Journal
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
Volume 125, Issue 3, Pages 236-241Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijfoodmicro.2008.04.002
Keywords
beer-spoilage bacteria; Firmicutes; Phylum-specific detection; real-time PCR
Categories
Funding
- Natural Science and Engineering Research Council of Canada
- Molson Coors Brewing Company, Golden, Co
- University of Saskatchewan
- College of Medicine
- American Society of Brewing Chemists Foundation Coors Brewing Company
- Cargill Malt Scholarships
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Members of the bacterial Phylum Firmicutes occupy a wide range of habitats and can be either beneficial or detrimental in diverse settings, including food- and beverage- related industries. Firmicutes are responsible for the vast majority of beer-spoilage incidents and, as Such, they have a substantial financial impact in the brewing industry. Rapid detection and identification of a bacterium as a Firmicutes is difficult due to widespread genetic transfer and genome reduction resulting in phenotypic diversity in these bacteria. Here we describe a real-time multiplex PCR to detect and differentiate Firmicutes associated with beer-spoilage from non-Firmicutes bacteria that may be present as benign environmental contaminants. A region of the 16S rRNA gene was identified and predicted to be highly conserved amongst, and essentially specific for, Firmicutes. A real-time PCR assay using a hydrolysis probe targeting this region of the 16S rRNA gene was experimentally shown to detect ten genera of Firmicutes known to be beer spoilers, but does not cross-react with eleven of twelve non-Firmicutes genera which can periodically appear in beer. Only one non-Firmicutes species, Zymomonas mobilis, weakly reacted with the Firmicutes probe. This rPCR assay has a standard curve that is linear over six orders of magnitude of DNA, with a quantitation limit of DNA from < 10 bacteria. When used to detect bacteria present in beer, the assay was able to detect 50-100 colony forming units (CFU) of Firmicutes directly from 2.5 cm membranes used to filter 100 ml of contaminated beer. Through incorporation of a 4.7 cm filter and an overnight pre-enrichment incubation, the sensitivity was increased to 2.5-10 CFU per package of beer (341 ml). When multiplexed with a second hydrolysis probe targeting a universal region of the 16S rRNA gene, the assay reliably differentiates between Firmicutes and non-Firmicutes bacteria found in breweries. (c) 2008 Elsevier B.V. All rights reserved.
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