Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 162-169 |
| Number of pages | 8 |
| Journal | International Journal of Food Microbiology |
| Volume | 125 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2008 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- beer
- group-specific 16S rDNA based primer pair
- PCR-RFLP
- strictly anaerobic beer-spoilage bacteria
- real-time PCR
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Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia. / Juvonen, Riikka (Corresponding Author); Koivula, Teija; Haikara, Auli.
In: International Journal of Food Microbiology, Vol. 125, No. 2, 2008, p. 162-169.Research output: Contribution to journal › Article › Scientific › peer-review
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 -