Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Abstract

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (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 a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly 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 strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a 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.

FIG. 1.

Dendrogram based on UPGMA clustering with Pearson coefficient of OPA20-RAPD patterns from 60 O. oeni strains isolated from wines during spontaneous MLFs. Percent similarity is indicated at the branches; the dotted vertical line indicates the 80% similarity value for delineating 12 clusters.

FIG. 2.

OPA20-RAPD patterns obtained from LB221 and selected O. oeni strains. The arrow indicates a 391-bp RAPD fragment specific to LB221. Lanes: 1, LB221; 2, LB222; 3, LB224; 4, LB224; 5, LB225; 6, LA221; 7, LA224; 8, LB231; 9, LB232; 10, LB233; M, λ DNA EcoRI-HindIII Marker (MBI Fermentas, St. Leon-Rot, Germany).

FIG. 3.

Nucleotide sequence of the 391-bp OPA20-RAPD band obtained from strain LB221 (DDBJ/GenBank/EMBL accession number FN667619). Strain-specific primers (B391-f and B391-r) used for the first specificity assay by conventional PCR are underlined. Strain-specific nested primers (82-f and 141-r) used for QPCR assay are boxed.

FIG. 4.

Hybridization signals with the SCAR probe after digestion of LB221 DNA with the seven restriction enzymes and Southern blotting. Lanes 1 to 7 contained DNA digested with EcoRI, EcoRV, HindIII, BamHI, BglII, PvuI, and PstI, respectively.

FIG. 5.

Logarithmic-linear plot of the PCR assay amplification profile for 10-fold-diluted LB221 DNA. The number of log₁₀ genome copies is plotted versus C_T values. C_T values are the results from three replicates, and error bars (where visible) represent coefficients of variation.

FIG. 6.

Linear relationship between LB221 population densities (CFU/ml) detected by QPCR and by plating after serial dilution in LG medium (trial A, ⧫) and red wine (trial B, □). R² values are 0.99 for trial A and 0.92 for trial B.

FIG. 7.

Standard curves for strain LB221 obtained from three LG cultures (trial 1, ▵; trial 2, ⋄; trial 3, □) and three assays in red wine (trial 4, ×; trial 5, ▴; trial 6, ▪). The error bars show the standard deviation and are smaller than the symbols where not visible.