Development of a Sequence-Characterized Amplified Region Marker ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2010, p. 7765–7774 0099-2240/10/$12.00 doi:10.1128/AEM.00929-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 23

Development of a Sequence-Characterized Amplified Region Marker-Targeted Quantitative PCR Assay for Strain-Specific Detection of Oenococcus oeni during Wine Malolactic Fermentation䌤 Lisa Solieri* and Paolo Giudici Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Via Amendola 2, Padiglione Besta, 42100 Reggio Emilia, Italy Received 16 April 2010/Accepted 30 September 2010

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 5ⴕ and 3ⴕ 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 ⴛ 102 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. resistance to wine conditions (19, 63). Furthermore, loss of vitality was observed when strains isolated from wines and then cultivated in the laboratory were reinoculated into wine (19). Finally, the viability and dominance of O. oeni over an indigenous LAB population can be affected by several technological factors, such as cellar operations, wine type, low temperature, nitrogen and nutrient deficiencies, high ethanol content, the presence of organic acids and sulfites, and the yeast strains used in the previous alcoholic fermentation (2, 8, 34, 45). Therefore, the selection of novel O. oeni MLS is a laborintensive and time-consuming process based on physiological characterization of strains in different harsh conditions and evaluation of their dominance of the MLF in wine (7, 23). Rapid procedures for detecting the growth of inoculated O. oeni strains during MLF might shorten the selection procedure of novel MLS, increasing the reliability of the fermentation process and the wine quality. Methods for typing O. oeni strains include the study of patterns of total soluble cell proteins (11, 13), ribotyping (58), 16S and 23S rRNA spacer region analysis (26, 64), randomly amplified polymorphic DNA (RAPD)-PCR (3, 17, 43, 62), pulsed-field gel electrophoresis (PFGE) (22, 24, 27, 51, 65), differential display PCR (25), and amplified fragment length polymorphism (AFLP) analysis (6). Being easy to use, RAPD and multiplex RAPD assays have been employed to study O. oeni population dynamics in wine and to check which strains really are responsible for MLF (43, 44, 54, 62). However, these methods show shortcomings in reproducibility and require a high number of bacterial pure cultures for analysis.

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

* Corresponding author. Mailing address: Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Via Amendola 2, Padiglione Besta, 42100 Reggio Emilia, Italy. Phone: 39 0522 522057. Fax: 39 0522 522027. E-mail: [email protected]. 䌤 Published ahead of print on 8 October 2010. 7765

