Amplification Facilitators and Pre-Processing Methods for PCR Detection of Strictly Anaerobic Beer-Spoilage Bacteria of the Class Clostridia in Brewery Samples

摘要

The aim of this study was to evaluate easy pre-PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer-spoilage bacteria throughout the brewing process by end-point and real-time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end-point PCR mixture reduces the inhibiting effect of brewery sample extracts (3-10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri-polyphosphate-EDTA wash, and cross-flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one-hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a two-step centrifugation followed by physical disruption of cells. The detection limit of the procedure was 10~1-10~3 CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.