Detection of MDR1, CDR1 and ERG11 Gene Expression of Candida albicans by Single Tube Relative Multiplex RT-PCR Assay with Coamplification of 18s rRNA as Internal Control.



Background: The study of the correlation between the transcriptional overexpression of antifungal drug resistant genes and fluconzole-resistant Candida albicans isolates requires a rapid method for evaluation of contextual expression. Here, a relative multiplex RT-PCR (RMRP) assay with specific coamplification of 18s rRNA as an internal control is described for the parallel quantitative detection of expression of three C. albicans antifungal drug resistance genes (MDR1, CDR1 and ERG11). Methods:Gene specific primer pairs for MDR1, CDR1 and ERG11 and 18s rRNA were developed to enable specific amplification of the respective mRNAs. Specificity of all primer pairs was confirmed by matching the length of the amplicon with prediction from GenBank. The linear range of each gene was optimized so four single RT-PCR reactions could be integrated into one tube. Eleven clinical isolates of C. albicans with different fluconazole MICs were analyzed by RMRP. For each strain, MDR1, CDR1 and ERG11 expression was expressed as a ratio of the signal obtained for the gene specific amplicon by the signal obtained for the 18s rRNA amplicon. Results: Resistant isolates showed enhanced gene expression. This approach simultaneously eliminated RNA quality control issues for samples run in parallel while improving efficiency in the use of time and materials, and allowed multiplexed primers to behave similarly to uniplex RT reactions. Conclusions: To our knowledge, this is the first report on the use of RMRP to assay the expression of multiple antifungal drug resistance genes of C. albicans simultaneously. The RMRP described is a rapid and sensitive method for the detection of these genes, and may replace Northern blot.

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42nd Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 42nd