摘要
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 explore the relationship between the point mutations of ERG11 gene in Candida albicans and azole antifungal agent resistance.Methods Disc diffusion method was performed to screen preliminarily the clinical isolated drug-resistant strains of Candida albicans.Clinical and Laboratory Standards Institute M-27 was used to measure their minimal inhibitory concentration.Ten drug-resistant strains were randomly selected to be studied.Genome DNA of the Candida albicans was extracted.Three pairs of PCR primers overlapping all the sequence of ERG11 gene were synthesized.The fragments of ERG11 gene(P1,P2 and P3) were amplified and purified.Results The size of all the three PCR fragments was consistent with that of expected.Sequencing results showed that the complete ERG11 genes were successfully amplified.The aligning analysis between acquired sequences and the standard sequence in GenBank(X13296) showed that both same-sense and missense mutations were existed in the resistant strains.Fifteen sites of missense mutation were proved in total of 27 base altered sites.Five sites including F126L,I166N,H183Q,S453F and N490K were newly probed.Conclusion Multiple sites of missense mutation exist in ERG11 gene of Azoles resistance Candida albicans and are multi-point mutation,which may be related with drug resistance.
出处
《中华实用诊断与治疗杂志》
2011年第9期870-872,875,共4页
Journal of Chinese Practical Diagnosis and Therapy