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PCR-denaturing gradient gel electrophoresis (DGGE) analysis and quantitative PCR (QPCR) have been demonstrated to be useful for analyzing food microbial communities owing to species-specific detection without cultivation. These cultureindependent techniques have been targeted on protein-encoding genes rpoB and mle to monitor O. oeni in wine (38, 47, 48, 55). Because these gene sequences exhibit relatively conserved sequences among closely related species, they do not allow the discrimination of inoculated and indigenous O. oeni strains at the subspecies level. RAPD-PCR can be applied to identify sequence-characterized amplified regions (SCAR) as molecular markers in order to design species- or strain-specific primer pairs. An increasing number of studies have applied SCAR marker-based PCR assays to enumerate LAB (15, 29, 32, 40, 53, 57) and yeasts (28, 49) in vivo. In this study, we developed a new SCAR-targeted QPCR assay to quantitatively monitor the growth of a single O. oeni strain in wine. This strategy may also be applicable to other O. oeni strains for which SCAR markers can be identified, in order to investigate their viability and capacity for implantation over indigenous LAB populations during MLF. MATERIALS AND METHODS Strains and culture conditions. Sixty O. oeni strains isolated from six wine samples during spontaneous MLF (54), the commercial strain AGF115 (provided by AEB, Brescia, Italy), and four LAB reference strains obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) were used in this study. The LAB reference strains were O. oeni DSM 20052T, Leuconostoc mesenteroides subsp. mesenteroides DSM 20343T, Pediococcus pentosaceus DSM 20336T, and Lactobacillus hilgardii DSM 20176T. O. oeni strains were grown on LG agar (15 g/liter) or broth medium, as previously reported (54). In the case of other LAB species, de Man-Rogosa-Sharpe medium (Oxoid, Milan, Italy) was used. The following yeast reference strains were obtained from Centraalbureau voor Schimmelcultures (CBS; Utrecht, Netherlands): Saccharomyces cerevisiae CBS 1171T, Saccharomycodes ludwigii CBS 821T, Hanseniaspora osmophila CBS 106T, Candida stellata CBS 157T, and Dekkera bruxellensis CBS 2499. The reference strains of acetic acid bacteria (AAB) used in this study were Acetobacter aceti DSM 3508T and Acetobacter pasteurianus DSM 3509T. Yeasts and AAB were grown on YPD medium (1% [wt/vol] yeast extract, 1% [wt/vol] peptone, 1% [wt/vol] dextrose) and GYC medium (5% [wt/vol] glucose, 1% [wt/vol] yeast extract, 2% [wt/vol] calcium carbonate), respectively, at 28°C. DNA extractions. For the standard PCR assays, DNA from pure LAB cultures was extracted according to the mechanical lysis method previously described (16); DNAs from yeasts and AAB were extracted as described by Querol et al. (41) and Gullo et al. (18), respectively. For the real-time PCR experiments, DNA from wine was prepared using a Wizard genomic DNA kit (Promega, Madison, WI) according to the manufacturer’s instructions, with modifications reported by Renouf et al. (47). DNA was resuspended in 50 ␮l of sterile water, and its concentration was quantified using a Nanodrop Nd 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and then confirmed by agarose gel visualization. RAPD analysis, cloning, and sequencing. OPA20-based RAPD analysis was performed following the protocol previously described (54). The gels were stained in ethidium bromide and photographed on a UV transilluminator. Photopositives were scanned into a computer and subsequently analyzed using Bionumerics software, version 2.5 (Applied Maths, Kortrijk, Belgium). RAPDPCR patterns were grouped with the Pearson product moment correlation coefficient and the unweighted-pair group method using arithmetic average linkages (UPGMA) cluster analysis. Potential RAPD markers were excised from 1.5% agarose gels with a sterile gel slicer, and the DNA was purified using a QIAquick gel extraction kit (Qiagen, Inc., Hilden, Germany). The purified DNA fragments were then ligated into the pCR4-TOPO vector and transformed into One Shot TOP10 chemically competent Escherichia coli cells using the TOPO TA cloning method according to the manufacturer’s instructions (Invitrogen, Paisley, United Kingdom). The screening of the transformed clones was performed by PCR analysis with the primer pair T7 and T3. The inserted DNA

