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 (P erkin-Elmer Corp., Applied Biosystems, Foster City, Calif,), DNA from seven clinically important Aspergillus species was screened by RAPD-PC R to identify section-or species-specific amplicons, With the OPZ19 RA PD primer a 1,264-bp product was amplified from all A. fumigatus strai ns initially examined but not from other species. A partial DNA sequen ce of this product was used to design a specific primer pair, which ge nerated a single 864-bp fragment with DNA from 90 of 100 A. fumigatus isolates when a ''touchdown'' (65-->55 degrees C) annealing protocol w as used. The TaqMan system, a fluorogenic assay which uses the 5'-->3' endonuclease activity of Tag DNA polymerase, detected this 864-bp pro duct with DNA from 89 of these 90 A. fumigatus strains; 1 DNA sample g enerated an indeterminate result. With DNA from three morphologically typical A, fumigatus isolates, six white (''albino'') A. fumigatus iso lates, and five of six Neosartorya species (non-A, fumigatus members o f the section Fumigati), the 864-bp product was amplified differential ly at an annealing temperature of 56 degrees C but not with the touchd own annealing format. No amplicon was detected with DNA from 56 isolat es of heterologous Aspergillus, Penicillium, and Paecilomyces species or from Neosartorya fennelliae; TaqMan assay results were either negat ive (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR and TaqMan assay offers promise as a nucleic acid-based syst em that can be used for the identification of filamentous fungal isola tes and that requires no postamplification sample manipulations.