Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 60, Issue 3, Pages 325-333Publisher
ELSEVIER
DOI: 10.1016/j.mimet.2004.10.010
Keywords
lactic acid bacteria; Oenococcus oeni; wine; internal control; quantitative PCR; transcription; stress
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The expression gene pattern reflects, in part, mechanisms involved in adaptation to environmental conditions. Thus, we established and validated a method that enables relative transcript quantification in different conditions in the lactic acid bacteria Oenococcus oeni, notably in a technological medium. First, we determined an internal control in our conditions by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the SYBR(R) Green I technology. Among the seven presumed housekeeping tested genes, the ldhD gene was retained for further experiments. Then, the PCR reproducibility was verified in our conditions and the comparative critical threshold (2(DeltaDeltaCT)) method was applied to quantify the transcript level of genes. The quantification of transcript levels of several stress genes already studied in our laboratory by Nor-them blot after a heat shock and at the entry of stationary phase allowed us to validate this method. RT-qPCR appeared as a powerful tool to study O. oeni response in stress conditions and wine mimetic conditions. (C) 2004 Elsevier B.V. All rights reserved.
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