APPL. ENVIRON. MICROBIOL. fragments were sequenced with the same primer pair in an automatic sequencer (MWG Biotech Company, Ebersberg, Germany). To ensure the correctness of the sequences, at least two clones were sequenced. Sequences were assembled and edited using the DNAStar 4.05 software package (DNAStar, Inc., Madison, WI) and compared to the GenBank nonredundant database using the BlastN algorithm (1) from the National Center for Biotechnology Information (NCBI) network service (http://www.ncbi.nlm.nih.gov/BLAST/). The open reading frame (ORF) signatures within the SCAR sequence were detected using the NCBI ORF finder tool. Designing SCAR primers and PCR amplification. Two SCAR primers, named B391-f (5⬘-TGCGATCCAATTGTTAGTTGG-3⬘) and B391-r (5⬘-TGTTGCGA TCCCAGTTTTGA-3⬘), were designed using PRIMER 3 software (http: //primer3.sourceforge.net) and synthesized by MWG Biotech Company. The primer specificity was assessed in an amplification reaction with a final reaction mixture volume of 25 ␮l, as follows: 1⫻ Taq polymerase buffer, 200 ␮M deoxynucleotides, 2 mM MgCl2, 0.3 ␮M each SCAR primer, 0.625 U Takara Ex Taq DNA polymerase (Takara Kyoto, Japan), and 25 ng of extracted DNA. PCR amplification was performed in a Bio-Rad thermal cycler (MyCycler; Bio-Rad Laboratories) according to the following running conditions: an initial denaturation step for 5 min at 95°C, followed by 30 cycles of denaturation for 40 s at 94°C, annealing for 40 s at 55°C, and elongation for 40 s at 72°C, and a final elongation step at 72°C for 10 min. Southern blot analysis and compatible-end ligation inverse PCR (CELIPCR). DNA was digested with appropriate endonucleases (EcoRI, EcoRV, HindIII, BamHI, BglII, PvuI, and PstI; MBI Fermentas, St. Leon-Rot, Germany), electrophoresed on an 0.8% agarose gel, and analyzed by Southern hybridization according to standard procedures (50). A SCAR probe was prepared by PCR amplification using the SCAR primer pair designed as described above and labeled with digoxigenin (DIG)-labeled 11-dUTP using a DIG DNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany). Probe hybridizations were performed at 46°C. Detection was carried out by chemiluminescence using an antidigoxigenin antibody and CDP-Star (Roche Diagnostics GmbH, Mannheim, Germany) as recommended by the manufacturer. The CELI-PCR protocol (46) was modified as follows. To produce 5⬘GATC-3⬘ cohesive end fragments, 5 ␮g of DNA was double digested with 30 U of BamHI (AGATCT) and 15 U of BglII (AGATCC) in a 30-␮l reaction mixture volume for 3 h at 37°C, according to the manufacturer’s instructions (MBI Fermentas, St. Leon-Rot, Germany). DNA fragments were purified using a DNA Clean and Concentrator kit (Zymo Research, Orange, CA) and spectrophotometrically quantified. The ligation reaction was performed for 1 h at 22°C using T4 DNA ligase, as recommended by the enzyme suppliers (MBI Fermentas, St. Leon-Rot, Germany). One microliter of the ligation product was used as a template in CELI-PCR amplification with inverse primers B391-f and B391-r according to the amplification reaction protocol reported above. In the first 10 rounds of touchdown PCR, the annealing temperature range was from 66 to 56°C, followed by 30 rounds with an annealing temperature of 58°C. The CELIPCR products were ligated into the pCR4-TOPO vector, as previously reported; four plasmids were subsequently sequenced using the vector primer pair T7 and T3. The sequences were analyzed as described above. QPCR analysis. (i) QPCR primers and PCR conditions. Two nested primers named 82-f (5⬘-GGC ATT ATC CTT TGG TTG ATT CC-3⬘) and 141-r (5⬘CAG ATG CCA TGC GTG AAT TT-3⬘) were designed on the basis of the SCAR marker sequence using Primer Express software, version 2.0 (PE Applied Biosystems). Amplification and detection were performed with a 7300 fast realtime PCR sequence detection system (PE Applied Biosystems). Reactions were performed in 0.5-ml, thin-walled, optical-grade PCR tubes (PE Applied Biosystems) by adding the following components: 12.5 ␮l of 2⫻ SYBR green PCR master mix (PE Applied Biosystems), 5 ␮l of 10-fold-diluted DNA, and 150 nM each primer in a 25-␮l final volume. The thermal cycling conditions consisted of one cycle at 95°C for 10 min for the initial denaturation, followed by 40 cycles of a two-step cycling program (15 s of denaturation at 95°C and annealing and extension at 60°C for 40 s). Fluorescence was measured at the end of the annealing-extension phase of each cycle. A threshold value for the fluorescence of all samples was set manually. The reaction cycle at which the PCR product exceeded this fluorescence threshold was identified as the threshold cycle (CT). To determine the specificity of amplification, analysis of the product melting curve was performed after each amplification. Additional specificity analysis included product size verification by gel electrophoresis of samples after the QPCR assay. (ii) QPCR amplification sensitivity and efficiency. To determine the QPCR sensitivity, DNA from a selected strain was serially diluted 10-fold and amplified by QPCR as described above. A strain-specific calibration curve was obtained by plotting the CT values of the QPCR assays against the O. oeni genome copy

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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.

numbers. The O. oeni genomic equivalent was calculated according to the method of Wilhelm et al. (59) using the O. oeni genome size reported by Mills et al. (31). The amplification efficiency was estimated using the slope value according to the model of Pfaffl (36). (iii) Standard curves. Standard curves were obtained from cells inoculated both in LG medium and red wine. Three O. oeni cultures (2.2 ⫻ 108 CFU/ml) were serially diluted 10-fold in LG liquid medium, and their DNAs were extracted and amplified by the QPCR method. The same dilutions were also spread in duplicate on LG agar plates and incubated for 4 to 5 days at 30°C to obtain the number of CFUs per ml. Three aliquots of 50 ml of the red wine, purchased in a local supermarket and filtered through a 0.22-␮m membrane, were inoculated with the selected strain at the final concentration of 2.2 ⫻ 108 CFU/ml, serially diluted 10-fold, and submitted to plate counting and QPCR analysis as described above. External standard curves showing the relationships between the average CT values of QPCR and the log input cells were constructed both for LG medium and red wine cultures using linear regression analysis. Extrapolation of the regression to a CT value of 40 was used to estimate the theoretical minimum detection limit, according to the method of Brinkman et al. (5).

