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Food Microbiology 27 (2010) 19–23

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Food Microbiology

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Quantification of Listeria monocytogenes in minimally processed leafy vegetablesusing a combined method based on enrichment and 16S rRNA real-time PCR

Maria Aparecida de Oliveira a, Eliana Guimaraes Abeid Ribeiro a, Alzira Maria Morato Bergamini a,Elaine Cristina Pereira De Martinis b,*

a Instituto Adolfo Lutz – Laboratorio Regional de Ribeirao Preto, Rua Minas, 877, Campos Elıseos, 14085-410, Ribeirao Preto, SP, Brazilb Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Sao Paulo,Universidade de Sao Paulo, Av. do Cafe s/n, Monte Alegre, 14040-903 Ribeirao Preto, SP, Brazil

a r t i c l e i n f o

Article history:Received 9 March 2009Received in revised form3 July 2009Accepted 12 July 2009Available online 17 July 2009

Keywords:Listeria monocytogenesMinimally processed vegetablesReal-time PCR16S rRNAMicrobiological quality

* Corresponding author. Tel.: þ55 16 3602467; fax:E-mail address: edemarti@usp.br (E.C. Pereira De

0740-0020/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.fm.2009.07.003

a b s t r a c t

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eatfoods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of thoseproducts may favor the growth of psychrotrophic bacteria, including the pathogen Listeria mono-cytogenes. In this work, minimally processed leafy vegetables samples (n ¼ 162) from retail market fromRibeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immu-noassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminatedsamples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN)with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCRwith primers specific for L. monocytogenes using the commercial preparation ABSOLUTE� QPCR SYBR�

Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and nostatistical significant difference was found in comparison with the conventional culture method(p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

All over the world, public health agencies are concerned withfood safety assurance due to globalization of food markets,growing demand for minimally processed ready-to-eat (RTE)foods and increasing numbers of meals served outside home(Kennedy and Wall, 2007). RTE products are subjected to mildtreatments and are often stored under conditions that may favorthe growth of diverse spoilage and pathogenic microorganisms,such as Listeria monocytogenes (WHO/FAO, 2007). This psychro-trophic bacterium causes a disease with mild flu–like symptomsand rarely gastroenteritis in healthy adults, but it may manifest assevere infection in immunocompromised persons and duringpregnancy, leading to abortion, stillbirth or perinatal morbidity(Abadias et al., 2008; Aureli et al., 2000; Berrada et al., 2006;Donnelly, 2001; Gasanov et al., 2005).

Vegetables are highly susceptible to microbiological contami-nation due to lack of good agricultural practices by use of untreated

þ55 13 36024725.Martinis).

All rights reserved.

water, inappropriate organic fertilizers and fails during harvesting,handling and distribution (Francis et al., 1999). Minimal processingof leafy vegetables is intended to improve quality, microbiologicalsafety and to extend shelf life, but it can also cause mechanicalinjuries to the tissues. The minimal processing of vegetables maycause loss of water and color changes during steps of selection,washing, peeling, cutting, sanitizing, rinsing, drying and packaging(Francis et al., 1999; Soares and Geraldine, 2007)

High populations of L. monocytogenes have been reported forsome RTE products, including outbreak samples, but growth rate ofL. monocytogenes in vegetables is generally lower compared to milk,dairy and processed meat products (Chen et al., 2006). It is alsoimportant to consider that minimally processed vegetables areoften used as ingredients to prepare salads, which may have longerrefrigerated shelf lives, with risk of increasing of L. monocytogenespopulation (Aguado et al., 2004).

The quantification of L. monocytogenes in foods is generally doneby the classical Most Probably Number (MPN) method, whichrequires replicated dilution series of food in selective enrichmentbroth followed by plating on selective agar plates and various testsfor species identification (Berrada et al., 2006; Churchill et al., 2006;De Martinis et al., 2007; Duvall et al., 2006).

M. Aparecida de Oliveira et al. / Food Microbiology 27 (2010) 19–2320

Rapid and sensitive methods for enumeration of L. monocytogenesare important for microbiological safety throughout the foodproduction chain and in this last 10 years, a considerable number ofdetection methods using molecular tools have been proposed(Aguado et al., 2004; Amagliani et al., 2007; Aznar and Alarcon, 2002;Berrada et al., 2006; De Martinis et al., 2007; Duvall et al., 2006;Ingianni et al., 2001; Jung et al., 2003; Norton, 2002; O’Grady et al.,2008; Sommer and Kashi, 2003; Rosmanith et al., 2006).

