Methods for integrated air sampling and DNA analysis for detection of airborne fungal spores

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 mini ature 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 spo res to PCRs, disrupting spores (fracturing of spore walls to release the co ntents) using Ballotini beads, and disrupting spores followed by DNA purifi cation. Three P. roqueforti-specific assays were tested: single-step PCR, n ested PCR, and PCR followed by Southern blotting and probing. Disrupting th e 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, ro queforti, but this was never achieved when fewer than 1,000 spores were add ed to the PCR, By disrupting the spores, with or without subsequent DNA pur ification, it was possible to detect DNA from a single spore. When known qu antities of P, roqueforti spores were added to air samples consisting of hi gh concentrations of unidentified fungal spores, pollen, and dust, detectio n sensitivity was reduced. P. roqueforti DNA could not be detected using un treated or disrupted spore suspensions added to the PCRs. However, using pu rified DNA it was possible to detect 10 P. roqueforti spores in a backgroun d of 4,500 other spores. For all DNA extraction methods, nested PCR was mor e sensitive than single-step PCR or PCR followed by Southern blotting.