甲型副伤寒沙门菌MLVA分型方法的建立及应用

发布时间：2018-01-13 23:03

本文关键词：甲型副伤寒沙门菌MLVA分型方法的建立及应用　出处：《南华大学》2009年硕士论文　论文类型：学位论文

【摘要】：研究目的:建立甲型副伤寒沙门菌(Salmonella paratyphoid A,SPA)多位点可变数目串联重复序列分析(Multiple-Locus Variable number tandem repeat Analysis,MLVA)分子分型方法,并用于云南甲型副伤寒沙门菌的分型分析。材料和方法:21株SPA参考菌株购买于中国医学细菌保藏管理中心,194株应用于分型分析的SPA临床分离株分离自2005-2009年中国云南省玉溪市。应用系统测试法,根据国际测序株A-9150和A-12601的基因组测序结果,首先用TRF软件筛选和鉴定串联重复位点,然后基于序列和标本测试筛选多态性串联重复序列(Variable number tandem repeat,VNTR)。设计引物,PCR扩增VNTR,通过普通琼脂糖凝胶电泳、毛细血管电泳和测序确定VNTR。最后将筛选结果应用于194株临床分离株的分型,用Bionumerics软件效用均等的分类资料UPGMA分析方法对分析结果进行聚类分析,以分类图或者最小扩展树的形式展现聚类分析的结果。研究结果:应用TRF软件在A-9150、A-12601菌株中鉴定了1591、1512个重复位点,并发现TR27在2个基因组中重复数目呈现多态性。第一轮筛选出51个位点,它们的TRF报告得分大于80%,或者位点重复拷贝数大于3,或者同源性大于85%的位点。然后设计引物,用21株SPA现实菌株确定了12个位点具有扩增条带多态性。通过测序分析确定9个位点(指标)的PCR产物大小变化由VNTR重复数的不同引起,经过分型效能分析筛选,最终确定9个用于SPA MLVA分型的指标,即TR5、TR51、TR24、TR27、TR40、TR41、TR43、TR44、TR49。21个参考菌株分为14个MLVA型别。云南省玉溪市194株临床分离菌株分为9个MLVA型别,其中流行区域主要为红塔区,15型为其优势型别。194株进行最小生成树分析代表菌株为6株11型菌株以及2株16型菌株和2株14型菌株,而澄江县09620菌株(13型)、江川县08140(17型)、红塔区07085(18型)各为一类型。同时与参考菌株比较结果显示,菌株50433(保加利亚)、50508(广东省疾病预防控制中心)、50509(广东省疾病预防控制中心)的亲缘关系与红塔区较为疏远,50002(丹麦)、50504(大连生研所)的亲缘关系与新平县菌株较为疏远,保加利亚50433、丹麦-上海同济50002等菌株与194株中国云南玉溪的人源菌株在MLVA分型方面没有关联。以08140菌株作为基点分为三个簇,其中第一簇(05115、06036、09616、09622、09532、09639)为同一类型(76型),与08140菌株比较具有单一MLVA基因位点不同。第二簇与08140比较具有2个MLVA基因位点不同的评价菌株为50101、50507、50078、50672和50434;3个MLVA基因位点不同的菌株为50154;5个MLVA基因位点不同的是评价菌株50674。第三簇均为2个MLVA基因位点不相同,评价菌株分别为50501、50502、50506、50001、9150(ATCC)、50503、50084。结论:1)首次建立了甲型副伤寒沙门菌MLVA分型方法; 2)MLVA分型应用于甲型副伤寒沙门菌分型具有较好的应用价值,发现了云南省玉溪市甲型副伤寒沙门菌的流行优势菌株和流行地区; 3)红塔区SPA的暴发流行可能对整个玉溪市SPA的暴发流行起关键作用,应加强对红塔区SPA的监测; 4)云南省玉溪市应加强MLVA 15型甲型副伤寒沙门菌的预防,同时不容忽视其它型别菌株的监测。
[Abstract]:Objective: to establish Salmonella paratyphi A (Salmonella paratyphoid A, SPA) multilocus variable number tandem repeat analysis (Multiple-Locus Variable number tandem repeat Analysis, MLVA) molecular typing method for analysis and typing of Salmonella paratyphi A in Yunnan.
Materials and methods: 21 strains of SPA reference strains purchased from Chinese medical culture collection center, 194 strains should be used in typing analysis of clinical isolates of SPA isolated from Yunnan city of Yuxi province China 2005-2009. Application system test method, according to the results of A-9150 and A-12601 international genome sequencing sequencing strains, TRF software was used for screening and identification tandem repeat, then based on the sequence and sample test screening of tandem repeat polymorphisms (Variable number tandem repeat, VNTR). The primers were designed to amplify VNTR PCR, by agarose gel electrophoresis, capillary electrophoresis and sequencing VNTR. finally screening results applied to 194 clinical isolates from typing methods the results of cluster analysis by Bionumerics software utility equal classification data UPGMA analysis, show the results of cluster analysis to classification and minimal spanning tree form.