Many papers have been published on the detection of L. mono-cytogenes by PCR, using primers targeted to virulence and nonvirulence factors, including hemolysin (hly), invasion associatedprotein (iap) and 16S rRNA genes (Berrada et al., 2006; Gasanovet al., 2005; Niederhauser et al., 1992; Thimothe et al., 2004;Sommer and Kashi, 2003; Wang et al., 2007). The PCR methodbased on 16S rRNA genes for detection of L. monocytogenes presentsbetter sensitivity due to the presence of multiple copies of thegenes in the cell, to maintain adequate concentration of ribosomes(Alberts et al., 2002).

The aim of the present study was to evaluate the MPN methodcombined with RTi-PCR based on 16S rRNA genes for enumerationof L. monocytogenes in naturally and artificially contaminatedminimally processed leafy vegetables.

2. Materials and methods

2.1. Food samples

From September 2007 to August 2008, a total of 162 samples ofminimally processed leafy vegetables were randomly acquiredfrom six grocery stores in the city of Ribeirao Preto, Sao Paulo,Brazil. Ten different types of samples were examined: collard green(n ¼ 30), cabbage (n ¼ 28), lettuce (n ¼ 26), mixed parsley andspring onion bunches (n ¼ 22), chinese cabbage (n ¼ 13), arugula(n ¼ 6), chicory (n ¼ 11), wild chicory (n ¼ 13), spinach (n ¼ 9) andwatercress (n ¼ 4).

2.2. Detection of Listeria spp

Detection of Listeria spp. was accomplished with the immuno-assay Listeria Rapid Test (Oxoid Ltd, Basingstoke, Hampshire,England) according to manufacturer’s instructions (Oxoid, 2007).Aliquots of all samples were kept frozen and positive samples bythe immunoassay were further tested for L monocytogenes byconventional identification tests.

2.3. Enumeration of L. monocytogenes

L monocytogenes was enumerated in two naturally and sixartificially contaminated samples of leafy minimally processedvegetables, using the Most Probable Number (MPN) technique withclassical and real-time PCR detection (De Martinis et al., 2007;Hitchins, 2007).

For non-inoculated samples, each vegetable (50 g-portion) wasmixed in a sterile plastic bag for 2 min with 450 ml of BLEB ina piston homogenizer (MA 440, Marconi, Brazil). For experimentalinoculation studies, 50 g-portions of each vegetable were sampledin sterile plastic bags and added of 450 ml of Buffered ListeriaEnrichment Broth (BLEB, Oxoid Ltd, Basingstoke, Hampshire,England) and added of 1-ml inoculum prepared in phosphatebuffered saline (PBS) to yield ca. 1, 2 or 3 log10 CFU ofL. monocytogenes ATCC 19114 per gram of food.

Triplicate series of tubes for MPN enumeration were preparedto contain 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 and 0.000001 g offood. After incubation at 30

�C for 4 h, cultures were added of BLEB

selective supplement (Oxoid, SR 0141E) and further incubated

for 44 h at 30�C. Aliquots of enrichment broths were streaked on

plates containing Oxford (OX) and PALCAM (PAL) agars (OxoidLtd) and incubated for two days at 35

�C, for isolation of

presumptive Listeria spp. colonies. Three to five typical blackcolonies from each agar were picked for purification on TSA-YEplates and colonies with blue appearance under transiluminationwere selected for L. monocytogenes identification by biochemicaltests (Hitchins, 2007).

2.4. Extraction of L. monocytogenes DNA

DNA from cultures of each MPN tube was extracted by theboiling method without Triton X-100 (Hitchins et al., 2004). Forthat, samples were centrifuged for 15 min at 10

�C and 8000 g

(Sorvall, Legend� MACH 1.6/R, Germany) and the supernatantswere discarded. The pellets were res-suspended in 5 ml PBS and re-centrifuged for 10 min. The supernatants were discarded again andthe pellets were res-suspended in 2 ml of purified water (Milli-Q,Millipore, USA). The suspensions were heated for 10 min ina boiling water bath and immediately cooled on ice. After centri-fugation for 10 min, the supernatants were retained and the sedi-ments were discarded. The DNA extracted was stored at �20

�C

until further analysis.

