e., E

, P

Multiplex qPCR

ethesermteda medinewtald o

analyzed. A very low limit of detection (5 cfu/25 g after enrichment) for simultaneous identication ofined.

tuffs isood ch20 hue yeaxigenicrted, b

the most frequently identied (EFSA, 2012).

break in the USA due to Salmonella serovar Montevideo which caused in collaboration with International Standard Organization (ISO), has

,b, 2011a,b).luding, multiplex PCRion of food-poisoningsay greatly improved2009).

International Journal of Food Microbiology 164 (2013) 9298

Contents lists available at SciVerse ScienceDirect

International Journal o

evspecic and sensitive than traditional culture methods. These proce-dures enable the detection of sub-dominant populations, even inthe absence of a selective enrichment medium and in the presence

Although qPCR has many of these advantages with respect toculture methods, the development of robust and highly reproducibleassays requires optimization, standardization and a thorough evalua-infections and 29 deaths (CDC, 2011, 2012a,b).A trend toward molecular methods, mainly PCR-based methods,

for rapid detection of food-borne pathogens in food and feed microbi-ology, has been observed in recent years (Kagkli et al., 2011). Thesemethods, based on the detection of nucleic acids, are faster, more

(Hoorfar et al., 2003, 2004; ISO, 2005a,b, 2006aIn the past decade, improved PCR assays inc

and qPCR have been developed for the detectbacteria, being specicity and rapidity of the asat present (Kagkli et al., 2011; Kobayashi et al.,66 illnesses and 1 death. In 2011 another multistate outbreak affected8 people who were infected with E. coli O157. Moreover, in 2011 alarger multistate outbreak due to Listeria monocytogenes caused 146

proposed a general guideline for PCR testing, and the inclusion of anIAC is always required in the reaction mixture. In fact, a completeset of rules concerning these types of methods has been developedof other populations (Anderson et al., 2011;

Corresponding author. Microbiology and ToxinsCampus Univ. 16, 36310 Vigo PO, Spain. Tel.: +34 986 46

E-mail address: agcabado@anfaco.es (A.G. Cabado).

0168-1605/$ see front matter 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.ijfoodmicro.2013.03.024In June 2012 the Centersosted a multistate out-

recommended by different authors (Anderson et al., 2011; Hoorfaret al., 2003, 2004). The European Standardization Committee (CEN),for Disease Control and Prevention (CDC) p1. Introduction

Microbiological analysis of foodsmicrobial safety management in the fAs an example, in the year 2010, 99,0were reported in Europe. On the samlisteriosis was 1601. Regarding verotoa total of 4000 infections were repoan integrated part ofain (Kagkli et al., 2011).man salmonellosis casesr the number of humanEscherichia coli (VTEC),

eing serogroup O157 as

One major difculty for the application of PCR for foodborne path-ogen detection is the presence of compounds that inhibit the PCR re-action. These compounds can contaminate DNA templates extractedfrom food or environmental samples generating false-negative results(Hyeon et al., 2010). Some inhibitors that may affect different steps ofthe qPCR method include: phenolic compounds, fats and glycogen.For this reason, the implementation of an adequate internal ampli-cation control (IAC) in diagnostic PCR and qPCR has been strongly 2013 Elsevier B.V. All rights reserved.Validation these 3 pathogens was obtaIn-house validation of a multiplex real-timsimultaneous detection of Salmonella sppand Listeria monocytogenes

Alejandro Garrido, Mara-Jos Chapela, Beln Romn

a b s t r a c ta r t i c l e i n f o

Article history:Received 28 August 2012Received in revised form 24 February 2013Accepted 26 March 2013Available online 3 April 2013

Keywords:Salmonella spp.E. coli O157L. monocytogenes

A wide variety of qPCR mmonocytogenes detection. Thin simplex or in multiplex foed, and the number of validawas to develop and validateprobes. A modied broth moptimized. Efciency of theand multiplex analyses. A tothe evaluation of the metho

j ourna l homepage: www.e lsPostollec et al., 2011).

Area, ANFACO-CECOPESCA,9 303; fax: +34 986 469 269.

l rights reserved.PCR method forscherichia coli O157

aula Fajardo, Juan M. Vieites, Ana G. Cabado

ods currently exist for Salmonella spp., Escherichia coli O157 and Listeriamethods target several genes and use different detection chemistries, eitherats. However, the majority of these methods have not been carefully validat-methods that use multiplex qPCR is even lower. The aim of the present studyultiplex qPCR method from previously validated simplex qPCR primers and

um was selected and primary and secondary enrichment times were furtherly combined qPCR systemwas comprised between 91% and 108%, for simplexof 152 food and environmental, natural and spiked samples, were analyzed forbtaining values above 91% that were reached for all the quality parameters

f Food Microbiology

i e r .com/ locate / i j foodmicrotion performing prociency tests. These verications and assessmentsare difcult to achieve but they are needed for the adoption ofthese protocols in order to be used by the industry (Abdulmawjoodet al., 2004a; Wright et al., 2007). To be completely suitable for im-plementation as an analytical tool, the diagnostic accuracy of thePCR-based method must be thoroughly evaluated and demonstrated.

