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Updated: Jul 31, 2026
Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA
Published on: May 2, 2018
Methods for integrated air sampling and dna analysis for detection of airborne fungal spores
R H Williams1, E Ward, H A McCartney
1Plant Pathology Department, IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, United Kingdom.
Abstract:
Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.
Insights
Detecting airborne fungal spores like Penicillium roqueforti requires spore disruption for sensitive PCR analysis. Nested PCR combined with DNA purification offers the highest sensitivity for airborne fungal spore detection.
Area of Science:
- Environmental microbiology
- Molecular biology
- Aerobiology
Background:
- Airborne fungal spores pose health risks and impact indoor/outdoor environments.
- Accurate detection of specific fungal species in air samples is crucial for risk assessment.
- Current methods often lack sensitivity or require extensive sample processing.
Purpose of the Study:
- To develop and optimize integrated air sampling and PCR-based methods for detecting airborne fungal spores.
- To evaluate the impact of different DNA extraction methods on PCR sensitivity.
- To compare the efficacy of various PCR assays for fungal spore identification.
Main Methods:
- Air sampling using a miniature cyclone-type sampler to collect Penicillium roqueforti spores.
- Comparison of three DNA extraction methods: untreated spores, disrupted spores, and purified DNA.
- Evaluation of three PCR assays: single-step PCR, nested PCR, and PCR with Southern blotting.
Main Results:
- Spore disruption was essential for sensitive PCR detection, enabling detection of single spores.
- Nested PCR demonstrated higher sensitivity than single-step PCR or Southern blotting.
- Detection sensitivity decreased in complex air samples, but purified DNA allowed detection of 10 spores in a mixed background.
Conclusions:
- Integrated air sampling and PCR, particularly with spore disruption and nested PCR, provide a sensitive method for detecting airborne fungal spores.
- DNA purification is critical for maximizing sensitivity in complex environmental samples.
- The developed method offers improved detection capabilities for airborne fungal contaminants.