2.5. Real-time PCR assay

PCR amplification of DNA from extracts of each MPN enrichmentculture was done according to Wang et al. (2007) with primers for16S rRNA purchased from Invitrogen, Carlsbad, USA (L-1: 50-CACGTGCTACAATGGATAG-30 and L-2: 30-GATTAGGGTATTTTGA-TAAGA-50). The final reaction mixture of 25 ml was composed by12.5 ml of AbsoluteM QPCR SYBR� Green Mix (ABgene, BlenheimRoad, Epson, Surrey, UK), 4 ml of extract of DNA (or appropriatedilution, corresponding to ca. 39 ng of DNA) and 1.25 ml of primersolution (0.25 mM of each primer). The amplification of target DNAand detection of PCR products were performed with the real-timePCR system MiniOpticon� (Bio-Rad Laboratories, Hercules, CA,USA) equipped with the Opticon Monitor Analysis Software -version 3.1 (Bio-Rad Laboratories) for data acquisition and analysisof results. The amplification of the 16S rRNA genes was performedat 95

�C for 15 min followed by 45 cycles of 2 s at 95

�C, 10 s at 55

�C

and 10 s at 72�C. After the final amplification, the specificity of PCR

was checked by the melting temperature of amplicon (Tm), calcu-lated from a dissociation curve obtained by increasing thetemperature from 60 to 95

�C at a rate of 0.3

�C/s (De Martinis et al.,

2007). SYBR� Green allowed the continuous monitoring of ampli-con concentration by the fluorescence characteristic of the SYBR�

Green: DNA complex.Real-time PCR amplification products were also analyzed by

electrophoresis in agarose gel at 3% in TAE 1X (Tris base 0.04M,0001M EDTA, 0.04M sodium acetate, pH 8.5) and 0.5 mg/ml ofethidium bromide (Vetec, Brazil). Each reaction mixture (5 ml) plus3 ml of the loading buffer Bromophenol blue 0.25% (Vetec, Brazil)and 30% glycerol (Synth, Brazil) were applied in the gel. Electro-phoretic separation was performed at 110 V, 1.81 A (FB 200, FisherScientific, USA) for 1 h and 15 min, the gel was observed underultraviolet light and photos were taken for documentation ofresults (MiniBio UV, DNR Bio-Imaging Systems, Israel). Themolecular weight of the PCR product (70pb) was determined bycomparison with a standard of 50 bp molecular weight marker(Invitrogen, Carlsbad, USA).

2.5.1. Inclusivity and exclusivity tests for real-time PCR assayDNA of pure cultures were extracted as previously described

(Hitchins et al., 2004) and the bacterial strains used for inclusivity

Table 2Bacterial strains used in the tests of specificity of L. monocytogenes.

Species Strainsa Other descriptions

Staphylococcus aureus ATCC 25923 IAL 2432Staphylococcus epidermidis ATCC 12228Bacillus cereus ATCC 14579Bacillus subtilis ATCC 6633Enterococcus faecalis ATCC29212Rhodococcus equi ATCC 6939Salmonella Typhimurium ATCC 14028 INCQS 150Shigella sonnei CIP 6310 IAL 1585Proteus mirabilis CDC 305 IAL 022Escherichia coli ATCC 25922 IAL 339Pseudomonas aeruginosa ATCC 27853 IAL 1026Klebsiella pneumoniae ATCC 10031 IAL 520Enterobacter aerogenes CDC 1535 IAL 106Citrobacter diversus IAL 1816

a ATCC, American Type Culture Collection, EUA; IAL, Instituto Adolfo Lutz, SP,Brazil; CIP, Collection Institut Pasteur, Paris, France; CDC, Centers for Disease Controland Prevention; INCQS, Instituto Nacional de Controle de Qualidade em Saude, RJ,Brazil.

M. Aparecida de Oliveira et al. / Food Microbiology 27 (2010) 19–23 21

and exclusivity tests are listed in Tables 1 and 2. Listeria specieswere grown in 10 ml of TSB-YE (Oxoid, UK) while all other bacteriawere grown in brain heart infusion (BHI). Pure culture of L. mono-cytogenes ATCC 19114 and Listeria innocua ATCC 33090 were used aspositive and negative controls, respectively.

