1, 100 bp DNA ladder; Lane 2, PCR product (275 bp) for

www .e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Detection

of

E.

coli

O157:H7

and

Shigella

dysenteriae

toxins

in

clinical

samples

by

PCR-ELISA

Jafar

Amani

_,

_Askary

_Ahmadpour

_,

_Abbas

_Ali

_Imani

_Fooladi

,

Shahram

Nazarian

a_{AppliedMicrobiologyResearchCenter,BaqiyatallahUniversityofMedicalSciences,Tehran,Iran}

b_{ImamHossainUniversity,FacultyofScience,DepartmentofBiology,Tehran,Iran}

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Articlehistory:

Received14October2014 Accepted21February2015 Availableonline21April2015

Keywords:

Shigelladysenteriae

E.coliO157:H7

PCR-ELISA

Diagnosticmethods

a

b

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t

Shigatoxinproducing bacteriaare potentialcauses ofserioushumandisease suchas hemorrhagiccolitis,severeinflammationsofileocolonicregionsofgastrointestinaltract, thrombocytopenia,septicemia, malignantdisorders inurinaryducts, hemolyticuremic syndrome(HUS).Shiga toxin1(stx1),shigatoxin2(stx2),ora combinationofbothare responsibleformostclinicalsymptomsofthesediseases.Alotofmethodshavebeen devel-opedsofartodetectshigatoxinssuchascellculture,ELISA,andRFPLA,butduetohighcosts andlabortimeinadditiontolowsensitivity,theyhavenotreceivedmuchattention.Inthis study,PCR-ELISAmethodwasusedtodetectgenesencodingshigatoxins1and2(stx1and stx2).Todetectstx1andstx2genes,twoprimerpairsweredesignedforMultiplex-PCRthen PCR-ELISA.PCRproducts(490and275,respectively)weresubsequentlyverifiedby sequenc-ing.SensitivityandspecificityofPCR-ELISAmethodweredeterminedbyusinggenome serialdilutionandEnterobacteriastrains.PCR-ELISAmethodusedinthisstudyprovedtobe arapidandpreciseapproachtodetectdifferenttypesofshigatoxinsandcanbeusedto detectbacterialgenesencodingshigatoxins.

©2015ElsevierEditoraLtda.Allrightsreserved.

Introduction

Shigatoxinsbelongtoalargefamilyofbacterialtoxinswith two major groups, stx1 and stx2.1 _{These virulence factors} aremainlyproducedbyShigelladysenteriaeandShigatoxigenic group ofEscherichia colilike E. coli O157:H7 which are able tocauseinfectious diseases.2,3 _{ThetoxinisoneoftheAB}

5

toxins and hasbinding (B) and catalytic domains (A). The pentamericBsubunitofthetoxinisresponsibleforreceptor

∗ _{Correspondingauthorat:AppliedMicrobiologyResearchCenter,BaqiyatallahUniversityofMedicalSciences,VanakSq.MolasadraSt.,}

P.O.Box19395-5487,Tehran,Iran.

E-mailaddress:imanifouladi.a@gmail.com(A.A.ImaniFooladi).

bindingandintracellulartraffickingoftheholotoxins.4_The toxinbindstoGb3locatedoncellsurfacesandisintroduced by endocytic uptake. N-glycosidase activity of the A sub-unit inhibits protein synthesis in the cell and causes cell death.5_{Insomecellsthesetoxinsalsotriggercytokine} syn-thesis and induce apoptosis, which is caused by ribotoxic stress.6 _{The gene encoding toxin in Shigella dysenteriae is} chromosomal.HoweverstxgeneinE.coliO157:H7is associ-atedwithaprophage.7_{Differentsubtypesofshigatoxinare} identifiedasstx1,stx1c,stxfc,stx2,stx2e,stx2dandstx2g.8

http://dx.doi.org/10.1016/j.bjid.2015.02.008

Infections by shiga toxins producing bacteria have world-wideprevalenceandarewidespreadindevelopingcountries suchassouth-easternAsiancountries,Indiansubcontinent, SouthAfrica,centralAsia,andBangladesh.9_{InfectionofShiga} toxin producing bacteria is a major health concern even in developed countries all over the world. These bacteria are potentialcause ofdiarrhea,hemorrhagic colitis,severe inflammationsofileocolonic regionsofthegastrointestinal tract,thrombocytopenia,septicemia,centralnervoussystem (CNS)involvement,malignantdisordersinurinaryducts,and hemolyticuremic syndrome(HUS).10 _{urinarytractinfection} byEHECismainlygeneratedbyE.coliO157.Inchildrenunder fiveyearsoldandadultsover60,astheyhavethereceptor, itcauseskidneyfunctiondeficiencyandhasadeathrateof 5–10%.11 _{Cows,goatsand other animalscannaturally bea} sourceofstxproducingE.coliandotheranimalssuchascrabs alsoplayarole initstransfer.12 _{Transferbetweenhumans} canalsotakeplace.13_{Sinceshigatoxinscausemanydiseases,} especiallyinchildrenandimmunocompromisedelderly peo-ple,arapidandsensitivediagnosticmethodwithprognostic informationwould berather useful.So far,many different detectionmethodssuchascellculture,serologicaland molec-ularmethodssuchasRPLA,real-time,PCR,hybridizationhave beenutilizedtodetectshigatoxinsortheirrespectivegenes.14 Yet,allthesemethodshavetheirownshortcomingsasthey are time-consuming, quite costly and have limitations in handlingmanysamplessimultaneously.Atpresent,although molecularmethodssuchas PCRandhybridization, despite beingless time-consuming,less costly,andmoresensitive, theyare notsuitable astheyrelyon agarose electrophore-siswithcarcinogenethidiumbromide,amajorhealththreat forlabpersonnel,anddoesnotallowanalysisofmany sam-plesatatimeincaseofepidemicbreakouts.15_{Nevertheless,} real-timePCRdespite100%specificityandhighsensitivityhas notgainedmuchattentionbecauseofhighcostsof fluores-centmaterialandshortageofexpertpersonnel.16_Toovercome shortcomingsoftheaforementionedmethods,PCR-ELISAis anappropriatealternativeapproachfordetectingstxgenes, whichissafeandnon-radioactive.PCR-ELISAismore conve-nientforrapidand reliabledetection and quantificationof pathogen-specificgene sequences.17,18 _{Besideshavingbeen} usedinmedicalandfoodindustries,PCR-ELISAhasalsobeen usedintheveterinaryindustry.19