(iv) MLF in wine. To determine the effect of contaminating DNA on QPCR strain quantification, nonsterile and 0.22-␮m-membrane-filtered red wine (black Pinot variety) with a composition of 11.3% (vol/vol) ethanol, pH 3.3, 12.3 mg/ liter free SO2, and 4.2 g/liter L-malic acid was inoculated with the selected strain at a final concentration of 106 CFU/ml, as previously reported (54). Uninoculated and unfiltered red wine was used as a control. The wines were kept at 20°C for 40 days, and L-malate consumption was monitored with an enzymatic kit according to the manufacturer’s instructions (Boehringer, Mannheim, Germany). Ten-milliliter aliquots of each wine were collected at different times and subjected to DNA extraction and QPCR assay as described above. LAB and yeast populations were checked in uninoculated wine by serial dilution and counting by plating on LG agar or YPD medium, respectively. Statistics. Pro Fit 6.0.6 software (Quantum Soft, Switzerland) was used to fit a first-order function to the experimental data, aiming to obtain standard curves relating CT to log10 input CFU/ml. A robust algorithm was imposed as the numerical fitting method, and the goodness of fit was evaluated according to the method of Bouquet et al. (4). The reproducibility of the experimental trials was evaluated by error analysis, which was carried out through 500 fitting simulations,

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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, ␭ DNA EcoRI-HindIII Marker (MBI Fermentas, St. Leon-Rot, Germany).

assuming a P value of 0.05 and Gaussian-type error distribution for each experimental replicate. Student’s t test was used to evaluate differences among fitting parameters. Nucleotide sequence accession number. The SCAR marker sequence has been deposited in the EMBL nucleotide sequence database (www.ebi.ac.uk/embl/) under accession number FN667619.

RESULTS SCAR marker identification. To identify a strain-specific SCAR marker, we tested the OPA20 RAPD primer on 60 previously identified O. oeni strains (54). The UPGMA tree constructed by numerical comparison of the OPA20 RAPD patterns is shown in Fig. 1. Above 80% similarity, 9 of 12 clusters were formed from more than one strain, mainly isolated from the same wine sample (Fig. 1, clusters from M1 to M9). Three clusters contained only one strain, namely, LA114, LA224, and LB221. The OPA20 primer generated an approx-

imately 391-bp band specific to LB221 and absent in all other strains (Fig. 2). The 391-bp fragment represented a good candidate to generate a strain-specific SCAR marker and was cloned and sequenced (Fig. 3). The DNA consensus sequence derived from the cloned RAPD fragment contained 10-bp inverted-repeat sequences at both ends (5⬘-GTTGCGATCC-3⬘) and a G⫹C content (43%) higher than the average G⫹C content of the O. oeni PSU-1 strain chromosome (38%) (31). On the basis of this sequence, the primer pair B391-f and B391-r was designed at the ends of the RAPD fragment, containing a partial sequence of the progenitor RAPD primer plus a contiguous stretch varying from 9 to 13 bases. The SCAR primer pair was tested for specificity against all the isolated O. oeni strains, the commercial strain AGF115, and four LAB type strains, as well as against four yeasts and two AAB reference strains representative of the main oenological species. As shown by the results in Table 1, only DNA from strain LB221

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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.

was amplified using the SCAR primer pair B391-f and B391-r, resulting in an expected 391-bp PCR product (Table 1). The results indicated that the RAPD band was successfully converted into a SCAR marker and that this marker might be unique for LB221. SCAR marker characterization. To confirm whether the SCAR marker could be suitable for designing a strain-specific QPCR assay, its nucleotide sequence was submitted to BlastN homology search against the nonredundant GenBank database. The search results showed no significant match with previously determined sequences. Among the ORFs predicted by the ORF Finder tool, an ORF spanning nucleotide (nt) 51 to nt 390, encoding a 113-amino-acid sequence, showed a putative conserved domain belonging to the ArsB/NhaD permease superfamily. This peptide had an identity of 50% to diand tricarboxylate transporters (COG0471) from three Bacillus spp. (scores from 118 to 117 bits; E-values from 3e-25 to 5e-25) and P. pentosaceus (PEPE_0210; score 113 bits, E-value 8e-24). The gene PEPE_0210 belongs to the Lactobacillales-specific cluster of orthologous genes LaCOG01717. The orthologous

