Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia

Riikka Juvonen (Corresponding Author), Teija Koivula, Auli Haikara

Research output: Contribution to journalArticleScientificpeer-review

44 Citations (Scopus)

Abstract

The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism (KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 100–103 CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6–7 h and 2–3 h, respectively. Pre-PCR enrichment of beer samples for 1–3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1–2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.
Original languageEnglish
Pages (from-to)162-169
Number of pages8
JournalInternational Journal of Food Microbiology
Volume125
Issue number2
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

Fingerprint

Clostridium
beers
Restriction Fragment Length Polymorphisms
brewing industry
Real-Time Polymerase Chain Reaction
restriction fragment length polymorphism
quantitative polymerase chain reaction
Bacteria
Polymerase Chain Reaction
Zymophilus
Selenomonas
Megasphaera
Pectinatus
spoilage
methodology
sampling
brewing
melting point
DNA
spoilage bacteria

Keywords

  • beer
  • group-specific 16S rDNA based primer pair
  • PCR-RFLP
  • strictly anaerobic beer-spoilage bacteria
  • real-time PCR

Cite this

@article{b8133ae122c347d986d80c52c97af270,
title = "Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia",
abstract = "The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism (KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 100–103 CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6–7 h and 2–3 h, respectively. Pre-PCR enrichment of beer samples for 1–3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1–2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.",
keywords = "beer, group-specific 16S rDNA based primer pair, PCR-RFLP, strictly anaerobic beer-spoilage bacteria, real-time PCR",
author = "Riikka Juvonen and Teija Koivula and Auli Haikara",
year = "2008",
doi = "10.1016/j.ijfoodmicro.2008.03.042",
language = "English",
volume = "125",
pages = "162--169",
journal = "International Journal of Food Microbiology",
issn = "0168-1605",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia

AU - Juvonen, Riikka

AU - Koivula, Teija

AU - Haikara, Auli

PY - 2008

Y1 - 2008

N2 - The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism (KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 100–103 CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6–7 h and 2–3 h, respectively. Pre-PCR enrichment of beer samples for 1–3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1–2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.

AB - The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism (KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 100–103 CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6–7 h and 2–3 h, respectively. Pre-PCR enrichment of beer samples for 1–3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1–2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.

KW - beer

KW - group-specific 16S rDNA based primer pair

KW - PCR-RFLP

KW - strictly anaerobic beer-spoilage bacteria

KW - real-time PCR

U2 - 10.1016/j.ijfoodmicro.2008.03.042

DO - 10.1016/j.ijfoodmicro.2008.03.042

M3 - Article

VL - 125

SP - 162

EP - 169

JO - International Journal of Food Microbiology

JF - International Journal of Food Microbiology

SN - 0168-1605

IS - 2

ER -