Quantification of Candida albicans actin mRNA by the LightCycler system as a means of assessing viability in a model of cutaneous candidiasis.
The LightCycler system (two-step reverse transcription-PCR-fluorescent hybridization [LC RT-PCR-FH]) was used to quantify Candida albicans actin mRNA as a means of assessing its viability in a reconstituted skin model of cutaneous candidiasis following the application of an antimycotic. A 192-bp ACT exon fragment was ligated into the pCR2.1 plasmid vector, and dilutions of the cloned insert (pACT; 4.092 kb) were used as the standard reference template. The LC RT-PCR-FH system could detect 1 fg of pACT, equivalent to 2.2 copies of the plasmid. The ACT exon-based PCR primers and FH probes were C. albicans specific, and electrophoretic analysis of the LC RT-PCR-FH assay product showed a 174-bp band in agarose gel. The number of copies of C. albicans ACT mRNA per milligram of tissue decreased with increasing amounts of amorolfine applied to a C. albicans-infected skin model, showing a reduction in viability. Detection and quantification of ACT mRNA in tissue by the LC RT-PCR-FH assay corresponded with cultural isolation of C. albicans from samples. The ACT mRNA-targeted LC RT-PCR-FH assay represents a sensitive, specific, rapid, and quantitative means of assessing the viability of C. albicans in infected tissue. This method may also be useful in evaluating the therapeutic efficacies of antifungal drugs in the treatment of various forms of candidiasis and other fungal diseases.
Okeke
C N
CN
Department of Dermatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunko-ku, Tokyo 113, Japan.
Tsuboi
R
R
Ogawa
H
H
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D002176:Candida albicans / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D002179:Candidiasis, Cutaneous / Q000382:microbiology*; D005456:Fluorescent Dyes; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology
D000199:Actins; D005456:Fluorescent Dyes; D012331:RNA, Fungal; D012333:RNA, Messenger