Real-time PCR법을 이용한 Candida glabrata의 Azole 내성 유전자 발현 분석:Northern 교잡법과의 비교
Comparison of Real-Time Quantitative PCR with Northern Hybridization for Quantification of CgCDR1 and CgCDR2 Gene Expression in Candida glabrata
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Background: One of main mechanisms responsible for acquired azole resistance of Candida glabrata is the increased drug efflux mediated ABS transporters, which are encoded by CgCDR1 and CgCDR2 genes. Objectives: We compared real-time reverse transcriptase PCR (RT-PCR) with northern hybridization for quantitative analysis of CgCDR1 and CgCDR2 expression in bloodstream isolates of C. glabrata. Methods: Nineteen blood isolates of C. glabrata were selected, including nine fluconazole susceptible (MIC ≤8 μg/ml), nine susceptible dose-dependent (S-DD, MIC 16~32 μg/ml), and one resistant (MIC 128 μg/ml), isolates. The expression of CgCDR1 and CgCDR2 was quantified using real-time RT-PCR with ROTOR Gene 3000 (Corbettet research, Austria). The results were compared with northern hybridization with sequence-specific probes. Results: Correlation of quantification results between real-time RT-PCR and northern hybridization yielded correlation coefficients of 0.92 for CgCDR1 and 0.82 for CgCDR2 gene. By both methods, no significant differences were observed in the levels of expression of CgCDR1 and CgCDR2 between fluconazole-susceptible isolates and S-DD isolates. In contrast, a strain with high fluconazole resistance (MIC 128 μg/ml) revealed a greater abundance of CgCDR1 by both methods, compared to the other isolates. Conclusion: This study show that real-time PCR method for C. glabrata RNA quantification correlates well with traditional northern hybridization and can be a valuable alternative to northern hybridization for rapid quantification of CgCDR1 and CgCDR2 genes in clinical isolates of C. glabrata.
Abstract
Background: One of main mechanisms responsible for acquired azole resistance of Candida glabrata is the increased drug efflux mediated ABS transporters, which are encoded by CgCDR1 and CgCDR2 genes. Objectives: We compared real-time reverse transcriptase PCR (RT-PCR) with northern hybridization for quantitative analysis of CgCDR1 and CgCDR2 expression in bloodstream isolates of C. glabrata. Methods: Nineteen blood isolates of C. glabrata were selected, including nine fluconazole susceptible (MIC ≤8 μg/ml), nine susceptible dose-dependent (S-DD, MIC 16~32 μg/ml), and one resistant (MIC 128 μg/ml), isolates. The expression of CgCDR1 and CgCDR2 was quantified using real-time RT-PCR with ROTOR Gene 3000 (Corbettet research, Austria). The results were compared with northern hybridization with sequence-specific probes. Results: Correlation of quantification results between real-time RT-PCR and northern hybridization yielded correlation coefficients of 0.92 for CgCDR1 and 0.82 for CgCDR2 gene. By both methods, no significant differences were observed in the levels of expression of CgCDR1 and CgCDR2 between fluconazole-susceptible isolates and S-DD isolates. In contrast, a strain with high fluconazole resistance (MIC 128 μg/ml) revealed a greater abundance of CgCDR1 by both methods, compared to the other isolates. Conclusion: This study show that real-time PCR method for C. glabrata RNA quantification correlates well with traditional northern hybridization and can be a valuable alternative to northern hybridization for rapid quantification of CgCDR1 and CgCDR2 genes in clinical isolates of C. glabrata.
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