in a SpectraMaxM5microplate reader (BioNova Cientca S.L., Spain).Concerning stress experiments, overnight enrichment cultures of

each target bacterium were transferred to a new tube, turbidity ad-justed to 2 McFarland with sterile TSB and incubated in a waterbath at 60 C for 1 h. After heating, tubes were placed in a beakerwith water and ice for 2 min. This thermal treatment showed a con-stant reduction of 4 log units in the viable bacterial counts (experi-mentally determined).

The growth data were tted to the following logistic model(Guerra et al., 2010):

y y0 K

1 eumax bt

where y and y0 correspond to the cell load (O.D. at 600 nm) attime t and zero, respectively; K is the global increase of cell loadattained in the stationary phase; max is the maximum rate ofgrowth (O.D. 600 nm h1) and b is the lag time (h). Kineticexperiments were repeated 4 times for either healthy or stressedbacteria.

2.2.2. Secondary enrichment optimizationFor the optimization of the secondary enrichment time healthy

and thermally stressed bacteria were prepared as in Sections 2.1

93A. Garrido et al. / International Journal of Food Microbiology 164 (2013) 9298Selectivity and the limit of detection are the most critical parameterswhich dene the accuracy of a PCR-based assay (Abdulmawjoodet al., 2004b).

Many previous PCR and qPCR methods for detection of Salmonellaspp., E. coli O157 and L. monocytogenes have been developed and ap-plied (Bhagwat, 2004; Calvo et al., 2008; Fratamico and DebRoy,2010; Gordillo et al., 2011; Liming and Bhagwat, 2004; Myint et al.,2006; Oravcova et al., 2007; Paddock et al., 2011; Rantsiou et al.,2008; Sharma and Dean-Nystrom, 2003) either in simplex or multi-plex formats (Elizaquivel and Aznar, 2008; Germini et al., 2009;Jofre et al., 2005; Kawasaki et al., 2005; Ruiz-Rueda et al., 2011;Singh et al., 2012). Recent studies have applied qPCR for multiplexsimultaneous detection of these 3 pathogens (Kim and Bhunia,2008; Omiccioli et al., 2009; Suo et al., 2010; Zhang et al., 2009) butan extensive validation has not been performed. In the developmentof the present study the modular approach for microbial PCRmethods validation was applied (Kagkli et al., 2011).

In this study a multiplex qPCR method for the simultaneous detec-tion of Salmonella spp., E. coli O157 and L. monocytogeneswhich fulllsall requirements for international validation and application in foodtesting laboratories was developed and validated. To achieve thisgoal previously inter-laboratory validated primers and probes wereselected and optimized in a multiplex format with an appropriateInternal Amplication Control (IAC). Adequate enrichment brothand DNA extraction method were chosen, and the overall methodwas pre-validated. Moreover, relative specicity, sensitivity, dynamicrange, qPCR efciency and limit of detection, as well as relative accu-racy, positive and negative predictive values and the kappa index ofconcordance of this procedure were in-house evaluated.

2. Materials and methods

2.1. Bacterial strains and culture media

For the evaluation of modied TA10 broth, Salmonella entericaCECT 4594 (serovar Typhimurium, serotype 4, 5, 12: i: 1, 2), E. coliO157 CECT 4267 and L. monocytogenes CECT 935 purchased by theSpanish Type Culture Collection (CECT) were used as referencestrains. The microorganisms were stored frozen at20 C. Fresh cul-tures used in the present study were obtained by inoculating 10 mLtubes of Tryptic Soy Broth (TSB, BioMrieux S.A., France) and incubat-ed at 37 C overnight.

Several Vibrio spp. strains were selected to test the method speci-city (Table 1). These bacteria were grown in 10 mL of AlkalinePeptone Water double strength (SAPW, Biolife Italiana S.r.l., Italy)and incubated at 37 C overnight.

Viable reference values of each pathogenic bacterium under studywere calculated. Microorganisms were grown as described above,ten-fold serially diluted in Buffered Peptone Water (BPW, Biokardiagnostics S.A., France) and plated on Tryptic Soy Agar (TSA, Biokardiagnostics S.A., France) for S. enterica and E. coli O157 and on TrypticSoy Yeast Extract Agar (TSYEA, Biolife Italiana S.r.l., Italy) forL. monocytogenes. Plates were incubated 18 h at 37 C.