O. oeni PSU-1 ORF is the hypothetical protein OEOE_1170. Sequence alignment analysis showed that both the SCAR marker sequence and the OEOE_1170 sequence overlapped two different portions of PEPE_0210 (data not shown). The copy number and position of the SCAR marker were studied by Southern blotting assay and CELI-PCR (46), respectively. Southern blot hybridization of the SCAR probe to digested DNA of LB221 yielded single bands for all the endonucleases tested (Fig. 4). This finding suggests that the LB221 genome could contain a single SCAR marker or, alternatively, a cluster, including copies of this element localized nearby. To investigate this last hypothesis, DNA regions flanking the 5⬘ and 3⬘ ends of the SCAR marker were amplified with a CELIPCR protocol. The amplified DNAs were cloned and sequenced, and the sequences were aligned with the annotated O. oeni PSU-1 genome database (http://www.genome.jp /kegg-bin/show_organism?org⫽ooe). The SCAR marker was found to be between the genes lysA (OEOE_0771) and dapD (OEOE_0772), encoding diaminopimelate decarboxylase and 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyl trans-

TABLE 1. Strains tested in this study and amplification results obtained by conventional PCR and QPCR Result for: Microorganism type and species

LAB Oenococcus oeni

Leuconostoc mesenteroides Lactobacillus hilgardii Pediococcus pentosaceus

Strain(s)

LB221 DSM 20052T, LB211, LB212, LB213, LB214, LB215, LA211, LA212 LA213, LA215, LZ211, LZ212, LZ213, LZ214, LZ215, LB222, LB223, LB224, LB225, LA221, LA224, LB231, LB232, LB233, LB234, LB235, LA231, LA232, LA233, LA234, LA235 LB114, LB116, LB1110, LA113, LA1111, LB121, LB122, LB123, LB124, LB126, LB128, LB1210, LA121, LA128, LB131, LB132, LB134, LB135, LB136, LB139, LB1310, LA131, LA134, LA136, LA137, LA139, LA1310, LA1311, LA1312, AGF115c DSM 20343T DSM 20176T DSM 20336T

Yeasts Saccharomyces cerevisiae Saccharomycodes ludwigii Hanseniaspora osmophila Candida stellata Dekkera bruxellensis

CBS CBS CBS CBS CBS

AAB Acetobacter aceti Acetobacter pasteurianus

DSM 3508T DSM 3509T

a b c

1171T 821T 106T 157T 2499

⫺, absence of PCR product; ⫹, presence of PCR product (391 bp). ⫺, absence of melting curve; ⫹, presence of melting curve (Tm, 76.02°C ⫾ 0.20°C). Primer pair 82-f/141-r was used. ⫺, O. oeni commercial strain.

Conventional PCR with primer paira:

QPCRb

B391-f/B391-r

82-f/141-r

⫹ ⫺

⫹ ⫺

⫹ ⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺

⫺ ⫺

⫺

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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 CT values. CT values are the results from three replicates, and error bars (where visible) represent coefficients of variation.

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.

ferase, respectively. A second copy of the SCAR marker lay between a short-chain alcohol dehydrogenase superfamily member-coding gene (OEOE_0703) and a putative 588-bp ORF (OEOE_0704). Therefore, in silico sequence analysis and previously reported PCR results showed that SCAR markers are suitable for designing strain-specific QPCR primers. QPCR assay. Two additional primers (Fig. 3, boxes), named 82-f and 141-r, were selected to quantitatively enumerate the LB221 population dynamics in wine with a QPCR assay. Initially, the effectiveness and specificity of these primers was evaluated by conventional PCR using purified DNA from different LAB and non-LAB strains, listed in Table 1. The results showed that the primer pair was specific only to strain LB221 and had no cross-reaction against nontarget microorganisms (Table 1). Subsequently, the QPCR assay was set up using the strainspecific primers and the nonselective double-stranded DNA binding dye SYBR green. The specificity of the system was assessed by melting curve analysis. The melting curve of the PCR product obtained from LB221 had an average melting temperature (Tm) of 76.02 ⫾ 0.20°C (mean ⫾ standard deviation). No other product was detected from other strains, either with melting curves or after migration of the PCR product on agarose gel (Table 1). The sensitivity of the DNA detection was determined by QPCR amplification of 10-fold-diluted DNA from LB221. We estimated the O. oeni genomic equivalent to be 1.95 ⫻ 10⫺15 g. As shown by the results in Fig. 5, the relationship between CT values and the genome copy number was linear over 7 log cycles from 2.02 ⫻ 107 to 20 genome copies per

reaction mixture volume. The amplification plot generated a slope of ⫺3.70, with a correlation coefficient of 0.99 and an efficiency of 86%. The quantification ability of the method was tested by correlating the log values of the CFU/ml concentration estimated by plate count and QPCR. A cell suspension was serially diluted 10-fold in LB medium (trial A) and in red wine (trial B) to create samples of known concentration. The LB221 population was determined in each trial by QPCR and correlated with that found by direct plate counting (Fig. 6). Our data demonstrate a linear relationship between the population densities of strain LB221 estimated in both LG medium and red wine by QPCR and plating on LG medium, obtaining R2 values of 0.92 and 0.99, respectively. Six standard curves correlating log10 CFU/ml and CT values

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, 䡺). R2 values are 0.99 for trial A and 0.92 for trial B.

