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Journal of Microbiological Methods 42 (2000) 175–184www.elsevier.com/ locate / jmicmeth

An evaluation of chelex-based DNA purification protocols for thetyping of lactic acid bacteria

*Giorgio Giraffa , Lia Rossetti, Erasmo NevianiIstituto Sperimentale Lattiero Caseario, Via A. Lombardo 11, 26900 Lodi, Italy

Received 6 March 2000; received in revised form 3 May 2000; accepted 30 May 2000

Abstract

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprintingexperiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA bychelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCRfingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA sampleswhich, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme andphenol–chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure wasverified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol–chloroform extraction.Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array oforganisms and/or biological materials. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Bacterial taxonomy; Diagnostic microbiology; DNA extraction; Polymerase chain reaction (PCR) fingerprinting; Lactic acidbacteria

1. Introduction trace within the system. Typing of individual strains,together with more sensitive tools for species identi-

Lactic acid bacteria (LAB) play an important role fication, represent a way to overcome this problemin the human and animal gastrointestinal tract as well (Dykes and von Holy, 1994).as in the production of many foods, feeds, and Development of molecular biology techniques hasbeverages (Stiles and Holzapfel, 1997). In many improved the knowledge on the taxonomy andfermented foods microbial starters are composed of ecology of LAB and other bacteria, while opening aartisan cultures, which are mixed microbial com- new interdisciplinary field, the molecular microbialmunities of LAB. All of these systems are complex ecology, to study microbial communities. Nowadays,and usually consist of mixed consortia of lactobacilli, molecular techniques provide an outstanding tool forthe individual components of which are difficult to microbial detection, identification, and typing. Sever-

al PCR-based DNA fingerprinting methods, includ-ing rDNA-RFLP analysis (or ARDRA), rDNA spacerlength polymorphism analysis, and arbitrarily-primed*Corresponding author. Tel.: 139-371-45011; fax: 139-371-PCR (or RAPD) have been claimed to produce35579.

E-mail address: [email protected] (G. Giraffa). species-specific and strain-specific fingerprints in

0167-7012/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0167-7012( 00 )00172-X

176 G. Giraffa et al. / Journal of Microbiological Methods 42 (2000) 175 –184

food-associated LAB (Klein et al., 1998; Tailliez et 2. Materials and methodsal., 1996, 1998; Moschetti et al., 1997, 1998; Van-damme et al., 1996; Van Reenen and Dicks, 1996). 2.1. Bacterial strains and growth conditionsBecause of the vast array of PCR-fingerprintingtechniques which have been described during past Strains of LAB used in this study are listed indecades, computer-assisted comparisons have re- Table 1. Bacterial strains were maintained as frozenplaced visual comparison of PCR-generated finger- stocks at 2 808C in the presence of 150 ml / lprints (Vauterin and Vauterin, 1992). These ad- glycerol as cryoprotective agent. Working culturesvances, coupled with the progressive introduction of were prepared through three transfers in MRS brothsimple and rapid methods of extracting DNA suitable medium (Biokar, Beauvais, France). Strains werefor PCR amplification from a variety of media, have cultivated at 308C (for Lactococcus lactis subsp.undoubtedly facilitated the diagnostic microbiology lactis and Lactobacillus plantarum) and 378C (forthus allowing the rapid screening of several strains Lactobacillus helveticus, Lactobacillus delbrueckiiand the more effective ecological studies of popula- subsp. bulgaricus, and Lactobacillus delbrueckiition dynamics and typing of LAB in complex food subsp. lactis) for 16–18 h. Purity was checked bysystems to be undertaken (Cocconcelli et al., 1995; plating on MRS agar medium and microscopicDrake et al., 1996; Vaneechoutte and van Eldere, examination. Confirmation of classification from1997). purified cultures before DNA extraction was per-

In the present study we compared different proto- formed by species-specific PCR according to prim-cols for rapid extraction of DNA from LAB culture ers, methods, and amplification conditions previouslylysates for use in PCR fingerprinting experiments by described (Table 1).chelex (iminodiacetid acid) ionic resin. Chelex is anionic resin that can bind compounds which inhibit 2.2. Phenol–chloroform extraction of total DNAPCR (Singer-Sam et al., 1989). Compatibility, re-peatability, reproducibility, and consistency of the Approximately 100–200 ml of each strain grown

7PCR-fingerprinting patterns obtained by one chelex- overnight in MRS broth ( | 10 CFU) were pelletedbased DNA extraction protocol were evaluated and by centrifugation at 12,500 3 g for 5 min. Thecompared with the classical phenol–chloroform pellets were washed twice with sterile water, orDNA extraction procedure. TE buffer (10 mM Tris–HCl–0.1 mM EDTA, pH0.1