Sample enrichment for qPCR analysis was done in TA10 broth, thecommercial name of No. 17 broth (Kamisaki-Horikoshi et al., 2011;Kawasaki et al., 2005). This broth was modied as previously de-scribed, being the nal composition as follows: tryptose 10.0 g/L,beef extract 5.0 g/L, yeast extract 5.0 g/L, sodium chloride 5.0 g/L,disodium phosphate 19.3 g/L, monopotassium phosphate 3.4 g/L(Garrido et al., 2013; Omiccioli et al., 2009).

2.2. Enrichment optimization

The enrichment optimizationwas carried out in 2 consecutive steps.First, selected brothwas evaluatedmeasuring growth kinetics by absor-

bance to ensure the suitability of the broth for the growth of the 3 targetbacteria. The second step consisted in the optimization of secondaryenrichment time for reduction of the previously reported 8 h (Garridoet al., 2013) using thermally stressed and non-stressed bacteria.

2.2.1. Growth kineticsThe growth kinetics study was performed not only to verify the

suitability of modied TA10 (mTA10) for the growth of the bacteriaof interest, both healthy and stressed, but also to evaluate a reduc-tion in the minimum primary enrichment time determined in a previ-ous study (Garrido et al., 2013). Then, the secondary enrichmentwould be further optimized (see Section 2.2.2). 200 L of mTA10medium was inoculated with 2 L of the corresponding bacterium,rendering a nal viable count of 10100 cfu, following ISO 11133-2:2003 recommendations regarding productivity inocula (ISO, 2003).Growth curves were constructed by measuring bacterial growth of S.enterica, E. coli O157 and L. monocytogenes at 600 nm, every 15 minfor 24 h at 35 C, in a Falcon 96 well plate. Measurements were done

Table 1Bacterial strains selected for specicity.

Microorganism Strain invA rfbE prfA IAC

V. cholerae CECT 514 (O1) +V. cholerae CCUG 47460 (O139) +V. parahaemolyticus CCUG 43362 +V. parahaemolyticus CCUG 43363 +V. parahaemolyticus CCUG 43364 +V. parahaemolyticus CCUG 43365 +V. parahaemolyticus CECT 5271 +V. parahaemolyticus CECT511 +V. parahaemolyticus CAIM 58 +V. alginolyticus a +V. alginolyticus CECT 586 +V. alginolyticus CAIM 342 +V. mimicus BCCM/LMG 7896 +V. mimicus CECT 4218 +V. vulnicus CECT 4608 +V. vulnicus CECT 529 +V. vulnicus CECT 4869 +V. vulnicus CAIM 611 +

CECT: Spanish Type Culture Collection, CCUG: Culture Collection University ofGteborg, CAIM: Collection of Aquatic Important Microorganisms, BCCM/LMG:Belgian Co-Ordinated Collections of Micro-Organisms.IAC: Internal Amplication Control. +/: indicates positive/negative qPCR signal.

a Strain isolated in our laboratory.and 2.2.1.

Twenty-ve grams of boiled frozen mussels was spiked with 10100 cfu of either stressed or non-stressed bacteria and then 225 mLof mTA10 was added. This mixture was homogenized for 1 min in astomacher at normal speed and incubated 22 2 h at 35 C. After in-cubation, 1 mL of the enrichment broth was transferred to a tubecontaining 10 mL of fresh mTA10 medium which was re-incubatedat 35 C. Aliquots from time 0 to 8 were taken every hour and ana-lyzed by multiplex qPCR.

2.3. DNA extraction methods

2.3.1. DNA extraction from Vibrio spp. pure culturesDNA extraction of all Vibrio spp. strains selected for this study was

done as previously reported (Blanco-Abad et al., 2009; Garrido et al.,2012a). Briey, overnight cultures of each strain were sedimented bycentrifugation, pellets were washed and re-suspended in TrisEDTA(TE) 1 and boiled for 10 min. Finally, they were centrifuged againand supernatant, containing DNA, was stored at 20 C until use.

probe for Salmonella spp., 75 nM primers and 45 nM probe forE. coli O157; 500 nM primers and 250 nM probe for L. monocytogenes.Regarding the IAC, 75 nM primers, 45 nM probe and 8 102 copiesof IAC DNA were added per reaction.

2 L of template DNA was added per reaction tube. StratageneMx3005p thermocycler (Agilent Technologies, Inc., USA) was pro-grammed with the following thermal prole: 3 min at 95 C for theactivation of the polymerase (Hot Start), followed by 40 cycles, eachone consisting on a denaturation step of 15 s at 94 C andannealing-extension step at 64 C for 60 s.

