P-16
Real-time RT-PCR detection of beer-spoilage lactic acid bacteria. Reverse transcriptase PCR (RT-PCR) is a potentially sensitive, specific and
rapid in vitro technique for amplification of ribonucleic acid (RNA)
molecules. RT-PCR could also allow specific detection of viable cells, since RNA
is generally less stable in dead cells than DNA. Recently, the development of
real-time PCR technology has greatly facilitated the PCR analysis, making
simultaneous amplification, detection and quantification of target nucleic acid
molecules possible. Here we report the application of real-time RT-PCR for the
detection of RNA in beer-spoilage lactic acid bacteria (LAB) belonging to
species Lactobacillus brevis, Lactobacillus lindneri and
Pediococcus damnosus. Moreover, various methods were compared and
optimised for the isolation of RNA. Elongation transcription factor messenger
RNA (Ef-Tu mRNA) and ribosomal RNA (rRNA), which are abundantly and
constitutively expressed by metabolizing bacterial cells, were used as primer
targets. The species and group specific primers were previously published or
designed in the course of the study. The amplification and detection of
amplicons was carried out using a one-step RT-PCR kit, SyBrGreen chemistry and a
capillary-based LightCycler instrument that allows ultra-rapid thermal cycling.
A conventional RNA extraction method based on mechanical disruption of cells in
the presence of a chaotropic agent and a total RNA isolation kit from Qiagen
both produced reasonable yields of good-quality RNA from LAB. In optimal
magnesium concentration, the RT-PCR assays for rRNA and Ef-TU mRNA were able to
detect 2-6 fg and 2-6 ng purified RNA per reaction, respectively. The initial
RNA concentrations correlated well with the onset of exponential amplification
(Cp values; r > -0.98) over 3-8 log-units, confirming the suitability of the
technology for RNA quantification. The reproducibility of Cp values was good.
Preliminary experiments with beer indicated that the assays could be used to
detect the contamination of beer by LAB. The target RNAs were still detectable
in killed lactobacilli cells three weeks after a mild heat-treatment.
Experiments to study the degradation of cellular RNA after other killing
treatments are on-going. The use of a rapid-cycle real-time format simplifies
and speeds up the RT-PCR analysis and reduces the risk for false positive PCR
results. Further improvements, especially in the sample treatment step, are
needed before the methodology can be implemented in a brewery QC laboratory.
Riikka Juvonen received a MSc in Food Microbiology from the University of
Helsinki in 1997. She began employment with Technical Research Centre of Finland
in 1995 as a trainee. After her graduation she received a position as a research
scientist and was focusing on the rapid detection of beer-spoilage organisms.
Since then, she has functioned as a project leader and participated in several
national and international project. She has received supplementary courses,
e.g., on nucleic acid sequence analyses, molecular biological and microscopical
techniques, project management and communication skills. She is currently
working on her PhD degree.
RIIKKA JUVONEN, Tiina Partanen, Auli Haikara, and Teija Koivula. Technical
Research Centre of Finland, Biotechnology, P.O. Box 1500, FIN-02044 VTT,
Finland.