Table 1Strains investigated in the present study

aOrganism Collection number PCR identification

Expected amplicons References(size in bp)

TLactococcus lactis subsp. lactis ATCC 19435 933 Corroler et al. (1998)Lactococcus lactis subsp. lactis ISLCPT5 (cheese isolate) 933 Corroler et al. (1998)

TLactobacillus helveticus ATCC 15009 About 200 Tilsala-Timisjarvi and Alatossava (1997)Lactobacillus helveticus LH30 (cheese starter) About 200 Tilsala-Timisjarvi and Alatossava (1997)

TLactobacillus delbrueckii subsp. bulgaricus ATCC 11842 1065 Torriani et al. (1999)Lactobacillus delbrueckii subsp. bulgaricus LB2 (yoghurt) 1065 Torriani et al. (1999)

TLactobacillus delbrueckii subsp. lactis ATCC 12315 1600 Torriani et al. (1999)Lactobacillus delbrueckii subsp. lactis LL5 (cheese whey) 1600 Torriani et al. (1999)

TLactobacillus plantarum ATCC 14917 250 Quere et al. (1997)a A superscript T denotes a type strain; the other strains come from the collection of Istituto Sperimentale Lattiero Caseario of Lodi, Italy

(sources of isolation are indicated in parentheses).

G. Giraffa et al. / Journal of Microbiological Methods 42 (2000) 175 –184 177

8.0), in a clean 1.5-ml microcentrifuge tube and same quantity of DNA from different DNA ex-repelleted by centrifugation. Total DNA was extraction procedures to each PCR reaction tube. DNAtracted from washed cell pellets by a standard integrity (e.g. no degraded DNA) before PCR andalkaline lysis method. Washed cell pellets were after quantification was visualised by agarose gelresuspended in 500 ml of TES buffer (50 mM Tris– electrophoresis in the presence of DNA mass lad-HCl–1 mM EDTA–6.7% saccharose, pH 8.0). Fol- ders.lowing addition of 10 mg of lysozyme (Sigma-Aldrich, Milan, Italy), tubes were incubated at 378C 2.5. PCR fingerprinting amplificationfor 30 min. In a second step, 125 ml of sodiumdodecyl sulfate (20%) were added, followed by an Total DNA from different strains was used as aincubation at 378C for 15 min. DNA was extracted template for PCR fingerprinting using as a primer thethree times with equal volumes of phenol and M13 minisatellite core sequence (Huey and Hall,chloroform and precipitated with cold (2208C) 1989) with sequence 59-GAGGGTGGCGGTTCT-39.isopropanol. After centrifugation at 12,500 3 g for Amplification conditions consisted of an initial de-30 min at 48C, 1 ml of ETOH (70%) was added to naturation step of 948C for 120 s, followed by 40pelleted DNA. After a brief spin at 12,500 3 g for 5 cycles of: 948C for 60 s, 458C for 20 s, and 728C formin at 48C, purified DNA was dried and resuspended 120 s; a final elongation step of 728C for 10 min wasovernight at 48C in 150 ml of TE buffer (10 mM performed. PCR was performed in 50-ml amplifica-Tris, 1 mM EDTA, pH 8.0). Ribonuclease A (10 tion mixtures in a Perkin Elmer, mod. 9700 thermob-mg/ml; Sigma-Aldrich) was added to resuspended lock (Perkin Elmer) with 50 mM Tris–HCl (pH 8.3),DNA and, after incubation at 378C for 1 h, DNA 2.0 mmol / l of primer (Celbio, Milan, Italy), 2.5samples were briefly stored at 2 208C until use. ng /ml of total DNA (i.e. between 10 and 20 ml of

chelex-based DNA preparations), 3.0 mM MgCl ,2

2.3. Chelex-based extraction of total DNA 1.25 U of Taq polymerase (Perkin Elmer), and 200mM of each dNTP. PCR profiles were visualised

