Application of differential display-PCR technique in fluconazole-resistance gene expression of Candida / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the application of differential display-2PCR(DD-PCR) in research on gene expression of Candida.</p><p><b>METHODS</b>Resistance to fluconazole was induced in a Candida albicans isolate 435 from vagina by culturing in YEPD broth with increasing fluconazole concentration in vitro, and the resistant isolate 435-2 (MIC=128 microg/ml ) was obtained after 80 days of incubation. Comparisons between 435 and 435-2 either in fluconazole-containing medium or in drug-free medium were performed with the modified DD-PCR including amplification with long primers, silver staining, reverse dot blot and non-radiographic labeling techniques.</p><p><b>RESULTS</b>Three differential displayed bands were found which showed high homology to alcohol dehydrogenase 1 (ADH1), TOP2 and CDR1, respectively. The up-regulating expression of ADH1 and CDR1 associated with fluconazole resistance was further identified by RT-PCR.</p><p><b>CONCLUSION</b>The up-regulating expression of ADH1 and CDR1 was associated with fluconazole resistance in Candida albicans, ADH1 might be a candidate of novel fluconazole resistant gene.</p>