Development of SCAR marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation.

Control over malolactic fermentation (MLF) is a hard goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starter (MLS) in a stressful environment such as wine. In this study we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based random amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled the strain-specific detection without cross amplification of other O. oeni strains and wine species of lactic acid bacteria (LAB), acetic acid bacteria and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detects as low as 2.2 x 10(2) CFU per ml of red wine, with good quantification effectiveness, as shown by correlation of QPCR and plate counting. Therefore the cultivation-independent monitoring of a single O. oeni strain in wine based on SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF