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.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Williams RH,Ward E,McCartney HAdoi
10.1128/AEM.67.6.2453-2459.2001keywords:
subject
Has Abstractpub_date
2001-06-01 00:00:00pages
2453-9issue
6eissn
0099-2240issn
1098-5336journal_volume
67pub_type
杂志文章abstract::The bacterium Rhodopseudomonas palustris grows with the aromatic acid benzoate and the alicyclic acid cyclohexanecarboxylate (CHC) as sole carbon sources. The enzymatic steps in an oxygen-independent pathway for CHC degradation have been elucidated, but it was unknown how the CHC operon (badHI aliAB badK) encoding the...
journal_title:Applied and environmental microbiology
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journal_title:Applied and environmental microbiology
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
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journal_title:Applied and environmental microbiology
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journal_title:Applied and environmental microbiology
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更新日期:1983-01-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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更新日期:2006-02-01 00:00:00
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更新日期:1987-08-01 00:00:00
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更新日期:2013-04-01 00:00:00
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更新日期:1999-11-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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更新日期:1990-03-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.59.7.2161-2165.1993
更新日期:1993-07-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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更新日期:2006-12-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.64.10.3718-3723.1998
更新日期:1998-10-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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更新日期:1999-04-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.42.2.187-191.1981
更新日期:1981-08-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.62.8.3053-3056.1996
更新日期:1996-08-01 00:00:00