Reverse transcription-3 ' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model

The presence of viable cells of Candida albicans, in broth or in a reconstr ucted living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and sec reted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified b y reverse transcription-3' rapid amplification of cDNA ends-nested polymera se chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA wer e amplified from total RNA extracts of C. albicans grown in yeast carbon ba se-albumin broth or in living skin equivalent tissue; only the former was a mplified from Sabouraud broth-grown organisms. Primer pairs targeted for AC T1 and SAP2 were Candida genus-specific and C. albicans-specific, respectiv ely. The sensitivity limits of the assay were 100 fg of total RNA or 10 cel ls of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP 2 mRNA were not detected in total RNA extracts. Non-amplification of the mR NA correlated with the absence of C. albicans growth in Sabouraud agar cult ures of living skin equivalent samples. Reverse transcription-3' rapid ampl ification of cDNA ends-nested polymerase chain reaction of the mRNA encodin g specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of a n antimicrobial therapy, and in detecting mRNA expressed in very little amo unts in tissue.