2.6. Calculations and statistical analyses

MPN values and 95% confidence intervals were obtained for thetwo methods (conventional and real-time PCR) from publishedtabulations with the proportions of positive tubes of each leafyvegetable portion tested (Blodgett, 2006). The difference betweenvalues for each pair of results comprised within the same confi-dence intervals were considered not significant. Descriptive andstatistical analyses of the data were done with SPSS� SoftwareVersion 15.0. Normality was verified by Shapiro–Wilk test, followedby T-test for paired samples and a probability value of less than 5%was considered to be significant.

3. Results and discussion

In the present study, Listeria spp. was detected in 6 (3.7%) out of162 samples tested: L. innocua was found in four samples, whileL. monocytogenes was isolated from two samples (1 collard greenand 1 bunch of parsley and spring onion). Other results on detec-tion of L. monocytogenes in vegetables consumed in Brazil arescarce. In a publication by Porto and Eiroa (2001), 250 samples ofvarious leafy vegetables were analyzed, and L. monocytogenes wasdetected in 3.2% of lettuce, parsley and watercress samples. Froderet al. (2007) examined 181 samples of minimally processed leafyvegetables, collected in the city Sao Paulo, Brazil, and found onlyone sample of spinach (0.6%) positive for L. monocytogenes.

However, no results on quantification of L. monocytogenes inBrazilian leafy vegetables were found in indexed literature.According to Rodrıgues-Lazaro et al. (2007), the enumeration ofpathogenic microorganisms is necessary for quantitative riskassessment. The conventional MPN method for quantification ofL. monocytogenes has the advantage of enabling detection of the

Table 1Listeria strains used in this study to evaluate Presence/Absence and quantification ofListeria sp.

Species Strainsa Other descriptions

Listeria monocytogenes, sorotipo 4b ATCC 19115 IAL 632L. monocytogenes, sorotipo 4a ATCC 19114 IAL 631L. monocytogenes, sorotipo 1/2a IAL 633L. monocytogenes ATCC 19122L. monocytogenes IALRP 167 meL. monocytogenes IALRP 27 meL. monocytogenes IALRP 26 meL. monocytogenes IALRP H 21L. monocytogenes IALRP H 23Listeria ivanovii IAL 1983Listeria welshimeri CIP 8149 IAL 1819Listeria welshimeri FCFRP LwVa1Listeria innocua IAL 1984Listeria innocua ATCC 33090 INCQS 500 354Listeria innocua IALRP H 133Listeria innocua IALRP H 140Listeria innocua IALRP H 158Listeria seeligeri CIP 100100 IAL 1820Listeria seeligeri FCFRP LsVa 1Listeria grayi FCFRP LgVa1

a ATCC, American Type Culture Collection, EUA; IAL, Instituto Adolfo Lutz, SP,Brazil; IALRP, Instituto Adolfo Lutz de Ribeirao Preto, SP, Brazil; CIP, CollectionInstitut Pasteur, Paris, France; INCQS, Instituto Nacional de Controle de Qualidadeem Saude, RJ, Brazil; FCFRP, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto –USP, SP, Brazil.

target pathogen even when it is present in low numbers(<100 CFU/g) in food matrices with a large number of backgroundflora. On the other hand, MPN is laborious and requires several daysfor confirmation of results, hampering the processing of severalsamples on the same day (Gasanov et al., 2005; Giaccone andOttaviani, 2007; Ingianni et al., 2001; Norton, 2002; Rodrıgues-Lazaro et al., 2007).

In this study, the real-time PCR method developed byDe Martinis et al. (2007) was applied to samples of leafy greens,with good specificity for primers L-1 and L-2. However, Aznar andAlarcon (2002) obtained discordant results with the same pair ofprimers and they reported that Listeria non-monocytogenes strainsyielded PCR products identical to the amplicon of primers L-1 andL-2. Controversies in results on specificity of primers L-1 and L-2 inliterature, encouraged further tests on inclusivity and exclusivity,with different strains of L. monocytogenes (N ¼ 9), strains of otherspecies of Listeria (N¼ 11), Gram positive and Gram negative genera(N ¼ 14) (Tables 1 and 2). The specificity of real-time PCR productswas also confirmed in this study by agarose gel electrophoresis,showing only one amplicon of 70pb, specific for 16S rRNA genes ofL. monocytogenes (results not presented). Based on experimentallyinoculated product with MPN quantification, the real-time PCRassay was capable of detecting 1–5 CFU per 50 g with no interfer-ence from the microflora present in these food samples.

