Isolation of an intron-containing partial sequence of the gene encoding dermatophyte actin (ACT) and detection of a fragment of the transcript by reverse transcription-nested PCR as a means of assessing the viability of dermatophytes in skin scales

An internal partial sequence of the gene encoding actin (ACT), 725 to 762 b p in length, was amplified by PCR from the genomic DNA extract of 12 specie s of dermatophytes and sequenced. An intron that is 56 to 93 bp in length w as located along the ACT fragment of all of the dermatophytes at codon posi tion 301 (-3) (a codon number followed by "-3" indicates that the intron di rectly follows the codon) with reference to the amino acid sequence of huma n alpha -smooth muscle actin. A primer pair that annealed to exon sequences flanking the ACT-associated intron produced a dermatophyte-specific 171-bp amplicon by reverse transcription-nested PCR (RT-FCR) of dermatophyte ACT mRNA. PCR primer pairs with antisense sequence based on the ACT intron sequ ence were species specific for dermatophytes, suggesting a potential for us e in the identification of dermatophytes. The viability of dermatophytes in skin scales was subsequently assessed by the presence of ACT mRNA in total RNA extracted from a 48-h culture of scale samples in 250 mul of yeast car bon base broth, RT-nested PCR of dermatophyte-infected samples amplified an ACT fragment of the predicted size of 171 bp. The results of viability tes ting based on ACT mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales. This method is a potential tool for rapidly ass essing fungal viability in the therapeutic efficacy testing of antimycotics .