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FIG. 7. Standard curves for strain LB221 obtained from three LG cultures (trial 1, ‚; trial 2, 〫; trial 3, 䡺) and three assays in red wine (trial 4, ⫻; trial 5, Œ; trial 6, f). The error bars show the standard deviation and are smaller than the symbols where not visible.

were constructed with LB221 cell suspensions serially diluted 10-fold from 2.2 ⫻ 108 CFU/ml to 2.2 ⫻ 102 CFU/ml in both LG medium and red wine. This population range was selected according to the usual levels of O. oeni in wines. In both models (LG medium and red wine), QPCR showed successful amplification in all of the dilutions tested. The lack of goodness of fit for all six individual standard curves demonstrated the hypothesis of linearity for the relationship of CT values to log10 CFU/ml in both LG medium and red wine (Fig. 7). The assays were linear over 7 orders of magnitude, and minimum quantification limits were obtained to 2.2 ⫻ 102 CFU/ml in both LG medium and red wine (Fig. 7). Extrapolation of the regression lines to a CT value of 40 was used to estimate the theoretical minimum detection limits, which ranged from 59.79 ⫾ 0.08 to 105.22 ⫾ 0.07 CFU/ml in LG medium and from 83.96 ⫾ 0.10 to 229.85 ⫾ 0.09 CFU/ml in red wine (Table 2). The confidence intervals arising from error analysis provided high reproducibility of the experimental trials under our PCR conditions,

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allowing us to propose two predictive regression equations (RE), for LG medium and red wine (Table 2, indicated as REm and REw, respectively). The results suggest that the amplification of DNA from pure culture in LG medium and red wine was equally efficient, as evidenced by the similar slope curves during the PCR exponential phase. Monitoring of inoculated MLF by QPCR. The QPCR assay was tested in wine during MLF to evaluate the impact of large amounts of nontarget DNA coming from other wine microorganisms (LAB and yeasts) on they specificity of strain detection. For this purpose, nonsterile red wine showing an indigenous LAB population of around 104 CFU/ml and a yeast population of around 103 CFU/ml and the same red wine sterilized by filtration at 0.22 ␮m were inoculated with strain LB221 to perform MLF. The QPCR amplification was applied to DNA directly extracted from these samples, as well as from uninoculated and nonsterile wine used as a control, at different times. The CT values obtained were extrapolated from the standard curve (REw) of DNA from artificially inoculated red wine to estimate the LB221 population dynamic. As expected, uninoculated and unfiltered red wine showed neither amplification signals nor melting curves for all the times tested, confirming the QPCR strain specificity; no LAB growth or Lmalate depletion was observed (data not shown). The CT values obtained from inoculated red wines (with contaminating DNA) and 0.22-␮m-membrane-filtered red wine (without contaminating DNA) for each time are shown in Table 3. Statistical treatment using the Student t test did not show significant differences in the CT values obtained with or without contaminating DNA. In both cases, the CFU/ml values of strain LB221 remained constant, similar to the inoculum charge (ca. 106 CFU/ml), for the first 20 days of MLF and then increased slightly after 2 weeks, and finally, a decrease was observed. L-Malate consumption started 15 days after inoculation and was completed in 40 days in both wines (data not shown). DISCUSSION The goal of this study was to develop a strain-specific SCARQPCR assay to enumerate a single O. oeni strain in wine. To our knowledge, this is the first description of a culture-inde-

TABLE 2. Intercepts and slopes, amplification efficiency, and theoretical minimum CFU/ml detection limit of standard curves obtained from LG cultures and inoculated red winesa Confidence interval forb: Samples