2.6. qPCR method evaluation

2.6.1. qPCR efciencyQPCR efciency was calculated using overnight pure cultures of

S. enterica, E. coli O157 and L. monocytogenes incubated at 37 C in10 mL of TSB. DNA of each bacterium was extracted with thelysis-GuSCN method and measured with a NanoDrop 1000 spectro-

mon

ATGCGG

TCGGTAGAGGCAGGCA

GTCTTC

: ar

94 A. Garrido et al. / International Journal of Food Microbiology 164 (2013) 92982.3.2. DNA extraction from target bacteria and from food samplesGuanidium thiocyanate DNA extraction method (lysis-GuSCN)

(Kawasaki et al., 2005) was applied with slight modications previ-ously described (Garrido et al., 2013). This method was also usedfor the obtention of DNA from pure cultures of S. enterica, E. coliO157 and L. monocytogenes in the enrichment time optimizationexperiments and for the evaluation of the qPCR efciency.

2.4. Target genes, primers and probes for qPCR

Detection of each pathogenic bacteriumwas achieved by targetingspecic genes. For Salmonella spp. the invA gene was selected andprimers and probe previously validated by Cheng et al. were selected(Cheng et al., 2008, 2009). Detection of E. coli O157 was carried outtargeting the rfbE gene with the primers and probe developed byPerelle et al. (2004). Finally, detection of L. monocytogenes wasachieved targeting prfA gene by using primers and probe previouslydeveloped and validated (D'Agostino et al., 2004; Rossmanith et al.,2006; Simon et al., 1996). Internal control based on chimerical DNAwas selected to monitor qPCR reaction (Calvo et al., 2008). Genes,primers and probes used in this study are gathered in Table 2.

2.5. Multiplex qPCR detection method for Salmonella spp., E. coli O157and L. monocytogenes

The qPCR reaction was carried out in a nal volume of 50 L withthe following components: 30 L Brilliant III Ultra-Fast QPCR MasterMix (Agilent Technologies, Inc., USA), 300 nM primers and 50 nM

Table 2Primers and probes for multiplex qPCR detection of Salmonella spp., E. coli O157 and L.

Microorganism Gene Primer/probe Sequence (5 to 3)

Salmonella invA invA3F AACGTGTTTCCGTGCGTAinvA3R TCCATCAAATTAGCGGAGinvA Probe1 TGGAAGCGCTCGCATTGT

E. coli O157 rfbE rfbE F TTTCACACTTATTGGATGGrfbE R CGATGAGTTTATCTGCAArfbE probe AGGACCGCAGAGGAAAG

L. monocytogenes prfA LIP1 GATACAGAAACATCGGTTLIP2 GTGTAATCTTGATGCCATLIP3 CAGGATTAAAAGTTGACC

IAC IAC forward TCCAGGGCGAAAGTAAACIAC IAC reverse GGCGAGCCGTACGAACAIAC IAC probe CCCAGTTGGCTGATCACT

TYE665: is a trademark of IDT, it can be used as a substitute of Cy5. IABkFQ and IAbRQSp

absorbance specter.photometer. For simplex qPCR efciency calculation, DNA from eachbacterium was ten-fold serially diluted in sterile milli-Q water.When evaluating multiplex qPCR efciency, DNA from all 3 bacteriawere mixed in equal concentrations, ten-fold serially diluted and an-alyzed by adding 2 L of each dilution as template. All experiments,either for simplex or multiplex qPCR were performed in triplicate.

The Mx3005pro software automatically calculates the standardcurve for each run based on the Cycle threshold (Ct) of each standard.The formula from which the amplication efciency was calculatedwas e = 101/s 1 (Kawasaki et al., 2010), where s is the slopeof the standard curve.

2.6.2. Evaluation of the limit of detection (LOD) in food samples by qPCREvaluation of the LOD was performed as previously described

(Garrido et al., 2012a,b). Briey, 10 samples were inoculated withlow concentration of S. enterica, E. coli O157 and L. monocytogenes.As an acceptance criterion 90% of positive results must be achieved.

All 3 target bacteria were grown individually by an overnight incu-bation at 37 C in 10 mL TSB. Turbidity of all pathogens was adjustedto 0.52 McFarland with sterile TSB. Ten-fold serial dilutions weredone in BPW to reach a nal concentration ranging from 1.5 to6 cfu/mL. These dilutions were plated on TSA, for S. enterica andE. coli O157 and in TSYEA for L. monocytogenes, to get a referencevalue of viable bacteria after incubation at 37 C for 18 2 h. TheqPCR method consisted of the enrichment of 25 g of sample in225 mLmTA10 broth at 35 C for 22 2 h. After primary enrichment,1 mL was transferred to a tube containing 10 mL of newmTA10 brothand further incubated for a minimum of 5 h at 35 C. After incubation,1 mL was taken for DNA extraction, and 2 L as template for qPCR.

ocytogenes.

