Detection Of Point Mutations In Fluconazole-resistant Candida Albicans Isolates By Rolling Circle Amplification

[Objective] Clinical Candida albicans strains resistant and susceptible to fluconazole were collected, and were detected point mutation of ERG11 and TAC1 gene by rolling circle amplification (RCA). RCA results were compared with sequencing results, to develop an accurate, rapid and specific assay to detect point mutation; at the same time to better understand the relationship between mutation of ERG 11 or TAC1 and resistance to azoles.[Methods] C. albicans were collected from Clinical specimens and were determinated drug susceptibility to fluconazole, and 25 fluconazole-resistant strains and 21 fluconazole-susceptible strains were obtained; there were 10 known ERG11 mutations in eight fluconazole-resistant strains from USA. Extracted DNA, and acquired target ERG 11 gene and three fragments of TAC1 gene by PCR amplification, then purified to remove excess buffer, primers and dNTP. After the processes of connection through the padlock probe, exonuclease digestion and hyperbranched rolling circle amplification, RCA assay was used to detect point mutation in ERG 11 of the resistant and susceptible strains and in TAC1 of the resistant strains. At the same time target fragments were sequenced after purification, and the results of RCA were compared with sequencing resuluts.[Results] In eight fluconazole-resistant strains with known mutations, RCA assay correctly detected mutations which were consistent with known point mutations; with application of RCA assay, the RCA signal were detect in a mixture containing a minimum of 5% target template. Of all clinical strains, RCA assay showed excellent concordance with DNA sequencing. For ERG11 gene,24 of 25 fluconazole-resistant strains were detected missense mutations resulting in 20 kinds of amino acids substitutions (prevalence 96%), they were E266D (n=11), V488I (n=8), D116E (n=8), K128T (n=7), G464S(n=4), K143R(n=3), G448E(n=3), G307S(n=3), F145L(n=3), V437I(n=3), F449S(n=2), K108E(n=2), D153E(n=2), G465S(n=1), R467K(n=1), S405F(n=1), Y132H(n=1), F126L(n=1), D278E(n=1), G450V(n=1). Among them, G450V was a new mutation which was not reported before. One strain was not detected any mutation; 18 of 23 fluconazole-susceptible strains were detected 5 kinds of amino acid substitutions (prevalence 78%), they were E266D (n=15), D116E (n=11), V488I (n=7), K128T (n=3), V437I (n=2). Mutations both emerged in resistant and susceptible strains were D116E, E266D, K128T, V437I and V488I. For TAC1 gene, among 33 fluconazole-resistant C. albicans strains (including 8 resistant strains from USA), there were 5 strains containing two mutations, namely T225A (n=1) and A736V (n=4), of which 4 strains from the United States.[Conclusion] Using RCA assay to detect point mutation in fluconazole-resistant C. albicans, padlock probe correctly detected mutations of ERG11 gene and TAC1 gene in the test strains; accurate results also were obtained by RCA assay with some mutations which were closer in the location. It demonstrated that using RCA assay to detect point mutation has good specificity and sensitivity, RCA is a good assay which can detect point mutation accurately and rapidly. The number of mutations or distribution patterns of ERG11 and TAC1 mutations have no obvious correlation with the MICs to azoles, but may vary with the geographic regions. There were close relation between mutations of ERG11 and resistance to azoles. Further investigation should be carried out to better understanding the relationship between mutations of TAC1 gene and resistance to fluconazole. Multiple molecular mechanisms lead to the resistance to azoles in C. albicans, so in the future further explore should be done to understand molecular mechanisms of resistance to azoles.