沙门菌属分离株MLST分析及甲型副伤寒沙门菌MLVA实验方法的建立与评价

发布时间：2018-04-25 06:03

本文选题：沙门菌 + 甲型副伤寒沙门菌　；参考：《中国疾病预防控制中心》2009年硕士论文

【摘要】： 沙门菌血清型复杂,其基因组差异需要深入了解,本研究利用中国沙门菌属分离株开展多位点序列分型(Multilocus Sequence Typing,MLST)研究,对MLST在不同血清型间及同一血清型内亚型的分型能力进行评价,同时评估MLST对鉴定沙门菌属血清型的参考价值,实验选择aroC,dnaN,hemD,hisD,purE,sucA,thrA作为沙门菌分离株MLST的分型基因,对我国参与全球沙门菌监测网(Global Salmonella Surveilance,GSS)的5省市2006-2007年分离的201株沙门菌菌株进行分型,并对所有菌株进行血清型鉴定和PFGE实验,MLST分析将45个血清型沙门菌分离株分为46个序列型(Sequence Type,ST),结果表明MLST分型结果与血清型分型结果有明显的对应关系,二者联合应用可以作为PFGE实验结果的有效补充,沙门菌动物来源和人来源沙门菌的MLST型具有既相互重叠又具有明显界限的特点。目前甲型副伤寒沙门菌在我国和东南亚发病较多,本研究探索建立用于甲型副伤寒沙门菌的多位点可变数目串联重复序列分析(Multiple Locus Variable Numbers Tandem RepeatAnalysis,MLVA)方法并对其应用价值进行评价。从甲型副伤寒沙门菌测序株ATCC9150的全基因组序列中确定所有可变数目串联重复位点(Variable Numbers Tandem Repeat,VNTR)并设计引物,选择42株在分离时间、地点和PFGE带型上有差异的甲型副伤寒沙门菌菌株作为panel,从所有VNTR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菌株的MLVA实验方法,再对我国9个地区1998年-2008年分离的97株甲型副伤寒菌株进行MLVA和PFGE实验,将这两种分型方法的实验结果进行比较分析,评价MLVA用于甲型副伤寒菌株分型的价值。本实验建立了基于7个VNTRs位点的甲型副伤寒沙门菌MLVA实验方法,与PFGE(SpeI酶切)实验结果比较,其分型能力要弱于PFGE,但MLVA能够反映甲型副伤寒沙门菌的流行病学上的差异和关联性,且与PFGE联合应用可以提供较PFGE跟高的分辨力,可以作为甲型副伤寒沙门菌分子分型的备选方法。
[Abstract]:The serotype of Salmonella is complex, and the genomic differences need to be deeply understood. In this study, multilocus Sequence typing was carried out by using Chinese Salmonella isolates. The typing ability of MLST between different serotypes and the same serotype was evaluated, and the reference value of MLST in identifying Salmonella serotype was evaluated. 201 strains of Salmonella isolated from 5 provinces and cities of China participating in Global Salmonella Surveillance system (GSS) in 2006-2007 were genotyped. All the strains were identified by serum type identification and PFGE analysis. 45 isolates of Salmonella serotype were divided into 46 serotypes, the results showed that there was a significant correspondence between the MLST typing results and the serotype typing results. The combined application of the two methods can be used as an effective supplement to the results of PFGE experiments. The MLST type of Salmonella from both animal and human sources has the characteristics of overlap and obvious limits. At present, salmonella paratyphoid A is more prevalent in China and Southeast Asia. In this study, a multiple Locus Variable Numbers Tandem repeat analysis (MLVAA) method was established for salmonella paratyphoid A and its application value was evaluated. All variable Numbers Tandem repeats (VNTRs) were identified from the whole genome sequence of ATCC9150, and 42 strains were selected for isolation. As a member of the panel, Salmonella paratyphoid A strains with different loci and PFGE bands were used to screen polymorphic loci from all VNTR, and to establish a MLVA assay for paratyphoid A strains by using these loci. 97 paratyphoid A strains isolated from 9 regions of China from 1998 to 2008 were tested by MLVA and PFGE. The results of the two methods were compared and analyzed to evaluate the value of MLVA in the typing of paratyphoid A strains. In this study, a MLVA assay based on 7 VNTRs loci was established. Compared with the results of PFGE(SpeI digestion, its typing ability was weaker than that of PFGE(SpeI, but MLVA could reflect the epidemiological differences and correlations of Salmonella paratyphoid A. Combined with PFGE can provide higher resolution than PFGE and can be used as an alternative method for molecular typing of Salmonella paratyphoid A.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

【引证文献】