本文關(guān)鍵詞：甲型副傷寒沙門(mén)菌MLVA分型方法的建立及應(yīng)用　出處：《南華大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甲型副傷寒沙門(mén)菌 多位點(diǎn)可變數(shù)目串聯(lián)重復(fù)序列分析 多態(tài)性串聯(lián)重復(fù)序列 分子分型

【摘要】：研究目的:建立甲型副傷寒沙門(mén)菌(Salmonella paratyphoid A,SPA)多位點(diǎn)可變數(shù)目串聯(lián)重復(fù)序列分析(Multiple-Locus Variable number tandem repeat Analysis,MLVA)分子分型方法,并用于云南甲型副傷寒沙門(mén)菌的分型分析。 材料和方法:21株SPA參考菌株購(gòu)買于中國(guó)醫(yī)學(xué)細(xì)菌保藏管理中心,194株應(yīng)用于分型分析的SPA臨床分離株分離自2005-2009年中國(guó)云南省玉溪市。應(yīng)用系統(tǒng)測(cè)試法,根據(jù)國(guó)際測(cè)序株A-9150和A-12601的基因組測(cè)序結(jié)果,首先用TRF軟件篩選和鑒定串聯(lián)重復(fù)位點(diǎn),然后基于序列和標(biāo)本測(cè)試篩選多態(tài)性串聯(lián)重復(fù)序列(Variable number tandem repeat,VNTR)。設(shè)計(jì)引物,PCR擴(kuò)增VNTR,通過(guò)普通瓊脂糖凝膠電泳、毛細(xì)血管電泳和測(cè)序確定VNTR。最后將篩選結(jié)果應(yīng)用于194株臨床分離株的分型,用Bionumerics軟件效用均等的分類資料UPGMA分析方法對(duì)分析結(jié)果進(jìn)行聚類分析,以分類圖或者最小擴(kuò)展樹(shù)的形式展現(xiàn)聚類分析的結(jié)果。 研究結(jié)果:應(yīng)用TRF軟件在A-9150、A-12601菌株中鑒定了1591、1512個(gè)重復(fù)位點(diǎn),并發(fā)現(xiàn)TR27在2個(gè)基因組中重復(fù)數(shù)目呈現(xiàn)多態(tài)性。第一輪篩選出51個(gè)位點(diǎn),它們的TRF報(bào)告得分大于80%,或者位點(diǎn)重復(fù)拷貝數(shù)大于3,或者同源性大于85%的位點(diǎn)。然后設(shè)計(jì)引物,用21株SPA現(xiàn)實(shí)菌株確定了12個(gè)位點(diǎn)具有擴(kuò)增條帶多態(tài)性。通過(guò)測(cè)序分析確定9個(gè)位點(diǎn)(指標(biāo))的PCR產(chǎn)物大小變化由VNTR重復(fù)數(shù)的不同引起,經(jīng)過(guò)分型效能分析篩選,最終確定9個(gè)用于SPA MLVA分型的指標(biāo),即TR5、TR51、TR24、TR27、TR40、TR41、TR43、TR44、TR49。21個(gè)參考菌株分為14個(gè)MLVA型別。云南省玉溪市194株臨床分離菌株分為9個(gè)MLVA型別,其中流行區(qū)域主要為紅塔區(qū),15型為其優(yōu)勢(shì)型別。194株進(jìn)行最小生成樹(shù)分析代表菌株為6株11型菌株以及2株16型菌株和2株14型菌株,而澄江縣09620菌株(13型)、江川縣08140(17型)、紅塔區(qū)07085(18型)各為一類型。同時(shí)與參考菌株比較結(jié)果顯示,菌株50433(保加利亞)、50508(廣東省疾病預(yù)防控制中心)、50509(廣東省疾病預(yù)防控制中心)的親緣關(guān)系與紅塔區(qū)較為疏遠(yuǎn),50002(丹麥)、50504(大連生研所)的親緣關(guān)系與新平縣菌株較為疏遠(yuǎn),保加利亞50433、丹麥-上海同濟(jì)50002等菌株與194株中國(guó)云南玉溪的人源菌株在MLVA分型方面沒(méi)有關(guān)聯(lián)。以08140菌株作為基點(diǎn)分為三個(gè)簇,其中第一簇(05115、06036、09616、09622、09532、09639)為同一類型(76型),與08140菌株比較具有單一MLVA基因位點(diǎn)不同。第二簇與08140比較具有2個(gè)MLVA基因位點(diǎn)不同的評(píng)價(jià)菌株為50101、50507、50078、50672和50434;3個(gè)MLVA基因位點(diǎn)不同的菌株為50154;5個(gè)MLVA基因位點(diǎn)不同的是評(píng)價(jià)菌株50674。第三簇均為2個(gè)MLVA基因位點(diǎn)不相同,評(píng)價(jià)菌株分別為50501、50502、50506、50001、9150(ATCC)、50503、50084。 結(jié)論:1)首次建立了甲型副傷寒沙門(mén)菌MLVA分型方法; 2)MLVA分型應(yīng)用于甲型副傷寒沙門(mén)菌分型具有較好的應(yīng)用價(jià)值,發(fā)現(xiàn)了云南省玉溪市甲型副傷寒沙門(mén)菌的流行優(yōu)勢(shì)菌株和流行地區(qū); 3)紅塔區(qū)SPA的暴發(fā)流行可能對(duì)整個(gè)玉溪市SPA的暴發(fā)流行起關(guān)鍵作用,應(yīng)加強(qiáng)對(duì)紅塔區(qū)SPA的監(jiān)測(cè); 4)云南省玉溪市應(yīng)加強(qiáng)MLVA 15型甲型副傷寒沙門(mén)菌的預(yù)防,同時(shí)不容忽視其它型別菌株的監(jiān)測(cè)。
[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.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378

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