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

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.Conventional methods for identifying and enumerating airborne fungi and other microorganisms rely on microscopic or cultural techniques and, as a consequence, are time-consuming and laborious. Additionally, microscopy is unreliable for detection of the small, nondescript spores produced by many fungi, while cultural techniques are unsuitable for detection of spores that are slow growing, or nonculturable in vitro and the choice of medium may influence which species can grow. These difficulties have restricted the use of routine air sampling in the study of plant, animal, and human diseases.Recently, however, molecular methods have been used in the development of diagnostic tests for a variety of fungi involved in plant diseases (8,25,26,28,30). While the potential of these techniques for detection of airborne spores has been recognized for some time (12,14), there have been few reports on progress in this area. The use of immunoassay in the detection of airborne plant pathogenic fungi has been investigated (10,20,21). However, the application of these methods is restricted by the difficulties of developing antibodies showing the required specificity. DNA-based detection methods offer greater potential for sensitive and specific detection, and some progress has been made in the detection of airborne bacteria using these techniques (1, 2, 9, 15, 17). Progress with fungi has been slower, although dete...

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