Modications Reference

Cheng et al. (2008)

5-/56-FAM//3BHQ_1/-3

TCAA Perelle et al. (2004)GAT GGAATTAAGG 5-/5TYE665//3IAbRQSp/-3C Rossmanith et al. (2006)G

5-/5HEX//3IABkFQ/-3 Calvo et al. (2008)

G 5-/5TexRd-XN//3BHQ_2/-3

e trademarks of IDT and stands for Iowa Black Flurophore Quencher, they differ in their

3.2.2. Secondary enrichmentEvaluation of results was carried out taking into account, the Ct

values obtained for each sample, and also the nal uorescence sig-nal. Results obtained for non-stressed S. enterica and E. coli O157showed consistent positive values at all times analyzed, includingthe direct food matrix, obtaining Ct values lower than 25 and naluorescence higher than 8000 and 2000 respectively. Only a slightdelay was observed at time 0, where a Ct value of 24 was recorded.In contrast, all other samples showed values lower than 22. In the

Table 3Growth kinetic results for thermally stressed and non-stressed bacteria.

Microorganism Thermaltreatment

K max b

S. enterica NS 0.437 0.016 0.607 0.055 14.7 0.4S 0.463 0.040 0.530 0.037 15.7 1.6

E. coli O157 NS 0.312 0.050 1.014 0.243 12.6 0.4S 0.376 0.038 0.723 0.211 12.5 0.8

L. monocytogenes NS 0.155 0.011 0.926 0.155 18.0 0.9S 0.156 0.020 0.782 0.210 17.3 2.4

95A. Garrido et al. / International Journal of Food Microbiology 164 (2013) 92982.6.3. Estimation of relative sensitivity, relative specicity, relative accu-racy, positive and negative predictive value and index kappa of concor-dance of the qPCR method

With the data obtained from the LOD, additional spiked and blindsamples (previously analyzed by traditional plating or alternativemethods as VIDAS BioMrieux S.A., France), relative sensitivity (SE),relative specicity (SP), relative accuracy (AC), positive and negativepredictive values (PPV and NPV) and kappa index of concordance ()of the method were calculated by comparing the results obtained byqPCR with the expected results. All mentioned parameters were cal-culated as previously described (Anderson et al., 2011; Tomas et al.,2009). All samples were kept in the same conditions as receiveduntil analysis (room temperature, refrigerated or frozen).

2.7. Statistical analysis

Data obtained for stressed and non-stressed bacterial kinetics re-garding K, max and b, were compared applying MannWhitneyU-test. Analysis was performed with SPSS 15.0 software (SPSS Inc.,Chicago, IL, USA).

3. Results

3.1. Specicity

The qPCR analysis did not show positive signal with any of the 3targets (invA, rfbE and prfA) for any of the Vibrio spp. strains selected,as expected. Correct amplication of the optimized IAC selected wasobserved with all strains. Results are shown in Table 1.

3.2. Enrichment optimization

3.2.1. Growth kineticsEvaluation of the optimization of the enrichment broth was ac-

complished using stressed and non-stressed bacteria to ensure suit-ability of the broth for the recovery of the microorganisms ofinterest. Growth curves demonstrated that the enrichment broth issuitable for all 3 bacteria selected in the present study.

Statistical analysis with MannWhitney U-test showed no sta-

K: global increase of cell load attained in the stationary phase. max: maximum rateof growth. b: lag time measured in hours. NS: non-stressed, S: thermally stressedtistical differences (p > 0.05) between thermally stressed and non-stressed bacteria for all variables analyzed (K, max and b).Data are summarized in Table 3.

Table 4qPCR simplex and multiplex efciencies.

Simplex

Efciency Slope R2

S. enterica 107.1 2.5 3.163 0.052 0.998 E. coli O157 104.7 4.2 3.212 0.095 0.998 L. monocytogenes 98.5 8.6 3.370 0.206 0.997

All values are averages of three independent replicates.case of L. monocytogenes, even though direct detection was observedfrom food matrix, more consistent results were observed after 5 hof secondary enrichment, since all samples showed Ct values lowerthan 35 with a nal uorescence signal higher than 1000.

When aliquots taken from stressed samples were analyzed, forS. enterica and E. coli O157 a general delay in the Ct value was ob-served although the same results were obtained with non-stressedbacteria. Regarding L. monocytogenes, a generalized delay in Ct valueswas obtained, thus not making possible a reliable detection before 5 hof secondary enrichment.

