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目的探讨白色假丝酵母菌ERG11基因突变与唑类抗真菌药物耐药的关系。方法纸片扩散法初步筛选临床分离的耐唑类抗真菌药物的白色假丝酵母菌,测定初筛耐药株对氟康唑和伊曲康唑的最低抑菌浓度,随机选择10株白色假丝酵母耐药株,提取基因组DNA。利用DNASTAR软件辅助设计3对PCR引物,以提取的目的 DNA为模板,分别从前(P1)、中(P2)、后(P3)3段扩增ERG11基因全序列。扩增后的PCR产物经纯化后测序,将测序结果与GenBank中已知标准序列(X13296)进行比较分析。结果电泳结果显示,获得的3段PCR产物大小与预期结果一致。测序结果显示成功获得10株白色假丝酵母菌ERG11全基因序列。与GenBank中标准株序列(X13296)比较,耐药株均存在同义突变和错义突变,共27个碱基突变位点。15个位点是错义突变,其中F126LI、166N、H183Q、V437I、S453F和N490K为新发现的突变位点。结论耐唑类抗真菌药物的白色假丝酵母菌ERG11基因有多个发生错义突变的位点,且为多点突变,可能与其耐药有关。
Objective To investigate the relationship between Candida albicans ERG11 gene mutation and azole antifungal drug resistance. Methods The primary screening of clinical Candida albicans resistant to azole antifungal agents by disk diffusion method was carried out to determine the minimum inhibitory concentrations of fluconazole and itraconazole on the primary screening drug resistant strains. Resistant strains of yeast, extraction of genomic DNA. Using DNASTAR software to design three pairs of PCR primers, the complete sequence of ERG11 gene was amplified from the former (P1), middle (P2) and later (P3) segments by using the extracted target DNA as a template. The amplified PCR product was purified and sequenced. The sequencing results were compared with the known standard sequence in GenBank (X13296). Results The electrophoresis results showed that the size of the obtained 3-stage PCR products was consistent with the expected results. Sequencing results showed that 10 strains of Candida albicans ERG11 gene were successfully obtained. Compared with the standard strain of GenBank (X13296), there were both synonymous mutations and missense mutations in the resistant strains, with 27 base mutation sites. Fifteen loci were missense mutations, of which F126LI, 166N, H183Q, V437I, S453F and N490K were newly discovered mutation sites. Conclusions There are multiple sites of missense mutation in ERG11 gene of Candida albicans resistant to azole antifungal agents, and they are multipoint mutations that may be related to their drug resistance.