[Application of differential display-PCR technique in fluconazole-resistance gene expression of Candida].
To investigate the application of differential display-2PCR(DD-PCR) in research on gene expression of Candida.



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.



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.



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.
Zhu
Yu-ning
YN
The Affiliated Obstetrics and Gynecology Hospital, College of Medicine, ZhejiangUniversity, Hangzhou 310006, China. zyn@zju.edu.cn
Lu
Shi-ming
SM
D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects / Q000235:genetics*; D025141:Drug Resistance, Fungal / Q000235:genetics*; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods
D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D015725:Fluconazole; D010088:Oxidoreductases