Intercept

Slope

Ec

Minimum detection limit (CFU/ml ⫾ SD)d

Standard curves

LG medium

1 2 3 REme

46.72 ⫾ 0.16 47.52 ⫾ 0.49 47.53 ⫾ 0.14 46.85 ⫾ 1.03

⫺3.78 ⫾ 0.02 ⫺3.78 ⫾ 0.08 ⫺3.72 ⫾ 0.04 ⫺3.71 ⫾ 0.17

0.84 ⫾ 1.45 ⫻ 10⫺5 0.84 ⫾ 1.76 ⫻ 10⫺4 0.86 ⫾ 5.28 ⫻ 10⫺5 0.86 ⫾ 9.5 ⫻ 10⫺4

59.79 ⫾ 0.08 97.28 ⫾ 0.25 105.22 ⫾ 0.07 70.42 ⫾ 0.54

Red wine

4 5 6 REw

47.12 ⫾ 0.19 47.48 ⫾ 0.53 49.34 ⫾ 0.17 47.65 ⫾ 1.59

⫺3.70 ⫾ 0.04 ⫺3.83 ⫾ 0.08 ⫺3.96 ⫾ 0.02 ⫺3.76 ⫾ 0.19

0.86 ⫾ 4.62 ⫻ 10⫺5 0.82 ⫾ 1.89 ⫻ 10⫺4 0.79 ⫾ 1.07 ⫻ 10⫺5 0.85 ⫾ 1.17 ⫻ 10⫺3

83.96 ⫾ 0.10 89.76 ⫾ 0.28 229.85 ⫾ 0.09 109.08 ⫾ 0.82

A P value of ⬍0.05 was considered significant. Goodness of fit was below 10⫺6 for all calculated linear regressions. E, amplification efficiency. d Theoretical minimum detection limit was calculated at a CT value of 40. e Regression equations (RE) were estimated by simultaneously fitting data from the three replicates in LG medium (REm) and red wine (REw), respectively. a b c

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TABLE 3. Comparison of mean CT values obtained by QPCR from wines inoculated with strain LB221 with and without nontarget wine microorganismsa CT (mean ⫾ SD) value with: Time (days)

0 3 5 7 11 13 21 23 27 30 32 34 37 40 a

LB221 and nontarget DNA

LB221 DNA only

24.19 ⫾ 0.02 23.72 ⫾ 0.53 24.07 ⫾ 0.12 24.12 ⫾ 0.01 24.05 ⫾ 0.11 23.71 ⫾ 0.11 23.60 ⫾ 0.12 23.63 ⫾ 0.07 23.05 ⫾ 0.14 23.04 ⫾ 0.12 22.65 ⫾ 0.32 22.76 ⫾ 0.04 22.92 ⫾ 0.05 24.22 ⫾ 0.15

24.25 ⫾ 0.10 23.71 ⫾ 0.52 24.07 ⫾ 0.11 24.12 ⫾ 0.01 24.06 ⫾ 0.12 23.72 ⫾ 0.12 23.57 ⫾ 0.11 23.63 ⫾ 0.07 23.06 ⫾ 0.15 22.91 ⫾ 0.02 22.74 ⫾ 0.25 22.86 ⫾ 0.21 22.97 ⫾ 0.12 24.18 ⫾ 0.02

A P value of ⬍0.05 was considered significant.

pendent QPCR detection system that accomplishes fast O. oeni typing during MLF at strain level. MLF is one of the most difficult steps to control in winemaking (19, 34). Several studies have provided evidence that the ability of a selected O. oeni strain to take over spontaneous MLF is affected by several microbiological and technological factors which should be evaluated in a screening program for selecting new MLS (2, 8, 23). These analyses require reliable and rapid detection systems for strain discrimination. Traditionally, the time-consuming procedures of isolation by plating, species identification, and typing by PCR-based methods are used for monitoring O. oeni population dynamics during MLF (7, 54). Recently, several culture-independent methods have been developed and applied to quantify wine yeasts (10, 20, 21, 28, 37, 42, 56) and LAB (10, 33, 38, 45, 55, 66), but these techniques do not permit the direct monitoring of a selected strain in wine at the subspecies level. Strain-specific SCAR markers can be developed from PCR-based typing technologies, such as RAPD, AFLP, simple sequence repeat-PCR (SSR-PCR), and inter-simple sequence repeat-PCR (ISSR-PCR). Due to increased specificity, SCAR amplifications are less sensitive to changes in reaction conditions and are more reproducible than traditional typing methods. Strain-specific SCAR markers have been widely and successfully used to monitor bacteria (14, 39, 61) and yeasts (9, 30) in environmental samples. Recently, PCR-based methods with strain-specific primer sets have been set up for quantification of Lactobacillus probiotics in feces (12, 15, 29). In contrast, no SCAR marker was developed for detecting O. oeni strains in wine. We used the OPA20-RAPD technique to find differentially amplified fragments for the SCAR primer design. A 391-bp RAPD fragment differentiated LB221 from the other O. oeni strains tested and was selected as the target for tracking the fate of this strain in wine. Two primer pairs designed in this study enabled the strain-specific detection of LB221 and failed to amplify DNA from other wine microorganisms. BlastNbased sequence analysis showed no significant nucleotide sequence homology of the SCAR marker with any other known database sequence, further confirming the strain specificity.