For real-time PCR, theoretically estimated value of Tm for theamplicon obtained with L-1 and L-2 primers is 80.55

�C

(De Martinis et al., 2007; Wang et al., 2007) and similar Tm valueswere obtained in tests with DNA from pure cultures of L. mono-cytogenes (79.0–79.4) and leafy vegetables (78.2–80.1). Accordingto the manufacturer, with Absolute� QPCR SYBR� Green Mix theTm may vary with the equipment and the software used. In thepresent study, different reaction mixtures and primer concentra-tions influenced Tm values (results not presented), indicating thatcareful optimization is needed to allow the selective detection ofL. monocytogenes. This requirement may explain the discrepanciesin specificity observed in one out of four studies (this study, Aznarand Alarcon, 2002; De Martinis et al., 2007; Wang et al., 2007). Inthis sense, the enrichment–real-time PCR method used is advan-tageous for L. monocytogenes enumeration.

In this study, it was observed that smaller numbers of positiveMPN tubes could occur in the presence of large quantities of sample(10 and 1 g of food). The main problem was the interference of thegreen pigment (chlorophyll), especially for vegetables withstronger pigmentation (wild chicory, parsley, spring onions and

Table 4Result of the quantification of L. monocytogenes by the MPN technique (conventionaland real-time PCR methods) in artificially contaminated minimally processed leafyvegetables.

Leafy vegetables Experimentallyinoculated log10

(CFU/g)a

Conventionalmethod log10

(MPN/g)b

Real-time PCRc

log10(MPN/g)b

Chinese cabbage 1.0 1.4 (0.6–2.1) 0.4 (�0.4–1.1)1.9 2.0 (1.2–2.6) 2.4 (1.6–3.1)2.8 3.4 (2.6–4.1) 4.0 (3.2–4.6)

Cabbage 1.0 2.0 (1.2–2.6) 2.0 (1.2–2.6)1.5 2.6 (1.8–3.3) 2.6 (1.8–3.3)2.3 3.6 (2.8–4.3) 3.6 (2.8–4.3)

Collard green 1.0 1.6 (0.8–2.3) 1.6 (0.8–2.3)1.7 2.9 (2.2–3.6) 2.9 (2.2–3.6)2.9 3.4 (2.6–4.1) 4.4 (3.6–5.1)

Lettuce 1.0 1.6 (0.8–2.1) 1.4 (0.6–2.1)1.8 2.8 (1.2–3.6) 2.8 (1.2–3.6)2.6 3.6 (2.8–4.3) 2.6 (1.8–3.3)

Mixed bunchof parsley plusspring onion

1.1 1.6 (0.8–2.3) 0.4 (�0.2–1.3)2.1 2.6 (1.8–3.3) 0.6 (�0.2–1.3)3.0 4.0 (3.2–4.6) 3.0 (2.2–3.6)

Wild chicory 1.0 1.6 (0.8–2.3) 1.0 (0.2–1.7)1.8 2.6 (1.8–3.3) 2.0 (1.1–2.7)2.9 2.6 (1.8–3.3) 3.0 (2.2–3.6)

a Population in the inoculum determined by enumeration on plates containingtriptic soy agar plus 0.6% yeast extract (TSA-YE).

b MPN and confidence intervals from Blodgett (2008).c Real-time PCR developed with ABSOLUTE� QPCR SYBR� Green Mix (ABgene,

UK) and primer 16S rRNA (De Martinis et al., 2007).

M. Aparecida de Oliveira et al. / Food Microbiology 27 (2010) 19–2322

collard green). In those cases, it was necessary to dilute the DNAextracts before analysis (1:50) by PCR to improve L. monocytogenesdetection. Despite this, for some samples, irregularly shaped DNAamplification curves and Tm slightly different from the calculatedtheoretical value were observed. According to Blodgett (2008), forsome microorganisms in some food matrices, there seem to befactors that reduce the efficiency of detection of the targetorganism in classical MPN cultures, such as competitive microor-ganisms or inhibitory compounds. Amagliani et al. (2007) investi-gated the quality of the DNA extract obtained from samples of foodsby measuring the absorbance at 260 and 280 nm, and calculatedthe ratio A260/A280 showing that one of the main problems of thePCR application for detection of microorganisms in food is thepresence of inhibitory substances in the sample, which may be co-extracted with DNA and inhibit amplification reaction thus leadingto false negative results.

