR) primers were designed for TR, select 33 strains in the separation of time and place as much as possible dispersion, PFGE band with paratyphoid salmonella strains were taken as evaluation samples (panel), screening of polymorphic loci from all TR, the use of these sites can be used to establish the MLVA experimental method of Salmonella paratyphi A. 209 strains of Salmonella paratyphi A strains in 1998 12 China isolated in -2010 were analyzed by MLVA and PFGE, the two kinds of classification methods are compared with experimental results analysis and evaluation of MLVA for Salmonella paratyphi A type of value. In the detection of 33 isolates, found only 7 VNTR loci. The copy number changes of the 7 VNTRs loci based on 209 strains of Salmonella paratyphi A isolates. For MLVA analysis, and PFGE (this study optimized SpeI enzyme digestion and electrophoresis parameters) experimental results, found that the classification ability was significantly weaker than that of PFGE, MLVA and distinguish the 21 types of PFGE and distinguish 65 types. Also suggested that in Salmonella paratyphi A, especially in a regional epidemic spread VNTR is not easy to occur, the genome copy number changes. We believe that MLVA is not suitable for a region (country or region) and the molecular epidemiology investigation and monitoring of Salmonella paratyphi A strains of popular, but in the genetic analysis of the clones identified can play a role in the.MLVA operation more convenient than PFGE, short time, the process is easy to standardization, some of the same PFGE of Salmonella paratyphi A strains with MLVA furtheranalysis, combined with PFGE can provide more than PFGE high resolution, so it can be used as an auxiliary molecular typing. The method, which is used in the epidemiological investigation of paratyphoid A, needs to be supplemented in depth analysis of the results of the PFGE typing.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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