Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction

Abstract

Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal-shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high-throughput PCR analyses of mutants in 96- or 384-well formats in a very short period of time without any concern about sample cross-contamination. This method is currently being used to validate the protein-coding gene and non-coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.

Keywords: Aspergillus fumigatus; DNA extraction; filamentous fungi; spore PCR.

Figure 1

PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). (A) Phusion High‐Fidelity DNA polymerase (New England Biolabs). (B) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

Figure 2

Thermal shock is crucial for successful PCR amplification from fungal spores. A. fumigatus spores at three different concentrations (8 × 10⁸/ml, 1 × 10⁸/ml, and 5 × 10⁷/ml) were prepared as described in Basic Protocol 1. 3 µl of spore suspension were used as the DNA template for PCR. (A) PCR result from spore suspension subjected to thermal shock of 95°C for 15 min and −80°C for 10 min prior to PCR, as described in Basic Protocol 2. No thermal shock was done for spore suspensions from Panels B and C. Subsequently, a PCR was run with an initial denaturation step of 95°C for 1 min (A), 5 min (B), or 15 min (C) with primers ITS1 and D2 (expected PCR product ∼1.2 kb) and LongAmp Taq DNA polymerase with PCR conditions described in Basic Protocol 2. P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

Figure 3

To verify the correct integration of the hygromycin B phosphotransferase (hph) marker in 45 different A. fumigatus putative gene knockout mutants, spore PCR was carried out with the primer pairs P1/hphsqR2 (A; amplification of the DNA spanning the upstream flanking region of the gene of interest and hph) and P4/hphsqF2 (B; amplification of the DNA spanning between the hph and the downstream flanking region of the gene of interest). Primers P1 and P4 are complementary to the upstream and downstream flanking regions of the gene of interest, respectively. Primers hphsqF2 and hphsqR2 target the sequence of the hph used to replace the genes of interest. The expected PCR band sizes for the correct knockout mutants are ∼1.5 kb. P: positive PCR control amplified from genomic DNA (50 ng) of a known A. fumigatus knockout strain; N: negative control (no DNA). (C) Spore PCR on the supernatant of the 45 A. fumigatus gene knockout mutants validated in Panels A and B using primer pair ITS1/D2. This PCR confirms the amplification of fungal rDNA and that the size of the correct amplification product is ∼1.2 kb. P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

Figure 4

Spore PCR of the AYG1 gene and its flanking regions to test the range of PCR products effectively amplified using different concentrations of A. fumigatus wild‐type spores. The expected PCR band sizes are as follows: 622 bp (1), 1546 bp (2), 2241 bp (3), 3005 bp (4), 3744 bp (5), 4515 bp (6), and 5259 bp (7); P: positive control for the PCR reaction using genomic DNA (50 ng) of the A. fumigatus wild‐type strain as template, with the expected band size of 5259 bp; N: negative control (no DNA).

Figure 5

PCR amplification from a range of spore concentrations. 1 µl of the spore supernatant from different known concentrations of A. fumigatus spores was used in the PCR reaction with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). The spore concentration in the spore suspension is indicated as number of spores per milliliter. 8 × 10⁸ spores/ml and 3.9 × 10⁵ spores/ml are, respectively, the highest and lowest concentrations tested. P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

Figure 6

Spore PCR using the supernatant from spore suspensions of different filamentous fungi with primers ITS1/D2 (expected PCR band size is ∼1.2 kb) and ITS1/ITS4 (expected PCR band size ∼600 bp). Two different spore concentrations were tested (i.e., 5 × 10⁷/ml and 1 × 10⁷/ml). 1 µl of the supernatant was used in the PCR reaction with the LongAmp Taq DNA polymerase. Positive PCR controls were amplified from DNA (50 ng) of the A. fumigatus wild‐type strain with primers ITS1/D2 (P1) and ITS1/ITS4 (P2). N: negative control (no DNA).

Figure 7

Spore PCR using the supernatant of a spore suspension of A. niger at different concentrations. Primers ITS1/D2 and ITS1/ITS4 were used. P: positive PCR control amplified from the A. niger genomic DNA (50 ng) wild‐type strain; N: negative control (no DNA).