Utility of random amplified polymorphic DNA PCR and TaqMan automated detection in molecular identification of Aspergillus fumigatus

Abstract

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.

FIG. 1

DNA from Aspergillus spp. amplified in duplicate either by RAPD-PCR with the OPZ19 primer (A) or by conventional PCR with the derived primers Z19-276 and Z19-660 (B). Lanes 1 and 10, A. fumigatus CDC MAS92-229; lanes 2 and 11, A. fumigatus CDC MAS92-564; lanes 3 and 12, A. fumigatus NRRL 163 (Thom 1911 ex type); lanes 4 and 13, A. fumigatus CDC-B5051; lanes 5 and 14, A. flavus MAS92-30; lanes 6 and 15, A. terreus MAS92-640; lanes 7 and 16, A. niger MAS92-52; lanes 8 and 17, A. fumigatus MAS93-678; lanes 9 and 18, no-template control. BP, base pairs.

FIG. 2

DNA from Aspergillus spp. amplified with primers Z19-276 and Z19-660 by either a conventional annealing protocol (A) or a touchdown protocol (B). Lanes 1 and 10, A. fumigatus CDC MAS92-229; lanes 2 and 11, A. fumigatus NRRL 163; lanes 3 and 12, N. spinosa NRRL 5034; lanes 4 and 13, N. fennelliae NRRL-A22212; lanes 5 and 14, A. flavus MAS92-30; lanes 6 and 15, A. flavus MAS92-230; lanes 7 and 16, A. niger MAS92-641; lanes 8 and 17, A. niger MAS92-243; lanes 9 and 18, no-template control. BP, base pairs.

FIG. 3

DNA from members of Aspergillus section Fumigati (A. fumigatus or Neosartorya spp.) amplified by RAPD-PCR with the OPZ19 primer (A), by conventional PCR with primers Z19-276 and Z19-660 (B), or by touchdown PCR with primers Z19-276 and Z19-660 (C). Lanes 1, A. fumigatus var. ellipticus NRRL 5109; lanes 2, A. fumigatus NRRL 163; lanes 3, N. spinosa NRRL 5034; lanes 4, A. fumigatus var. helvola NRRL 2244; lanes 5, N. aureola NRRL 2244; lanes 6, A. fumigatus var. sclerotiorum NRRL 6137; lanes 7, N. fischeri NRRL 181; lanes 8, N. glabra NRRL 2163; lanes 9, N. pseudofischeri NRRL 3496; lanes 10, A. fumigatus var. acolumnaris NRRL 5587; lanes 11, N. fischeri NRRL A7223; lanes 12, N. fennelliae NRRL A22212; lanes 13, A. fumigatus CDC 95-11279 (albino); lanes 14, A. fumigatus CDC 95-11277 (albino); lanes 15, A. fumigatus CDC MAS92-229; lanes 16, A. fumigatus CDC MAS92-564; lanes 17, A. fumigatus CDC B5051; lanes 18, A. fumigatus CDC 1809; lanes 19, no-template control. BP, base pairs.

FIG. 4

PCR for detection of the 864-bp A. fumigatus diagnostic product (FAM-labeled probe) and the 377-bp fungal rRNA control (HEX-labeled probe). Lane 1, no-template control; lane 2, plasmid 19B1 (FAM probe-only control); lane 3, A. flavus CDC MAS92-30 (HEX probe-only control); lane 4, A. fumigatus CDC MAS92-229 (FAM-positive control); lane 5, A. fumigatus MAS92-564 (FAM-positive control); lane 6, A. versicolor MAS92-490 (FAM- and HEX-indeterminate control); lane 7, A. flavus MAS92-230 (FAM-negative control); lane 8, plasmid 19B1 (HEX negative); lane 9, A. fumigatus MAS93-805; lane 10, A. terreus MAS93-997; lane 11, A. flavus MAS93-1040; lane 12, A. fumigatus var. ellipticus NRRL 5109; lane 13, A. fumigatus NRRL 163; lane 14, N. spinosa NRRL 5034; lane 15, A. fumigatus var. helvola NRRL 174; lane 16, N. aureola NRRL 2244; lane 17, A. fumigatus var. sclerotiorum NRRL 6137; lane 18, N. fischeri NRRL 181; lane 19, N. glabra NRRL 2163.

FIG. 5

Representative results from a TaqMan PCR experiment performed with the templates shown in Fig. 4. Results are expressed as normalized FAM result (A. fumigatus-specific probe) versus normalized HEX result (rRNA control probe). Arrows indicate the positions of the positive and negative cutoffs (0.8 and 0.35, respectively) for this experiment. Diamonds indicate TaqMan assay results for control templates: 1, A. flavus MAS92-230 (FAM negative, HEX positive); 2, A. versicolor MAS92-490 (FAM and HEX indeterminate); 3, plasmid 19B1 (FAM positive, HEX negative); 4, A. fumigatus CDC MAS92-229 (FAM and HEX positive); 5, A. fumigatus CDC MAS92-564 (FAM and HEX positive).