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 bp in length, was amplified by PCR from the genomic DNA extract of 12 species of dermatophytes and sequenced. An intron that is 56 to 93 bp in length was located along the ACT fragment of all of the dermatophytes at codon position 301 (-3) (a codon number followed by "-3" indicates that the intron directly follows the codon) with reference to the amino acid sequence of human 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-PCR) of dermatophyte ACT mRNA. PCR primer pairs with antisense sequence based on the ACT intron sequence were species specific for dermatophytes, suggesting a potential for use 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 microl of yeast carbon base broth. RT-nested PCR of dermatophyte-infected samples amplified an ACT fragment of the predicted size of 171 bp. The results of viability testing based on ACT mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales. This method is a potential tool for rapidly assessing fungal viability in the therapeutic efficacy testing of antimycotics.
Okeke
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Department of Dermatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Tsuboi
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R
Kawai
M
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Hiruma
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Ogawa
H
H
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D003883:Arthrodermataceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001483:Base Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D003881:Dermatomycoses / Q000382:microbiology*; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012867:Skin / Q000382:microbiology*
D000199:Actins; D017931:DNA Primers; D004271:DNA, Fungal; D012333:RNA, Messenger