Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/58095
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Type: Journal article
Title: Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing
Author: Wang, H.
Kong, F.
Sorrell, T.
Wang, B.
McNicholas, P.
Pantarat, N.
Ellis, D.
Xiao, M.
Widmer, F.
Chen, S.
Citation: BMC Microbiology, 2009; 9(1):1-12
Publisher: BioMed Central Ltd.
Issue Date: 2009
ISSN: 1471-2180
1471-2180
Statement of
Responsibility: 
Huiping Wang, Fanrong Kong, Tania C Sorrell, Bin Wang, Paul McNicholas, Namfon Pantarat, David Ellis, Meng Xiao, Fred Widmer and Sharon CA Chen
Abstract: BACKGROUND Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. RESULTS The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate. CONCLUSION The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.
Keywords: Humans; Candida albicans; Fluconazole; Fungal Proteins; DNA, Fungal; Antifungal Agents; Sensitivity and Specificity; Amino Acid Substitution; DNA Mutational Analysis; Drug Resistance, Fungal; Base Sequence; Point Mutation; Polymorphism, Single Nucleotide; Molecular Sequence Data
Rights: © 2009 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
RMID: 0020092413
DOI: 10.1186/1471-2180-9-167
Appears in Collections:Molecular and Biomedical Science publications

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