Results: the application of TRF software in A-9150, A-12601 strains identified 15911512 duplicated loci, TR27 was found in 2 genomic repeat number of polymorphism. The first round of 51 loci screened, they report the TRF score greater than 80%, or the site copy number is greater than 3, or more than 85% homologous loci and then designed primers, 12 loci amplified bands with polymorphism were determined by 21 strains of SPA isolates determined by sequencing analysis. The reality of 9 loci (PCR index) product size change by the repeat numbers of VNTR caused by different types of performance, after screening, and ultimately determine the 9 for the SPA MLVA type the index, namely TR5, TR51, TR24, TR27, TR40, TR41, TR43, TR44, TR49.21 reference strains were divided into 14 MLVA types. Yunnan city in Yuxi Province, 194 isolates were divided into 9 MLVA types, the endemic area of Hongta District, 15 for its advantage Type.194 strains were the minimum spanning tree analysis of representative strains of 6 strains of type 11 strains and 2 strains of type 16 strains and 2 strains of type 14 strains, and 09620 strains of Chengjiang county (type 13), Jiangchuan 08140 (type 17), Hongta 07085 (type 18) for each type. At the same time compared with reference strains the results showed that 50433 strains (Bulgaria), 50508 (Guangdong Center for Disease Control and prevention), 50509 (Guangdong Center for Disease Control and prevention) relationship with Hongta district more alienated, 50002 (Denmark), 50504 (Dalian Institute) with the relation of Xinping county is 50433 strains from Bulgaria, no association strains from Denmark - Shanghai Tongji 50002 strains and 194 strains of Chinese Yunnan in Yuxi MLVA type. 08140 strains as the base is divided into three clusters, the first cluster (051150603609616096220953209639) for the same type (type 76), compared with the 08140 strains with a single MLVA Second different loci. 08140 clusters and 2 strains compared with the evaluation of different sites of MLVA gene were 50101505075007850672 and 50434; 3 MLVA gene loci of different strains was 50154; 5 MLVA loci is third different strains of 50674. clusters were evaluated 2 MLVA loci is not the same as the evaluation of the isolates were 505015050250506500019150 (ATCC, 5050350084.)
Conclusion: 1) the MLVA classification method of Salmonella paratyphi A was established for the first time.
2) MLVA typing has a good application value in typing of Salmonella paratyphi A, and the prevalent predominant strains and epidemic areas of Salmonella paratyphi A in Yuxi city of Yunnan province were found.
3) the outbreak of SPA in Hongta district may play a key role in the outbreak of SPA in the whole city, and the monitoring of SPA in Hongta area should be strengthened.
4) the prevention of Salmonella type a type a paratyphoid type A (MLVA 15) should be strengthened in Yuxi, Yunnan Province, and the monitoring of other types of strains should not be ignored.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

【参考文献】