title: Nested PCR assays for detection of Blastomyces dermatitidis DNA in paraffin-embedded canine tissue.
abstract: A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family ONYGENACEAE: We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.
authors.affiliation: Institute for Tropical Medicine, University Hospital Tübingen, Tübingen, Germany. ralf.bialek@med.uni-tuebingen.de
authors.forename: Ralf
authors.initials: R
authors.lastname: Bialek
authors.forename: Anna Cascante
authors.initials: AC
authors.lastname: Cirera
authors.forename: Tanja
authors.initials: T
authors.lastname: Herrmann
authors.forename: Christian
authors.initials: C
authors.lastname: Aepinus
authors.forename: Valerie I
authors.initials: VI
authors.lastname: Shearn-Bochsler
authors.forename: Alfred M
authors.initials: AM
authors.lastname: Legendre
mesh_terms: D000818:Animals; D001758:Blastomyces / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000032:analysis*; D004285:Dogs; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D012337:RNA, Ribosomal, 18S