3.3. qPCR method evaluation

3.3.1. qPCR efciencyThree replicates were done for the efciency evaluation of each

simplex and multiplex qPCR methods. All 3 microorganisms returnedcorrect amplication efciencies, between 91.8% and 107.1%, withhigh correlation coefcients, above 0.997, for simplex qPCR andmultiplex qPCR. These efciency results were successfully obtainedcovering 5 orders of magnitude. Results for each individual bacteriumas well as the multiplex format, are detailed in Table 4.

3.3.2. Evaluation of the limit of detection (LOD)Viable bacteria reference values were calculated as described in

Section 2.1. Reference values obtained indicated a level of viableS. enterica, E. coli O157 and L. monocytogenes of 5 cfu/mL for eachpathogen. All 10 spiked samples returned clear positive results after5 h of secondary enrichment, with Ct values lower than 25 forS. enterica and E. coli O157, with nal uorescence higher than2000. Ct values were lower than 35 with nal uorescence higherthan 1000 for L. monocytogenes. IAC showed consistent positive Ctvalues between 30 and 35. Thus the LOD for the method wasestablished at 5 cfu/25 g for all 3 microorganisms.

3.3.3. Estimation of relative sensitivity, relative specicity, relativeaccuracy, positive and negative predictive value and index kappa ofconcordance of the qPCR method

Few discrepant results concerning Salmonella spp. and L. mono-cytogenes were obtained. Out of the 152 samples analyzed only 5negative deviations (ND) were obtained for Salmonella spp.; and 3positive deviations (PD) and 6 NDwere observed for L. monocytogenes.No discrepant results were detected for E. coli O157. Results obtainedfor all food samples analyzed are listed in Table 5.

Method quality parameters were calculated from all spiked andblind samples previously mentioned, obtaining values above 90% forthe 3 pathogens. Results are listed in Table 6.

Multiplex

Efciency Slope R2

0.001 91.8 1.6 3.536 0.044 0.999 0.0010.001 96.4 3.5 3.413 0.090 0.999 0.0010.004 93.9 2.7 3.478 0.073 0.999 0.001

Table 5Results obtained for blind and spiked samples in the evaluation of the method.

Food type Bld/Spk N Salmonella spp.

PA PD NA ND

Bivalves 6/20 26 15 0 6 5Cephalopods 4/0 4 0 0 4 0Fish 19/2 21 0 0 21 0Crustaceans 4/0 4 0 0 4 0Meat 12/5 17 2 0 15 0Water 6/0 6 0 0 6 0Byproductsb 11/0 11 1 0 10 0

les i

96 A. Garrido et al. / International Journal of Food Microbiology 164 (2013) 92984. Discussion

qPCR development has revolutionized molecular biology and anextensive number of applications have been carried out, both for re-search and clinical diagnosis (Raymaekers et al., 2009). qPCR offersadditional speed, sensitivity and specicity over traditional PCRmethods (Chapela et al., 2010; Elizaquivel et al., 2010). Its applicationin multiplex format increases the number of pathogens that can beanalyzed in the same reaction, and reduces costs (Elizaquivel andAznar, 2008; Ruiz-Rueda et al., 2011). However, there is some uncer-tainty about the reliable performance of qPCR due to the lack ofharmonized or validated protocols (Bustin et al., 2009; Rossmanithand Wagner, 2011). It has been shown that a cautious selection ofthe enrichment medium, combined with a proper DNA extractionmethod improves the detection of food-borne pathogens by qPCR(Martin et al., 2012; Taskila et al., 2012). The majority of these qPCRapplications are noncommercial and in-house developed open assays,being standardization and quality assurance required for moleculardiagnostics (Raymaekers et al., 2009).

Even though in recent years many PCR and qPCR methods havebeen developed, only a few have undergone a complete validationfor its application in food laboratories (Abdulmawjood et al., 2003;Lofstrom et al., 2010; Tomas et al., 2009). This number is evenlower and not always easily available when a multiplex format isdesired (Kawasaki et al., 2010). This lack of validated PCR and qPCRmethods could be due to the fact that this type of methods are notrecognized as ofcial, neither in Europe nor in many other parts ofthe world, where traditional culture methods are still the gold stan-dard in food microbiology. Furthermore, microbiological legislation

Vegetables 4/1 5 1 0 4 0Ready-to-eat 13/30 43 21 0 22 0Prociency test 4/0 4 2 0 2 0Boiled musselsa 1/10 11 10 0 1 0

a Samples used for LOD calculation.b Byproducts analyzed consisted on shmeal. N: represents the total number of samp

ment, PD: positive deviation, NA: negative agreement, ND: negative deviation.from many countries do not even consider the possibility of applyingPCR-based methods, or any other alternative, as ofcial, for pathogensscreening as it can be outlined from current European legislation andits amendments ((EC), 2005, 2007, 2010; (EU), 2011).

