Development of a Sequence-Characterized Amplified Region Marker-Targeted Quantitative PCR Assay for Strain-Specific Detection ofOenococcus oeniduring 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.Malolactic fermentation (MLF) is a secondary fermentation which decreases the acidity, enhances the sensorial properties, and increases the microbiological stability of wine (23). Often, this step occurs naturally after completion of alcoholic fermentation. However, when MLF is carried out by indigenous lactic acid bacteria (LAB), the process can be unpredictable and start randomly, many months after the end of alcoholic fermentation, leading to wine spoilage and the production of biogenic amines. Moreover, when Lactobacillus and Pediococcus species are responsible for spontaneous MLF, the wine quality decreases due to the production of off-flavor (8, 23).To overcome these drawbacks, Oenococcus oeni malolactic starters (MLS) were used owing to their ability to successfully withstand multiple adverse wine conditions and to produce well-balanced wine (8, 34). Although progress has been made in selecting and preparing MLS, the induction of malolactic fermentation (MLF) by direct inoculation with selected O. oeni strains is not always guaranteed (19). Several factors contribute to the unpredictable nature of inoculated MLF. O. oeni is known to be a fastidious, slow-growing bacterium (23), auxotrophic for several amino acids, while other amino acids are needed for optimal growth (19,23). This species is highly heterogeneous, with a considerable intraspecific variation in resistance to wine conditions (19,63). Furthermore, loss of vitality was observed when strains isolated from wines and then cultivated in the laboratory were ...

Multiplex variable number of tandem repeats for Oenococcus oeni and applications

“…This has been confirmed by DNAeDNA homology levels and the similarities observed in gene maps of different strains (Dicks et al, 1990;Zé-Zé et al, 2000). O. oeni strains may be differentiated by several DNA fingerprint profiles, such as: restriction endonuclease analysis-pulsed-field gel electrophoresis (REA-PFGE), patterns of low-frequency restricted genomes (Kelly et al, 1993;Gindreau et al, 1997;Larisika et al, 2008), random amplification of polymorphic DNA (RAPD) (Reguant and Bordons, 2003;Solieri and Giudici, 2010), amplified fragment length polymorphism (Cappello et al, 2008), or ribotyping analyses (Zavaleta et al, 1997;de las Rivas et al, 2004). The assumption of genetic homogeneity was repeatedly challenged by the detection of groups of strains, often two major groups with distinct molecular and/or phenotypic/metabolic characteristics, leading authors to suggest that O. oeni may have two subspecies (Peynaud and Domercq, 1968;Tenreiro et al, 1994).…”

Development of a SCAR (Sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum

“…The designed primers for the SCAR contained the complete sequence of the original RAPD primer S116 plus additional nucleotides. Considering SCAR primers are longer than RAPD primers, higher annealing temperatures will be required, and the amplification of a single fragment can be directed with a specific size from the target DNA (Solieri and Giudici 2010). The optimized SCAR was less sensitive than RAPD to various reaction conditions.…”

Detection and identification of microorganisms in wine: a review of molecular techniques

“…Connell et al [22] adapted this same technique for the detection of D. bruxellensis in winery air. [5,22,40,55,113] Yeast targets Direct Species to strain [18,49,61] QPCR Bacterial targets Direct Currently species but depending on the target gene they may be strain specific [45,78,106] Yeast targets Direct Currently species [27,45,47,89,93,108] Both papers consider this method to be a form of in situ hybridization, however, in this case it is not a direct detection method, as the probes are used on an enriched culture grown on media. Even though the technique takes less time than traditional indirect methods, growth of the organism is still needed.…”

“…Ruiz et al [99] used RAPD-PCR to determine which of 22 strains isolated from a Spanish winery were best able to implant and conduct malolactic fermentation (MLF). Finally, Solieri et al [106] isolated and sequenced a band created via RAPD-PCR to create a QPCR method to follow a specific O. oeni strain during the course of the fermentation. The method works well for bacteria, but as mentioned earlier, MLST appears to be more discriminatory [26].…”