Evaluation of reverse-transcription PCR detection of 16S rRNA and tuf mRNA for viable/dead discrimination of beer-spoilage lactic acid bacteria

Riikka Juvonen (Corresponding Author), Tiina Partanen, Teija Koivula

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

Abstract

The aim of this study was to evaluate the feasibility of using 16S rRNA and elongation factor (tuf) mRNA as targets for selective detection of viable beer-spoilage lactic acid bacteria (LAB) by reverse-transcription PCR (RT-PCR). One-step real-time RT-PCR was applied to monitor the target transcripts in Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Three methods were compared for total RNA extraction. To study the correlation between viability and RNA detection, log-phase cultures were inactivated using brewery disinfectant or heating. RT-PCR, denaturing agarose gel electrophoresis, and cultivation were performed before and at various times after the inactivation treatments. A method combining bead-beating with traditional RNA purification provided good RNA yield from LAB. The 16S rRNA and tuf mRNA targets were detected in inactivated cells by RT-PCR throughout a 21-day storage period. Cultivable cells could not be recovered at any postinactivation time. The 16S rRNA degraded with time, and its degradation appeared to be initially faster after disinfection than after heating. The overall degradation patterns between the strains were similar. Our results indicate that tuf mRNA and 16S rRNA can be stable in LAB cells inactivated under conditions they may encounter during the brewing process. Hence, the RT-PCR analysis of these target transcripts may not be a reliable technique for selective detection of viable LAB in brewery samples.
Original languageEnglish
Pages (from-to)101-106
Number of pages6
JournalJournal of the American Society of Brewing Chemists
Volume68
Issue number2
DOIs
Publication statusPublished - 2010
MoE publication typeA1 Journal article-refereed

Fingerprint

beers
spoilage
Reverse Transcription
lactic acid bacteria
Lactic Acid
reverse transcriptase polymerase chain reaction
ribosomal RNA
Bacteria
Polymerase Chain Reaction
Messenger RNA
RNA
brewing industry
Heating
Lactobacillus lindneri
Pediococcus damnosus
Lactobacillus brevis
Pediococcus
heat
Peptide Elongation Factors
degradation

Keywords

  • Beer
  • Lactobacillus
  • Pediococcus
  • RNA
  • RT-PCR
  • viability

Cite this

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title = "Evaluation of reverse-transcription PCR detection of 16S rRNA and tuf mRNA for viable/dead discrimination of beer-spoilage lactic acid bacteria",
abstract = "The aim of this study was to evaluate the feasibility of using 16S rRNA and elongation factor (tuf) mRNA as targets for selective detection of viable beer-spoilage lactic acid bacteria (LAB) by reverse-transcription PCR (RT-PCR). One-step real-time RT-PCR was applied to monitor the target transcripts in Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Three methods were compared for total RNA extraction. To study the correlation between viability and RNA detection, log-phase cultures were inactivated using brewery disinfectant or heating. RT-PCR, denaturing agarose gel electrophoresis, and cultivation were performed before and at various times after the inactivation treatments. A method combining bead-beating with traditional RNA purification provided good RNA yield from LAB. The 16S rRNA and tuf mRNA targets were detected in inactivated cells by RT-PCR throughout a 21-day storage period. Cultivable cells could not be recovered at any postinactivation time. The 16S rRNA degraded with time, and its degradation appeared to be initially faster after disinfection than after heating. The overall degradation patterns between the strains were similar. Our results indicate that tuf mRNA and 16S rRNA can be stable in LAB cells inactivated under conditions they may encounter during the brewing process. Hence, the RT-PCR analysis of these target transcripts may not be a reliable technique for selective detection of viable LAB in brewery samples.",
keywords = "Beer, Lactobacillus, Pediococcus, RNA, RT-PCR, viability",
author = "Riikka Juvonen and Tiina Partanen and Teija Koivula",
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Evaluation of reverse-transcription PCR detection of 16S rRNA and tuf mRNA for viable/dead discrimination of beer-spoilage lactic acid bacteria. / Juvonen, Riikka (Corresponding Author); Partanen, Tiina; Koivula, Teija.

In: Journal of the American Society of Brewing Chemists, Vol. 68, No. 2, 2010, p. 101-106.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Evaluation of reverse-transcription PCR detection of 16S rRNA and tuf mRNA for viable/dead discrimination of beer-spoilage lactic acid bacteria

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AU - Partanen, Tiina

AU - Koivula, Teija

PY - 2010

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N2 - The aim of this study was to evaluate the feasibility of using 16S rRNA and elongation factor (tuf) mRNA as targets for selective detection of viable beer-spoilage lactic acid bacteria (LAB) by reverse-transcription PCR (RT-PCR). One-step real-time RT-PCR was applied to monitor the target transcripts in Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Three methods were compared for total RNA extraction. To study the correlation between viability and RNA detection, log-phase cultures were inactivated using brewery disinfectant or heating. RT-PCR, denaturing agarose gel electrophoresis, and cultivation were performed before and at various times after the inactivation treatments. A method combining bead-beating with traditional RNA purification provided good RNA yield from LAB. The 16S rRNA and tuf mRNA targets were detected in inactivated cells by RT-PCR throughout a 21-day storage period. Cultivable cells could not be recovered at any postinactivation time. The 16S rRNA degraded with time, and its degradation appeared to be initially faster after disinfection than after heating. The overall degradation patterns between the strains were similar. Our results indicate that tuf mRNA and 16S rRNA can be stable in LAB cells inactivated under conditions they may encounter during the brewing process. Hence, the RT-PCR analysis of these target transcripts may not be a reliable technique for selective detection of viable LAB in brewery samples.

AB - The aim of this study was to evaluate the feasibility of using 16S rRNA and elongation factor (tuf) mRNA as targets for selective detection of viable beer-spoilage lactic acid bacteria (LAB) by reverse-transcription PCR (RT-PCR). One-step real-time RT-PCR was applied to monitor the target transcripts in Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Three methods were compared for total RNA extraction. To study the correlation between viability and RNA detection, log-phase cultures were inactivated using brewery disinfectant or heating. RT-PCR, denaturing agarose gel electrophoresis, and cultivation were performed before and at various times after the inactivation treatments. A method combining bead-beating with traditional RNA purification provided good RNA yield from LAB. The 16S rRNA and tuf mRNA targets were detected in inactivated cells by RT-PCR throughout a 21-day storage period. Cultivable cells could not be recovered at any postinactivation time. The 16S rRNA degraded with time, and its degradation appeared to be initially faster after disinfection than after heating. The overall degradation patterns between the strains were similar. Our results indicate that tuf mRNA and 16S rRNA can be stable in LAB cells inactivated under conditions they may encounter during the brewing process. Hence, the RT-PCR analysis of these target transcripts may not be a reliable technique for selective detection of viable LAB in brewery samples.

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