Total DNA from washed cell pellets of each strain after overnight electrophoresis (1.5 V/cm) in Seakemwas extracted by three chelex-based procedures GTG agarose gels (1.5% w/v; FMC Bio Products,according to: (A) the DNA extraction protocol SPA, Milan, Italy) and staining with ethidium bro-described for Lb. helveticus by Drake et al. (1996) mide. A 1-kbp plus DNA Ladder (Life Technologiesand using as the chelex reagent that sold from Sigma Italia, Milan, Italy) was used as a DNA molecular(Sigma-Aldrich); (B) the protocol described for the weight marker.preparation of genomic DNA from bacteria using asthe extraction reagent the InstaGene matrix (Bio-Rad 2.6. Data analysisLaboratories, Milan, Italy); and (C) the method forGram positive (and acid-fast) bacteria described in The photographs of the gels were scanned (Scanjetthe MicroSeqE protocol (Perkin Elmer, Monza, 6100 C/T, Hewlett Packard, Milan, Italy), and theItaly) which uses the DNA extraction reagent in- resulting densitometric traces of the band profilescluded in the kit. In Fig. 1 are summarized the were analyzed with the pattern analysis softwareessential steps of the three chelex-based protocols package GelCompar Version 4.1 (Applied Maths,and the phenol–chloroform extraction procedure. Kortrjik, Belgium). Calculation of similarity of the

PCR fingerprinting profiles was based on the Pearson2.4. Evaluation of quantity and purity of DNA product-moment correlation coefficient. The Pearson

correlation coefficient, which provides similarityQuantity and purity of DNA samples from alkaline based upon densitometric curves, was chosen be-

extraction procedures were checked by optical read- cause it is generally more appropriate to evaluateing at 230, 260, 280, and 320 nm, as described by similarities between PCR fingerprinting profiles. ASambrook et al. (1989). This enabled us to add the dendrogram was deduced from the matrix of simi-


Fig. 1. Schematic presentation of the four protocols, i.e. the three chelex-based methods and the phenol–chloroform method, used for DNAextraction.


larities by using the unweighted pair group method the final strain identification is only reliable if theusing arithmetic average (UPGMA) clustering algo- variations caused by differences in growth con-rithm (Vauterin and Vauterin, 1992). The quality of ditions and DNA extraction parameters are foundthe cluster analysis (i.e. the consistence of the to be within the ranges of repeatability andclusters) was verified by calculating the cophenetic reproducibility of the amplification conditions andcorrelation value (in percent) for each dendrogram DNA stability evaluated previously (Pot et al.,using the GelCompar software. This value, which is 1994). Consistency was estimated by runningcalculated for the whole dendrogram, provides an DNA samples from amplifications of duplicatedestimation of the faithfulness of a cluster analysis by DNA extracts of each strain to determine overlap.calculating the correlation between the dendrogram- Duplicated DNA extracts (DDE) came from twoderived similarities and the matrix similarities. The different cultures of the particular strain, grown asminimum level of repeatability of the amplification described above.conditions was calculated by running DNA samplesfrom duplicated amplifications of each DNA extract.To limit problems of repeatability (and reproducibil- 3. Results and discussionity) all the samples to be compared were processedat the same time. PCR fingerprinting profiles using the M13

For each strain, concordance between genotypes minisatellite sequence consisted of DNA bands sizedobtained using the three chelex-based extraction between |300 and 2000 bp (Fig. 2, PCR profiles ofmethods and conventional phenol–chloroform ex- four type strains shown as example). The clustertraction was determined. It was performed by com- analysis applied to all the DNA extracts usingparison and cluster analysis of the chelex-obtained Pearson as correlation coefficient allowed us toPCR fingerprinting profiles with those obtained by calculate a minimum repeatability of about 80% forphenol–chloroform extraction, used as controls. our PCR fingerprinting experiments, which corre-Comparisons were possible because by using Gel- sponds well to the minimum level of repeatability forCompar software the four methods were made a similar technique (i.e. the RAPD technique) ver-compatible, e.g. all data were obtained with one ified in a previous investigation (Tailliez et al.,single experimental procedure, processed with a 1996). Hence, it was deduced that only clusters withsingle, operator-independent, normalization proce- values of the correlation coefficient (expressed as adure and had the same format. In addition, the percentage value) above 80% were considered identi-normalization of the gels performed by the software cal.before the cluster analysis allowed to minimize The numerical analysis of the normalised PCRproblems of repeatability and reproducibility caused fingerprinting patterns of each strain (shown asby variability in the electrophoretic runs. dendrograms in Figs. 3 and 4) allowed to separate

Moreover, cluster analysis between profiles ob- profiles into distinct clusters. In Fig. 3 are showntained by the chelex-based protocol B (Fig. 1) and experiments to determine the best overlapping be-the controls allowed to evaluate the following param- tween chelex-based and conventional phenol–chloro-eters. form DNA extraction procedures. Seven out of nine

PCR fingerprinting profiles from DNAs extracted bychelex-based protocols B and/or C clustered together

