【摘要】：目的應用多位點序列分型(Multilocus Sequence Typing, MLST)對臨床分離白念珠菌進行分型研究,同時與ABC (ATP-binding cassette)分型、隨機擴增多態(tài)DNA技術(randomLy amplified polymorphic DNA, RAPD)結果比較,研究菌株分型與標本來源、藥物敏感性等之間的關系,建立國內白念珠菌MLST數(shù)據(jù)庫。 方法 1.收集臨床分離白念珠菌,進行體外藥物敏感性測定,選取10株氟康唑耐藥株,23株氟康唑敏感株,提取菌株DNA。 2.建立MLST分型方法,選擇7對管家基因進行PCR擴增,并將目的基因產(chǎn)物測序,然后將測序結果與標準序列比對后上傳至MLST網(wǎng)站數(shù)據(jù)庫,獲得相應的由7對管家基因組成的等位基因譜,最后將等位基因譜再次提交網(wǎng)站確認序列型(Sequence types, STs). 3.選取ABC分型和RAPD特定引物進行PCR擴增,產(chǎn)物經(jīng)瓊脂糖凝膠電泳后確定分型。 4.將MLST結果經(jīng)MEGA4.O生物信息學分析軟件分析后,采用非加權分組算術均方根法(UPGMA)制作最小生成樹,進一步分析ST與菌株信息、其他分型方法的關系。 結果 1.33株臨床白念珠菌通過MLST產(chǎn)生了33個STs,采用ABC方法分類為3型,采用RAPD方法引物RSD-12產(chǎn)生13型,引物OPE-18產(chǎn)生11型。 2.最小生成樹選取區(qū)分值0.0025作為分界線劃定組,產(chǎn)生8個組,顯示白念珠菌的MLST型別呈現(xiàn)多樣性,廣泛分布在各個進化支上。 結論 1. MLST具有較高的分辨力,是一種方便、快速的分子生物學方法,可用于流行病學和菌群多態(tài)性的研究。 2.聚類分析表明MLST分型結果與ABC分型有關系,但與菌株的標本采集來源、醫(yī)院來源、RAPD分型結果或抗真菌藥耐藥性無明顯相關。 3.初步建立了MLST分型方法和國內白念珠菌MLST數(shù)據(jù)庫。
[Abstract]:Objective to study the clinical isolates of Candida albicans by multilocus sequence typing (Multilocus Sequence Typing, MLST), and to compare the results of (randomLy amplified polymorphic DNA, RAPD) with ABC (ATP-binding cassette) typing and random amplified polymorphic DNA (DNA). The MLST database of Candida albicans was established by studying the relationship between strain typing and sample source, drug sensitivity and so on. Method 1. Clinical isolates of Candida albicans were collected and tested for drug sensitivity in vitro. Ten fluconazole-resistant strains and 23 fluconazole-sensitive strains were selected to extract DNA.. 2. MLST typing method was established, 7 pairs of housekeeping genes were selected for PCR amplification, and the target gene products were sequenced. The results were compared with standard sequences and uploaded to the MLST website database. The corresponding allelic spectrum consisting of 7 pairs of housekeeper genes was obtained. Finally, the allele spectrum was resubmitted to the website to confirm the sequence of (Sequence types, STs). 3. ABC typing and RAPD specific primers were selected for PCR amplification and the products were identified by agarose gel electrophoresis. 4. The MLST results were analyzed by MEGA4.O bioinformatics analysis software, and the minimal spanning tree was made by (UPGMA) with unweighted grouping arithmetic RMS method. The relationship between ST and strain information and other typing methods was further analyzed. Results 1. 33 strains of clinical Candida albicans produced 33 STs, types by ABC, 13 types by RAPD primer RSD-12 and 11 types by primer OPE-18. 2. 2. The minimum spanning tree selected 0.0025 as the dividing line and produced eight groups, which showed that the MLST types of Candida albicans showed diversity and were widely distributed in various evolutionary branches. Conclusion 1. MLST has high resolution and is a convenient and rapid molecular biological method, which can be used in epidemiology and microbial polymorphism research. 2. Cluster analysis showed that MLST typing was related to ABC typing, but not to sample collection, hospital origin, RAPD typing or antifungal drug resistance. 3. MLST typing method and MLST database of Candida albicans were established.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

【參考文獻】

相關期刊論文 前8條

1 李若瑜,李東明,余進,王愛平,王曉紅,萬哲,陳偉,涂平,馬圣清,朱學駿,王端禮;真菌與真菌病研究近況[J];北京大學學報(醫(yī)學版);2002年05期

2 徐偉文;多位點酶電泳法在分子流行病學中的應用[J];國外醫(yī)學(流行病學傳染病學分冊);1995年06期

3 付曉燕;胡克興;范黎;;脈沖場凝膠電泳技術及其在真菌學研究中的應用[J];微生物學通報;2006年01期

4 萬兵,閆杰,季明春,張利寧,申厚鳳,陳志琳;一種優(yōu)化的PCR方法——降落PCR擴增目的基因[J];江蘇臨床醫(yī)學雜志;2002年02期

5 班俊;醫(yī)院深部真菌感染的臨床研究進展[J];醫(yī)學文選;2001年02期

6 梅興宇;施偉民;霍克克;高飛;沈亮亮;陳德利;;71株白念珠菌臨床分離株微衛(wèi)星基因分型[J];中華傳染病雜志;2007年02期

7 趙心懋;徐英春;謝秀麗;王澎;張小江;王輝;陳民鈞;;隨機擴增多態(tài)性方法對17例院內感染念珠菌的感染方式研究[J];中華微生物學和免疫學雜志;2006年01期

8 王銘超;張麗娟;;深部真菌感染流行病學及治療新進展[J];中國煤炭工業(yè)醫(yī)學雜志;2008年06期

，

本文編號：2353601