Mutation Analysis Of ERG11 Gene In Clinical Isolated Candida Albicans Resistance To Azole Agents

Objective:To explore the mutations of ERG11 gene in clinical isolated Candida albicans resistance to Azole agents.Methods:Disc diffusion method was performed to preliminary screening the clinical isolated resistance strains of Candida albicans. Clinical and Laboratory Standards Institute (CLSI) M-27 was used to measuring their MIC 10 resistant strains (FLC MIC≥64ug/ml, ITC MIC≥1ug/ml) and 5 sensitive strains (FLC MIC≤8ug/ml, ITC MIC≤0.125ug/ml) were selected randomly to be studied. Genome DNA of the Candida albicans was extracted. Three pairs of PCR primers overlapping all the sequence of ERG 11 gene were synthesized. The fragments of ERG 11 gene (ERG 11.1, ERG 11.2 and ERG 11.3) were amplified and purified. Then the sequencing was operated by TaKaRa Biotechnology Co., Ltd. After that aligning analysis was performed according to the standard sequence (X13296) in GenBank to clear the mutant sites in the ERG11 genes of Azoles resistant strains.Results:The size of all the three PCR fragments was consistent with that of expected. Sequencing results showed that the complete ERG 11 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, while only same-sense mutation was found in sensitive strains.15 sites of missense mutation were proved in total of 28 base altered sites.10 sites F72L, F105L, D116E, A117G, Y132H, E266D, V437I, G464S, R467K and E488I had been reported previously in the resistant strains, and other five sites F126L, I166N, H183Q, S453F and N490K were newly probed.Conclusion:Multiple sites of missense mutation existed in ERG 11 gene of Azoles resistance Candida albicans, corresponding association maybe occurred between the mutation and their drug resistance. While the meaning of the 5 newly discovered mutant sites should be further investigated.