Genes selected for each pathogen in the present study werepreviously proposed as optimal by previous authors for individualdetection. The invasion gene, invA, involved in the internalization of

Table 6Summary of samples analyzed and results of quality parameters of the method.

PAa NAa PDa NDa SEb SPb ACb PPVb NPVb b

Salmonella spp. 52 95 0 5 91 100 97 100 95 93E. coli O157 29 123 0 0 100 100 100 100 100 100L. monocytogenes 71 72 3 6 92 97 96 96 92 94

a Values are the sum of those obtained for each type of food.b Values are expressed as percentage. PA: positive agreement, NA: negative agree-

ment, PD: positive deviation, ND: negative deviation, SE: relative sensitivity, SP: rela-tive specicity, AC: relative accuracy, PPV: positive predictive value, NPV: negativepredictive value, : kappa index of concordance.the bacterium in mammalian epithelial cells was selected for Salmo-nella spp. For E. coli O157 the rfbE gene, which is the fth gene ofthe rfb gene cluster, coding for the O-antigen and known to be specicfor E. coli O157, was used. Regarding L. monocytogenes, the prfAgene, which is the central virulence gene regulator was targeted(Abdulmawjood et al., 2003; Cheng et al., 2008; D'Agostino et al.,2004). In the present report inter-laboratory validated primers andprobes were selected for multiplex qPCR detection of Salmonellaspp., (Cheng et al., 2008, 2009) and L. monocytogenes, (D'Agostino etal., 2004; Rossmanith et al., 2006; Simon et al., 1996).

However, E. coli O157 primers and probe selected (Perelle et al.,2004) have not gone through a complete inter-laboratory validationprocedure, although a modular approach validation was previouslydescribed (Kagkli et al., 2011). Furthermore, the gene selected, rfbE,was also proposed as optimal for this bacterium detection, andselected in previous studies for PCR detection method validation(Abdulmawjood et al., 2003, 2004a,b). Even though of their specicityand extensive application, no previous study has tested their specic-ity against Vibrio spp., a genus that is known to posses the rfbE gene(Abdulmawjood et al., 2003). In the present study the specicity ofprimers and probe targeting rfbE gene, described by Perelle et al.(2004) was demonstrated against a panel of 18 Vibrio spp. from 5 dif-ferent species (Table 1). It was concluded from this and previousstudies (Kagkli et al., 2011) that primers and probe developed byPerelle et al. in 2004 are suitable for a deeper validation study.Concerning the IAC, the system was extensively applied in severalqPCR detection methods (Calvo et al., 2008; Garrido et al., 2012a,b).Overall the complete system with selected primers and probes wasconsidered reliable for specic detection of Salmonella spp., E. coli

E. coli O157 L. monocytogenes

PA PD NA ND PA PD NA ND

0 0 26 0 17 0 6 30 0 4 0 0 0 4 00 0 21 0 4 2 13 20 0 4 0 0 0 4 02 0 15 0 5 0 12 00 0 6 0 1 0 5 00 0 11 0 0 0 11 00 0 5 0 1 0 4 0

17 0 26 0 31 0 11 10 0 4 0 2 1 1 0

10 0 1 0 10 0 1 0

ncluding blind and spiked. Bld: Blind samples, Spk: spiked samples. PA: positive agree-O157 and L. monocytogenes with correct control of qPCR reaction byan exogenous IAC.

The enrichment broth selected (TA10) was previously developedand thoroughly evaluated (Kawasaki et al., 2005), proving suitablefor the recovery of these 3 bacteria from previously frozen foods(Kawasaki et al., 2009). From independent studies this broth gavebetter results than BLEB (Buffered Listeria Enrichment Broth), SEL(Kim and Bhunia, 2008), TSB and UPB (Universal Pre-enrichmentBroth) for the enrichment of L. monocytogenes in foods (Gehring etal., 2012; Kawasaki et al., 2005). Original formulation was modiedaccording to later studies where it was re-formulated and evaluatedagain in vitro and with different foods (Garrido et al., 2013;Horikoshi et al., 2008; Omiccioli et al., 2009).

The enrichment time was optimized by monitoring individualgrowth of each bacterium, both in optimal and heat-stressed condi-tions. No statistical differences (p > 0.05) were observed for any ofthe parameters analyzed, see Table 3 (the global increase, themaximum rate of growth and the lag time). These results indicatethat selected broth allows the recovery of thermally injured bacteria.

monocytogenes and Escherichia coli O157 H7 in non-spiked food products and eval-uation of real-time PCR as a diagnostic tool in routine food analysis. Food Control

97A. Garrido et al. / International Journal of Food Microbiology 164 (2013) 9298Considering the 3 parameters analyzed in this section, the lag timewas the most important. Reduction, or not increasing, in lag timeimproves the use of starter cultures and the recovering of bacteriafrom food and environmental samples (Robinson et al., 1998). Inthe present study this effect is enhanced by the addition of the5 hour-secondary enrichment.

