title: Real-time PCR detection of bacteria belonging to the Firmicutes Phylum.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, 2841 Royal University Hospital, Saskatoon SK, Canada S7N 0W8.
authors.forename: M
authors.initials: M
authors.lastname: Haakensen
authors.forename: C M
authors.initials: CM
authors.lastname: Dobson
authors.forename: H
authors.initials: H
authors.lastname: Deneer
authors.forename: B
authors.initials: B
authors.lastname: Ziola
mesh_terms: D001515:Beer / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D005374:Filtration; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S