• Reproducibility. The level of reproducibility of with controls at percent correlation coefficient valuesthe PCR fingerprinting patterns, which was de- higher than 80. Indeed, controls of only two strainsfined as the level of precision of the method over (L. helveticus LH30 and Lc. lactis subsp. lactistime, was evaluated by repeated running of DNA ISLCPT5) remained unclustered (Fig. 3). Converse-samples from duplicated amplifications of each ly, the most part (i.e. six out of nine) of the PCRDNA extract at time 0 (T0) and after 8 weeks of profiles obtained from DNA extracted with protocolstorage at 2208C (T1). A either remained unclustered or did not cluster with

• Consistency. In a given electrophoretic method, the controls (Fig. 3). The calculated value of the


Fig. 2. Agarose gel electrophoresis, shown as example, of amplified DNA of some of the strains used in the present study. DNA wasextracted by protocols A (lanes 2, 7, 12, and 17), B (lanes 3, 8, 13, and 18), C (lanes 4, 9, 14, and 19) and phenol–chloroform (lanes 5, 10,

T15, and 20) as described in Fig. 1. Lanes: 2–5, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 ; 7–10, L. delbrueckii subsp. lactisT T TATCC 12315 ; 12–15, L. helveticus ATCC 15009 ; 17–20, Lactococcus lactis subsp. lactis ATCC 19435 . Lanes 1, 6, 11, 16, and 21,

molecular size DNA marker 1-kbp plus DNA ladder (Life Technologies).

cophenetic correlation coefficient for this dendrog- prepared cultures between the procedure B (DDEram was 87.5%, indicating good reliability. In most profiles) and phenol–chloroform extraction (DDEcases, therefore, both B and C protocols allowed to control profiles). Except for L. helveticus LH30,obtain DNAs of a PCR quality similar to that of whose DDE control profiles grouped separately fromconventional DNA extraction, as indicated by identi- their corresponding DDE profiles at percent correla-cal profiles obtained after numerical analysis; the tion coefficient value of 78, the DDE and DDEprotocol B was chosen for further tests because it control profiles of the other strains were included inwas simpler, faster, and more practical than protocol either single clusters or merged into separate sub-C (Fig. 1). clusters at percent correlation coefficients higher than

The reproducibility of protocol B was tested by 80 (Fig. 4). The cophenetic correlation coefficientduplicated amplifications of DNAs at time 0 (T0) (about 90.0%) indicated a good reliability of thisand after 8 weeks of storage at 2208C (T1). A cluster analysis.pattern of reproducibility over time for almost all the It can also be observed that the PCR fingerprintingstrains was observed. Except for Lc. lactis subsp. analysis performed was confirmed to possess a good

Tlactis ATCC 19435 , the duplicate T0 and T1 capacity to resolve strains into expected taxa. Dis-profiles of each strain obtained by procedure B (T0 tinct clusters (and/or sub-clusters), correspondingand T1 profiles) and by phenol–chloroform extrac- well with species and subspecies established bytion (T0 and T1 control profiles) were usually species-specific PCR, were in fact obtained at per-grouped at percent correlation coefficient above the cent similarity values below 80 (Fig. 4).80% threshold (Fig. 4). Previous studies have demonstrated the impor-

A good consistency of the procedure B was also tance of colony age and other growth conditions asverified, as shown by comparing PCR fingerprinting major factors affecting the quality and reproducibil-profiles of DNA samples obtained using freshly ity of bacterial fingerprints obtained by PCR


Fig. 3. PCR fingerprinting profiles of strains and reference strains of various lactic acid bacteria obtained by DNA extracted with differentchelex-based protocols and with phenol–chloroform and corresponding dendrogram derived from the unweighted pair group average linkageof Pearson correlation coefficients (expressed as a percentage value). The similarity value of 80% is indicated by a vertical dotted line. Onthe right hand side of the figure are the strain numbers, the indication of the protocol used to obtain the corresponding profile, and the clusterdelineation by species. Abbreviations: L., Lactobacillus; Lc., Lactoccocus; subsp., subspecies; prot. A, prot. B, and prot. C, profiles obtainedwith DNA extracted by protocols A, B, and C as described in Fig. 1; control, profiles obtained with DNA extracted by phenol–chloroform asdescribed in Section 2.

