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Applied and Environmental Microbiology, June 2001, p. 2453-2459, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2453-2459.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

Roger H. Williams,dagger Elaine Ward,* and H. Alastair McCartney

Plant Pathology Department, IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, United Kingdom

Received 12 June 2000/Accepted 28 February 2001

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.

* Corresponding author. Mailing address: Plant Pathology Department, IACR-Rothamsted, Harpenden, Herts, AL6 2JQ, United Kingdom. Phone: 44 1582 763133. Fax: 44 1582 760981. E-mail: elaine.ward{at}bbsrc.ac.uk.

dagger Present address: Home Grown Cereals Authority, London, N1 9HY, United Kingdom.

Applied and Environmental Microbiology, June 2001, p. 2453-2459, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2453-2459.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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