【摘要】：目的應(yīng)用多位點(diǎn)序列分型(Multilocus Sequence Typing, MLST)對(duì)臨床分離白念珠菌進(jìn)行分型研究,同時(shí)與ABC (ATP-binding cassette)分型、隨機(jī)擴(kuò)增多態(tài)DNA技術(shù)(randomLy amplified polymorphic DNA, RAPD)結(jié)果比較,研究菌株分型與標(biāo)本來(lái)源、藥物敏感性等之間的關(guān)系,建立國(guó)內(nèi)白念珠菌MLST數(shù)據(jù)庫(kù)。 方法 1.收集臨床分離白念珠菌,進(jìn)行體外藥物敏感性測(cè)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(duì)管家基因進(jìn)行PCR擴(kuò)增,并將目的基因產(chǎn)物測(cè)序,然后將測(cè)序結(jié)果與標(biāo)準(zhǔn)序列比對(duì)后上傳至MLST網(wǎng)站數(shù)據(jù)庫(kù),獲得相應(yīng)的由7對(duì)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(rèn)序列型(Sequence types, STs). 3.選取ABC分型和RAPD特定引物進(jìn)行PCR擴(kuò)增,產(chǎn)物經(jīng)瓊脂糖凝膠電泳后確定分型。 4.將MLST結(jié)果經(jīng)MEGA4.O生物信息學(xué)分析軟件分析后,采用非加權(quán)分組算術(shù)均方根法(UPGMA)制作最小生成樹,進(jìn)一步分析ST與菌株信息、其他分型方法的關(guān)系。 結(jié)果 1.33株臨床白念珠菌通過(guò)MLST產(chǎn)生了33個(gè)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(gè)組,顯示白念珠菌的MLST型別呈現(xiàn)多樣性,廣泛分布在各個(gè)進(jìn)化支上。 結(jié)論 1. MLST具有較高的分辨力,是一種方便、快速的分子生物學(xué)方法,可用于流行病學(xué)和菌群多態(tài)性的研究。 2.聚類分析表明MLST分型結(jié)果與ABC分型有關(guān)系,但與菌株的標(biāo)本采集來(lái)源、醫(yī)院來(lái)源、RAPD分型結(jié)果或抗真菌藥耐藥性無(wú)明顯相關(guān)。 3.初步建立了MLST分型方法和國(guó)內(nèi)白念珠菌MLST數(shù)據(jù)庫(kù)。
[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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

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