(Coutinho et al., 1993; Wilson, 1997). Therefore, repeatability and reproducibility of the PCR finger-provided that bacterial growth conditions were opti- printing profiles were observed (data not shown).mised and bacterial purity was checked by species- The use of the PCR with arbitrary nucleotidespecific PCR, the procedure B: (i) provides stable primers to produce DNA fingerprints of cultivableDNA, giving over time reproducible PCR finger- bacterial strains has revolutionised population studiesprinting profiles; (ii) provides DNA giving PCR of microorganisms. Concerning LAB, there is now afingerprinting profiles similar to those obtained by large literature on procedures for analysing a varietyDNA extracted with a phenol–chloroform technique; of species (Andrighetto et al., 1998; Cocconcelli et(iii) has a high consistency, similar to that observed al., 1995; Corroler et al., 1998; Drake et al., 1996;using a phenol–chloroform technique, thus providing Klein et al., 1998; Moschetti et al., 1998; Tailliez eta reliable identification because the variations caused al., 1996, 1998; Torriani et al., 1999; Van Reenenby differences in growth conditions were found to be and Dicks, 1996). However, although most reportswithin the limits of repeatability and reproducibility involve the use of purified DNA as templates, recentof the procedure. studies describe the use of chelating agents, such as

The use of new stocks of freshly prepared M13 chelex or other synthetic resins, for rapid extractionprimer gave comparable results and no loss of of a DNA suitable for reproducible PCR amplifica-


Fig. 4. PCR fingerprinting profiles of strains and reference strains of various lactic acid bacteria obtained by DNA extracted withchelex-based protocol B (see Fig. 1) and phenol–chloroform extraction and corresponding dendrogram derived from the unweighted pairgroup average linkage of Pearson correlation coefficients (expressed as a percentage value). The similarity value of 80% is indicated by avertical dotted line. On the right hand side of the figure are the strain numbers, the indication of the DNA sample used to obtain thecorresponding profile, and the cluster delineation by species. Abbreviations: L., Lactobacillus; Lc., Lactoccocus; subsp., subspecies; T0,DNA extracted at time 0; T1, DNA extracted after 8 weeks of storage at 2208C (see Section 2); DDE, duplicated DNA extract; control,profiles obtained with DNA extracted by phenol–chloroform as described in Section 2.


tions from LAB (Cocconcelli et al., 1995; Drake et wider range of organisms (Singer-Sam et al., 1989;al., 1996). Walsh et al., 1991; Drake et al., 1996). Preliminary

The continuously accumulating set of PCR finger- experiments in our laboratory with different strainsprinting data and the construction of a reliable and PCR primers seem to confirm the suitability ofdatabase require a high degree of standardisation in this procedure to type a large number of LABexperimental methodology. If different protocols for isolates (unpublished results). Therefore, we proposeDNA extraction are used, concordance studies be- this as an easy protocol to extract DNA fromtween methods should be performed, whereas using cultivable LAB to be used as template for PCRa common protocol there probably exists as many fingerprinting. The protocol is also rapid, e.g. 40 minelectrophoretic variants in experimental procedures are needed to process 15 samples by the proposedas there are research groups. Even within a single InstaGene, chelex-based method and 240 min by thelaboratory, reproducibility of the DNA extraction phenol–chloroform purification method. We suggestprocedure, PCR assay, and running conditions have also a methodology to follow for demonstratingto be checked and corrected if necessary. Concerning reproducibility and consistency of PCR fingerprint-DNA extraction by chelex, a growing number of ing profiles obtained with differently extracted DNAchelex-based protocols and kits are now commercial- samples.ly available with different degrees of complexity andreproducibility, but concordance studies betweentechniques are still scarce. Therefore, comparative Acknowledgementsevaluation of these DNA extraction and purificationtechniques may be of interest. We are very grateful to Catia Ferri (University of

In the present study, factors affecting the quality Parma, Italy) for her technical assistance.and reproducibility of PCR fingerprinting patternsobtained from different DNA samples extracted fromMRS-broth-cultured LAB were investigated. We Referencescompared first different chelex-based protocols forPCR fingerprinting of LAB strains (either type- Andrighetto, C., De Dea, P., Lombardi, A., Neviani, E., Rossetti,strains or food isolates) belonging to food-associated L., Giraffa, G., 1998. Molecular identification and cluster

analysis of homofermentative thermophilic lactobacilli isolatedspecies with classical phenol–chloroform DNA ex-from dairy products. Res. Microbiol. 149, 631–643.traction technique. Generally, chelex-based protocols

Cocconcelli, P.S., Porro, D., Galandini, S., Senini, L., 1995.enabled to extract PCR-grade DNAs which, after Development of RAPD protocol for typing strains of lactic acidamplification, allowed to obtain electrophoretic pat- bacteria and enterococci. Lett. Appl. Microbiol. 21, 376–379.terns comparable with those obtained by classical Corroler, D., Mangin, I., Desmasures, N., Gueguen, M., 1998. An

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diversity and relationship within Lactococcus lactis as revealed

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