RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples.
Recently we showed the applicability and sensitivity of the RNA-based sandwich hybridisation assay (SHA) for detection of gram-negative cells in environmental samples [Leskelä, T., Tilsala-Timisjärvi, A., Kusnetsov, J., Neubauer, P., Breitenstein, A., 2005. Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay. J. Microbiol. Met. 62, 167-179.]. In this study the aim was to test and optimise this method for the detection of gram-positive cells from brewery yeast slurries that contain up to 10(9) yeast cells/ml. Eleven new oligonucleotide probes were designed for group-specific detection of different beer-spoiling lactic acid bacteria of the genera Lactobacillus and Pediococcus. Functionality of the designed probes was shown by testing individual and paired probes using in vitro transcribed 16S rRNA and crude cell extracts as samples. Various simple and fast cell disruption methods were evaluated for the efficient disruption of lactobacilli and pediococci. The applicability of the designed oligonucleotide probes and the SHA for detection of brewery contaminants was demonstrated using both artificial and actual yeast slurry samples from brewery fermentation tanks with either fluorimetric readout or an electric biochip analyser.
Huhtamella
Sanna
S
Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, Biocenter Oulu, University of Oulu, P.O. Box 4300, FI-90014 Oulu, Finland.
Leinonen
Marika
M
Nieminen
Timo
T
Fahnert
Beatrix
B
Myllykoski
Liisa
L
Breitenstein
Antje
A
Neubauer
Peter
P
D001515:Beer / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D005285:Fermentation; D005506:Food Contamination / Q000032:analysis; D007778:Lactobacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D010376:Pediococcus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S