Utility of random amplified polymorphic DNA PCR and TaqMan automated detection in molecular identification of Aspergillus fumigatus.
We developed a method for the identification of Aspergillus fumigatus fungal isolates by using random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) cloning and the TaqMan LS50B fluorogenic detection system (Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.). DNA from seven clinically important Aspergillus species was screened by RAPD-PCR to identify section- or species-specific amplicons. With the OPZ19 RAPD primer a 1,264-bp product was amplified from all A. fumigatus strains initially examined but not from other species. A partial DNA sequence of this product was used to design a specific primer pair, which generated a single 864-bp fragment with DNA from 90 of 100 A. fumigatus isolates when a "touchdown" (65-->55 degrees C) annealing protocol was used. The TaqMan system, a fluorogenic assay which uses the 5'-->3' endonuclease activity of Taq DNA polymerase, detected this 864-bp product with DNA from 89 of these 90 A. fumigatus strains; 1 DNA sample generated an indeterminate result. With DNA from three morphologically typical A. fumigatus isolates, six white ("albino") A. fumigatus isolates, and five of six Neosartorya species (non-A. fumigatus members of the section Fumigati), the 864-bp product was amplified differentially at an annealing temperature of 56 degrees C but not with the touchdown annealing format. No amplicon was detected with DNA from 56 isolates of heterologous Aspergillus, Penicillium, and Paecilomyces species or from Neosartorya fennelliae; TaqMan assay results were either negative (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR and TaqMan assay offers promise as a nucleic acid-based system that can be used for the identification of filamentous fungal isolates and that requires no postamplification sample manipulations.
Brandt
M E
ME
Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. mbb4@cdc.gov
Padhye
A A
AA
Mayer
L W
LW
Holloway
B P
BP
D001228:Aspergillosis / Q000382:microbiology*; D001232:Aspergillus fumigatus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015342:DNA Probes; D004271:DNA, Fungal / Q000302:isolation & purification; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D015342:DNA Probes; D004271:DNA, Fungal; D005456:Fluorescent Dyes