Table 1.
Environmental samples and TR34-PCR and TR46-PCR assay results.
Fig 1.
Detection limit of the TR34-PCR and TR46-PCR assays for Aspergillus fumigatus.
The DNA from a wild type isolate (1205) and TR-based resistant isolates (1467 and 1470) were tested from 50 fg to 1 fg per reaction. The least amount of DNA detected is indicated with an arrow. Controls in the PCR assays included isolates 1205 (wt), 1467 (TR34), and 1470 (TR46) as positive controls and sterile distilled water as the negative control (c-).
Fig 2.
Sensitivity of the TR34-PCR and TR46-PCR assays.
Assays were conducted for soil samples and autoclaved soil samples spiked with different quantities of conidia of A. fumigatus, including 100, 80, 60, 30, and 10 conidia per ml. The assays were also conducted on soil samples sterilized prior to spiking with conidia of A. fumigatus. Controls in the PCR assays included isolates 1205 (wt), and 1467 (TR34) or 1470 (TR46) as positive controls and sterile distilled water as the negative control (c-).
Fig 3.
Gel image of 16 of the environmental samples, including air, compost (comp), soil, and plant debris (debris), assayed by TR34-PCR and TR46-PCR.
Sample numbers above each lane correspond with samples in Table 1. DNA was extracted from each sample which was processed in duplicate for the nested PCR assays. Samples showing bands in one or both replicates were considered positive for A. fumigatus. The 2.5% agarose gel allows the differentiation of the wt allele from the TR34 and TR46 resistance alleles. Samples with the A. fumigatus wt allele for the cyp51A promoter yield PCR products of 100 bp and 103 bp for TR34-PCR and the TR46-PCR, respectively. Samples with the resistant alleles produce PCR products that are 134 or 146 bp for TR34-PCR or 137 or 149 bp for TR46-PCR. These samples are considered resistant (R) and undergo high-definition electrophoresis on a 4% gel to distinguish the TR34 and TR46 alleles. Samples without an amplification product were considered negative (N).
Fig 4.
The nested TR34-PCR and TR46-PCR assays amplify A. fumigatus wild type (wt) alleles and the TR34 and TR46 alleles.
Differentiation among these alleles can be accomplished with electrophoresis in 4% high-definition agarose.
Fig 5.
Fragment sizes of PCR products from the TR34-PCR and TR46-PCR assays.
Sizes are shown for the A. fumigatus cyp51A wt, TR46, and TR34 alleles based on the nested or second round PCR with the inner primers.