Real-time PCR detection of bacteria belonging to the Firmicutes Phylum

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.