Regarding this secondary enrichment, after analyzing the directfood matrix in mTA10 and 1 aliquot every hour, up to 8 h, it wasobserved that this step is not necessary to detect Salmonella spp.and/or E. coli O157. Although this step is highly recommended for de-tection of low numbers of L. monocytogenes, these results proved thatthe secondary enrichment time may be reduced from 8 h, as previ-ously reported (Garrido et al., 2013), to only 5 h. Furthermore, ifL. monocytogenes detection was not requested in the analysis, directdetection of Salmonella spp. and E. coli O157 may be achieved fromthe primary enrichment, even though it was observed that after 5 hof enrichment, Ct and nal uorescence results are clearer.

The DNA extraction method selected has been extensively applied,and its advantages over other protocols has also been reported previ-ously (Garrido et al., 2013; Germini et al., 2009; Horikoshi et al., 2008;Kawasaki et al., 2005, 2009, 2010; Mahmuda et al., 2007).

The analysis of the method developed indicated that the combina-tion of the primers and probes selected with the IAC, maintainedexpected efciency between 90% and 110% (Raymaekers et al.,2009) covering 5 orders of magnitude and a high correlation coef-cient above 0.997 (Table 4). All parameters analyzed to test themethod (SE, SP, AC, PPV, NPV and ) yielded values higher than90% demonstrating the high quality of the method developed.Out of the 152 samples analyzed only few discrepancies weredetected, 5 ND for Salmonella spp., which were associated eitherwith previously analyzed samples which were submitted to severalfreezing and thawing steps (previous authors have reported areduction in bacterial viability after freezing and thawing (Archer,2004; Keener et al., 2004)) or DNA extracts which were stored forlong periods of time (more than 5 months). The same situationhappened to the 6 ND found for L. monocytogenes. The 3 PD detectedfor L. monocytogenes, may be due to a lack of homogeneity in the sam-ples, since there are no previous reports indicating any cross reactiv-ity. Even though the highest numbers of deviations were found forL. monocytogenes, the results obtained for SE, SP and AC are in agree-ment with those previously reported for selected primers and probe,as no signicant differences were observed (original studies indicatedvalues of 94.2%, 100% and 97.5% respectively) (Rossmanith et al.,2006, 2010).

values indicated the delity of the method since valuesbetween 81% and 100% correspond to a very good concordance(Anderson et al., 2011; Cheng et al., 2009; DG, 1991).

5. Conclusions

The complete multiplex qPCR method developed and in-housevalidated in this study showed high sensitivity, specicity, accuracyand achieved a low limit of detection (5 cfu/25 g for each pathogen).This design constitutes a robust, fast and reliable method thatcan be applied for screening of Salmonella spp., E. coli O157 andL. monocytogenes even for thermally stressed bacteria, in a widevariety of foods and feeding stuffs. It is also suitable for extensiveapplication by the food industry, reducing time andmoney, comparedto other methods.

Acknowledgments

This work is nancially supported by the Secretary General forthe Sea of the Spanish Ministry of Agricultural, Land and Marine

Resources (MARM), by order ARM/1193/2009.22, 158164.Fratamico, P.M., DebRoy, C., 2010. Detection of Escherichia coli O157:H7 in food using

real-time multiplex PCR assays targeting the stx(1), stx(2), wzy(O157), and theAuthors also thank Victoria Docampo and Angeles Marcote fortechnical assistance.

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In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O15...1. Introduction2. Materials and methods2.1. Bacterial strains and culture media2.2. Enrichment optimization2.2.1. Growth kinetics2.2.2. Secondary enrichment optimization

2.3. DNA extraction methods2.3.1. DNA extraction from Vibrio spp. pure cultures2.3.2. DNA extraction from target bacteria and from food samples

2.4. Target genes, primers and probes for qPCR2.5. Multiplex qPCR detection method for Salmonella spp., E. coli O157 and L. monocytogenes2.6. qPCR method evaluation2.6.1. qPCR efficiency2.6.2. Evaluation of the limit of detection (LOD) in food samples by qPCR2.6.3. Estimation of relative sensitivity, relative specificity, relative accuracy, positive and negative predictive value ...

2.7. Statistical analysis

3. Results3.1. Specificity3.2. Enrichment optimization3.2.1. Growth kinetics3.2.2. Secondary enrichment

3.3. qPCR method evaluation3.3.1. qPCR efficiency3.3.2. Evaluation of the limit of detection (LOD)3.3.3. Estimation of relative sensitivity, relative specificity, relative accuracy, positive and negative predictive value ...

4. Discussion5. ConclusionsAcknowledgmentsReferences