Additionally, the in silico-translated SCAR sequence, comprised of nucleotides 51 to 390, had an identity of 50% with the amino terminal portion of the multidomain di- and tricarboxylate transporter PEPE_0210 from P. pentosaceus ATCC 25745. The in silico data and CELI-PCR and Southern blot results suggested that the SCAR marker could be a nonfunctional sequence present in two copies in the LB221 genome. SCAR-based QPCR assays provide the advantage of combining strain level resolution with in vivo quantitative detection. The standard curve constructed using the CT values obtained for DNA concentrations ranging from 2.02 ⫻ 107 to 20 genome equivalents per reaction mixture volume showed good linearity, confirming the suitability of the assay for the quantification of strain LB221. However, wine is a complex medium that contains various PCR inhibitors, such as polyphenols and tannins, which can produce false-negative results (60). Therefore, the inhibitory effect is stronger in red wines than in white wines (37). When the sensitivity of the QPCR method was tested using calibrated cell suspensions in both LG medium and red wine, regression curves correlating the CT values and the corresponding amount of template (log10 input CFU/ml) demonstrated the quantification linearity over seven orders of magnitude. As a result, the quantification was accurate in the range corresponding to 102 to 108 CFU/ml. Moreover, DNA amplification from LG medium cultures and inoculated red wines was equally efficient, as evidenced by the similar curve slopes during the exponential phase of the QPCR. This finding indicates that the wine had no significant impact on DNA extraction and subsequent QPCR performance. In contrast, other authors have reported differences when PCR was performed with DNA extracted from pure cultures or directly from wine (10, 33, 38). The quantification limit for the QPCR assay was around 220 CFU/ml, which is greater than that obtained for other quantitative assays in wine (33, 38). However, the minimum quantification cell number falls significantly below the amount of MLS used to start up MLF, i.e., between 104 and 106 CFU/ml, confirming the suitability of the method for monitoring strain dominance in inoculated wine. A shortcoming of the QPCR-based system could be that it detects DNA from both live and dead bacterial cells. This problem could be addressed by quantitative real-time reverse transcription-PCR (qRT-PCR), starting from mRNA (52). An easy-to-use alternative to the RNA-based quantification methods could be the DNA-intercalating dye ethidium monoazide bromide, which inhibits PCR amplification of DNA from dead cells (35), but this has not yet been further tested on LAB. In the QPCR assay developed in this work, the significant correlation between the results of the QPCR assay and those of the conventional plating method, occurring both with pure cultures and wine samples, proved the reliability of this protocol for the quantification of viable O. oeni populations. When testing the QPCR-based detection system on inoculated MLF in nonsterile red wine, the changes in the strain LB221 population were selectively monitored, while no amplification signal was detected in uninoculated wine. Comparison of the average CT values gained from nonsterile and sterile wines indicated that the presence of competing nontarget DNA from yeasts and other LAB species does not affect the quantitative strain enumeration. The results further confirmed that the method enables high-throughput quantitative analysis

VOL. 76, 2010

DETECTION OF O. OENI BY STRAIN-SPECIFIC QPCR

of a single O. oeni strain in wine contaminated with other LAB and yeast species. Being accurate and rapid, the direct detection of an inoculated O. oeni strain by the SCAR-based QPCR method described herein provides a significant advantage over culturing and RAPD typing of isolates for monitoring the MLF at strain level. The method might be an effective platform to develop similar strain-specific quantitative detection systems for O. oeni MLS. We propose the application of this strategy to track inoculated strains in wine and to assist in the selection of novel, more effective O. oeni starters in winemaking. ACKNOWLEDGMENTS We are most grateful to Luciana De Vero for her technical assistance in the management of the O. oeni culture collection and to Chiara Nicoletti for providing bacterial DNA. We thank Stefano Cassanelli for helpful discussion of the manuscript and Pasquale M. Falcone for assistance in statistical analysis.

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