关键词: 滚环扩增技术, 白念珠菌, 氟康唑，耐药, TAC1，点突变

Abstract:

Objective To detect point mutations of TAC1 gene in fluconazole-resistant Candida albicans isolates with rolling circle amplification （RCA）, develop an accurate, rapid and specific assay to detect single nucleotide polymorphisms （SNPs）, and to estimate the relationship between mutations of TAC1 gene and resistance to fluconazole. Methods A total of 33 fluconazole-resistant Candida albicans isolates, including 8 strains from America and 25 from Australia, were collected. Four TAC1-specific padlock probes were designed according to previously reported mutations. DNA was extracted from these tested strains, subjected to amplification of three targeted fragments of TAC1 gene with PCR. Then, RCA was performed to detect point mutations of TAC1 gene in resistant Candida albicans strains. At the same time, the target fragments underwent sequencing analysis, and the results of RCA were compared with those of sequencing. Results Two types of resistance-associated mutations were found in 5 out of the 33 fluconazole-resistant strains. Among the 5 strains, 4 were from America, 1 harbored T225A mutation and 4 carried A736V mutation. No related mutation was found in TAC1 gene of 4 fluconazole-sensitive isolates. Conclusions RCA assay could accurately and rapidly detect point mutations of genes. Further studies are required to clarify the relationship between TAC1 point mutations and fluconazole resistance.

Key words: Rolling circle amplification, Candida albicans, Fluconazole-resistant, TAC1, point mutation