Amplification facilitators and pre-processing methods for PCR detection of strictly anaerobic beer-spoilage bacteria of the class Clostridia in brewery samples

Riikka Juvonen (Corresponding Author), Auli Haikara

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

Abstract

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 101‐103 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.
Original languageEnglish
Pages (from-to)167-176
Number of pages10
JournalJournal of the Institute of Brewing
Volume115
Issue number3
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

brewing industry
Clostridium
processing technology
vinyl compounds
Bacteria
Polymerase Chain Reaction
Polyvinyls
Pyrrolidinones
bovine serum albumin
endpoints
Bovine Serum Albumin
sampling
brewers yeast
bacteria
brewing
extracts
DNA
beers
Anaerobic Bacteria
centrifugation

Keywords

  • beer
  • brewing process
  • facilitator
  • inhibition
  • PCR detection
  • sample treatment
  • strictly anaerobic beer-spoilage bacteria

Cite this

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title = "Amplification facilitators and pre-processing methods for PCR detection of strictly anaerobic beer-spoilage bacteria of the class Clostridia in brewery samples",
abstract = "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 101‐103 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.",
keywords = "beer, brewing process, facilitator, inhibition, PCR detection, sample treatment, strictly anaerobic beer-spoilage bacteria",
author = "Riikka Juvonen and Auli Haikara",
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N2 - 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 101‐103 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.

AB - 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 101‐103 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.

KW - beer

KW - brewing process

KW - facilitator

KW - inhibition

KW - PCR detection

KW - sample treatment

KW - strictly anaerobic beer-spoilage bacteria

U2 - 10.1002/j.2050-0416.2009.tb00365.x

DO - 10.1002/j.2050-0416.2009.tb00365.x

M3 - Article

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JO - Journal of the Institute of Brewing

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