Objective To detect the resistant mechanisms o f Candida albicans to fluconazole (FCZ) at molecular biology lev el, since the resistance me chanisms of azole antifungal agents have been the focus of attention these y...Objective To detect the resistant mechanisms o f Candida albicans to fluconazole (FCZ) at molecular biology lev el, since the resistance me chanisms of azole antifungal agents have been the focus of attention these years . Methods Thirty two FCZ resistant C.albicans were selected as our test strains (MICs≥64?μg/ml). With 14 α demethylase gene (ERG16 ge ne, target enzyme encoding gene of azoles) as our object, we chose six sets of prim ers from the ERG16 gene to amplify the interested fragments, and conducted South ern blot hybridization, restriction fragment length polymorphism (RFLP), and sin gle strand conformation polymorphism (SSCP) analysis for the fragments which wer e amplified by the six sets of primers, and pre resistant sensitive strains wer e used as controls. Three representative fragments, A66, D66 and E78, were selec ted to be cloned and sequenced. Results The polymerase chain reaction (PCR) amplification showed that s everal tested strains were negative for some primers. However, our Southern blot analysis reminded that their resistance did not result from the lack of targ et enzyme coding gene. SSCP analysis s howed that differences were noted between the resistant and sensitive strains an d inter resistant strains. Statistical analysis showed that the most variable s equence lied in the amplifier of the sixth pair of primer, and all the tested 32 strains showed positive results. In the 11 mutation points we found, five resu lted in amino acid alterations. It is likely that one or more mutational alterations (alone or in combination) might lead to the expression of an enzyme highly resi stant to the inhibitory action of FCZ which in turn is responsible for the FCZ r esistant trait in these strains. Conclusion One or more mutational alterations might lead to the azole r esistant trait in this strains.展开更多
文摘Objective To detect the resistant mechanisms o f Candida albicans to fluconazole (FCZ) at molecular biology lev el, since the resistance me chanisms of azole antifungal agents have been the focus of attention these years . Methods Thirty two FCZ resistant C.albicans were selected as our test strains (MICs≥64?μg/ml). With 14 α demethylase gene (ERG16 ge ne, target enzyme encoding gene of azoles) as our object, we chose six sets of prim ers from the ERG16 gene to amplify the interested fragments, and conducted South ern blot hybridization, restriction fragment length polymorphism (RFLP), and sin gle strand conformation polymorphism (SSCP) analysis for the fragments which wer e amplified by the six sets of primers, and pre resistant sensitive strains wer e used as controls. Three representative fragments, A66, D66 and E78, were selec ted to be cloned and sequenced. Results The polymerase chain reaction (PCR) amplification showed that s everal tested strains were negative for some primers. However, our Southern blot analysis reminded that their resistance did not result from the lack of targ et enzyme coding gene. SSCP analysis s howed that differences were noted between the resistant and sensitive strains an d inter resistant strains. Statistical analysis showed that the most variable s equence lied in the amplifier of the sixth pair of primer, and all the tested 32 strains showed positive results. In the 11 mutation points we found, five resu lted in amino acid alterations. It is likely that one or more mutational alterations (alone or in combination) might lead to the expression of an enzyme highly resi stant to the inhibitory action of FCZ which in turn is responsible for the FCZ r esistant trait in these strains. Conclusion One or more mutational alterations might lead to the azole r esistant trait in this strains.