Some researchers (Amagliani et al., 2007; Liu, 2008; Rodrıgues-Lazaro et al., 2007; Smith and Maxwell, 2007) highlighted theimportance of DNA extraction methods for improving the speci-ficity and increasing the sensitivity of PCR reactions. Amaglianiet al. (2007) tested 4 different methods of DNA extraction (3commercial methods, the phenol-chloroform extraction method,and the boiling water method). Their study showed that the boilingwater method was the most convenient method for DNA extrac-tion. However, the boiled extract of mozzarella enrichmentsappeared to contain some inhibitory material since amplicon yieldwas relatively low at the 1 CFU/g inoculum level. Nevertheless,according to the authors, the purity of the DNA recovered was morethan sufficient for subsequent application and isolated DNA wassatisfactory in PCR amplification.

Berrada et al. (2006) performed a study on the presence orabsence of L. monocytogenes, in 77 different types of salads servedin restaurants in Valencia (Spain), by real-time PCR and conven-tional methods. Those authors found three positive samples amongthe 77 analyzed, and reported that the results were similar for bothmethods. Rantsiou et al. (2008) also used the technique of real-timePCR to quantify L. monocytogenes in 66 different samples of foods.They detected four positive samples without pre-enrichment and,nine positive samples after 24–37 h enrichment. In the experimentsof the present study, to obtain better sensitivity, the quantificationof L. monocytogenes by real-time PCR was performed after a periodof 48 h of enrichment by MPN.

Among the 162 samples of vegetables evaluated in this study,L. monocytogenes was isolated from only two leafy minimally pro-cessed vegetables. In Table 3 it is shown that the results obtainedwith both methods of quantification were similar. Therefore, toconduct a comparison of conventional method of analysis and real-time PCR, six different leafy vegetables were experimentally inoc-ulated with L. monocytogenes populations ranging from 1 to 3log10 CFU per gram of food (Table 4) and results similar to theconventional MPN technique were obtained (95% confidenceinterval). Shapiro–Wilk test demonstrated that there was a normal

Table 3Result of the quantification of L. monocytogenes by the MPN technique (conventionalmethod and real-time PCR) in naturally contaminated minimally processed leafyvegetables.

Leafy vegetables Conventional methodlog10(MPN/g)a

Real-time PCRb

log10(MPN/g)a

Collard green 3.2 (2.5–3.6) 3.0 (2.2–3.7)Mixed bunch of parsley

plus spring onion�0.4(<�1.3–0.3) �1.4(<�2.3–0.71)

a MPN and confidence intervals from Blodgett (2008).b Real-time PCR developed with ABSOLUTE� QPCR SYBR� Green Mix (ABgene,

UK) and primer 16S rRNA (De Martinis et al., 2007).

distribution of quantitative results of both conventional and real-time PCR method (p ¼ 0.43). Moreover, T test for paired samplesshowed no significant differences for the two methods (p ¼ 0.12).

The choice of microbiological analytical method has to be basedon the objectives of the study, conditions of the laboratory(personnel, budget, equipments, etc.) and minimal generation oftoxic waste. Furthermore, for detection of small populations, vali-dation parameters of accuracy and limit of quantification shouldalso be considered. In this study, the MPN enrichment combinedwith the real-time PCR detection was fast and simple to perform, incomparison with the classical L. monocytogenes enumerationmethod. If only costs of consumable supplies are considered, thePCR method is also more advantageous economically.

In conclusion, the enumeration method based on real-time PCRproposed was comparable to the conventional culture method forthe enumeration of L. monocytogenes by MPN in leafy vegetables.

Acknowledgments

The authors are grateful to the Fundaçao de Amparo a Pesquisado Estado de Sao Paulo, Brazil for financial support (Process 2006/06401-3). Special thanks to Vanessa Maciel de Souza for herexcellent technical laboratorial support.

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