PCR-based detection of DNA from the human pathogen Blastomyces dermatitidis from natural soil samples.
Blastomyces dermatitidis is the dimorphic fungal agent of blastomycosis, a disease that primarily affects humans and dogs. The clinical appearance of this mycosis is well characterized, but there is still little known about its environmental niche, having been isolated from nature only 21 times. We have developed a PCR-based assay to detect B. dermatitidis from soil samples using primers specific to a portion of the promoter region of the BAD1 virulence gene. An internal standard control, pTJV2, was constructed to validate the results from soil samples. Amplification of this control indicated adequate removal of ambient soil inhibitors. The PCR detection limits for the control plasmid and B. dermatitidis genomic DNA were 0.1 and 500 femtograms, respectively. No PCR cross-reactivity was observed against bacteria, actinomycetes, and 13 other fungi that were genetically related or found in the same geographic areas. In spiked soil samples, this method was sensitive to 304 copies of pTJV2 DNA and 8,450 live B. dermatitidis yeast cells. Three of eight natural soil samples from a dog kennel near Lexington, KY in which dogs suffered from blastomycosis were positive using the described method, demonstrating its utility in detecting B. dermatitidis in its natural surroundings.
Burgess
J W
JW
Department of Microbiology, University of Wisconsin-La Crosse, Wisconsin, La Crosse, USA. burgess.joshua@mayo.edu
Schwan
W R
WR
Volk
T J
TJ
D001758:Blastomyces / Q000235:genetics* / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005656:Fungal Proteins / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011401:Promoter Regions, Genetic; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D004271:DNA, Fungal; D005656:Fungal Proteins; D006023:Glycoproteins