Inthis study,specific primersand probes forstx genes, amplificationandlabelingofproductsDIG-dUTP, hybridiza-tion ofstreptavidinwith biotinylatedprobes and detection with antibody against conjugated digoxigenin and peroxi-daseinmicrotiterplatesweredesigned.Weidentifiedspecific sequencesofstxgenesinalargenumberofsamplesusing smallamountssimultaneouslywithconsiderablesensitivity andshortturnaroundtime.

Materials

and

methods

Bacterialstrains

ShigelladysenteriaeandE.coliO157:H7strainswereprovided

fromShahedUniversity,Tehran,IranandPseudomonas

aeru-ginosa, Salmonellatyphimurium,Salmonellaparatiphi, Klebsiella

pneumoniaandVibriocholerastrainswereprovidedby

Baqiy-atallah University of Medical Sciences, Tehran, Iran. The strainswereverifiedbybiochemicalandimmunologic meth-ods.

ExtractionofbacterialgenomicDNA

ToextractgenomicDNAfrom ShigelladysenteriaeandE.coli O157:H7bacteriawereincubatedinliquidLBmediumfor18h in 37◦_{C.Bacterial culture was centrifuged in 3000rpmfor}

5min.Thepelletwasresuspendedin300␮LTEbufferfollowed bylysissolutioncontaining10␮Llysozyme(10mg/mL),200␮L SDS20%,3␮LproteinaseKandincubatedin37◦_Cfor60min.

DNAwaspurifiedbyextractionwithanequalvolumeof phe-nol/chloroform/isoamylalcohol(25:24:1)inthepresenceof5M sodiumperchlorate.A1/10volumeof3Msodiumacetateand 2volumesofabsoluteethanolwereaddedandincubatedin −20◦Cfor13h.Thegenomewaspelletedbycentrifugation, washed with70% ethanol and dried.Finally, DNA samples weredissolvedin100␮LTEbufferand,toeliminateRNA,3␮L RNaseAwasaddedandthetubeswereincubatedin37◦_Cfor

30min.

Theconcentration andpurityofthe DNAsampleswere determinedspectrophotometricallyatA260andA280by Nan-oDrop2000,ThermoScientific(USA).

Primerandprobedesign

Todesignprimers andthe nucleic-acid sequencingprobes, centralpartsofshigatoxingeneswereusedastemplate. Fea-turesinthedesignedprimerssuchasGCcontent;Tm,G,etc. were checkedbyDNASISand Oligo7softwares.Theprimer andprobesequencesareillustratedinTable1.The oligonu-cleotidesweresuppliedbyCinnaClone(IRAN).

Isolationandamplificationofstx1andstx2genesbyPCR

PCRreagentsinafinalvolumeof25␮Lincluded: 1␮L tem-plateDNA,0.5␮LTaqDNApolymerase(5U/␮L),1␮Lofeach primer(10pmol/␮L),1␮LdNTPmixture,2.5␮L10×PCRbuffer, 1.5␮LMgCl2(50mM)and16.5␮LsterileDDW.Thermalcycling

ofamplificationmixturewasperformedin30cycles.20 ThePCRprogramwas carriedout at94◦_Cfor3min

fol-loweddenaturingfor45sat94◦_{C,annealingfor45sat59}◦_C

and an extension for 1min at 72◦_{C. The final extension}

was at72◦_{C for5min. PCRproductswere electrophoresed}

in 1%agarosefollowed bystainingwithethidium bromide

(0.5␮g/mL) then visualized under ultraviolet light, and the

resultswererecordedbyphotography.PCRproductswere ver-ifiedbysequencing.ForlabelingofPCRproducts,thereaction ofPCRwas performedbydNTP mixturecontaining digoxi-geninlabelingmix(DigoxigenindNTPs,Roche,Germany)with thesamecondition.

DetectionofPCRproductsbyELISA

Table1–PCRprimersandhybridizationcaptureprobeforstx1andstx2genes.

Oligonucleotide Sequence Nucleotideposition Expectproductsize(bp)

Ahstx1F TTGTTTGCAGTTGATGTCAGAGG 210–233 275

Ahstx1R CAGGCAGGACACTACTCAACCTTC 676–700

Ahstx2F TTGCTGTGGATATACGAGGGC 214–235 490

Ahstx2R CGCCAGATATGATGAAACCAGTG 466–489

pStx1 Biotin-TGTTGCAGGGATCAGTCGTACG 422–444 –

pStx2 Biotin-CATATATCAGTGCCCGGTGTG 355–376 –

productofstx1 andstx2geneswere addedto90␮L1×SSC

bufferin1.5mLtubesandincubatedinboilingwaterfor10min andthen5minonice.Inthenextstep,10␮Lofstx1andstx2

probeswereaddedtoeachtubeandafter2hin60◦_C,100␮L

fromthishybridizationsolutionwereaddedtoeachwelland after1hin37◦_{C,itwaswashedwith20%BPSTbuffer3times.A}

1/1000solutionofanti-digoxigeninantibodyconjugatedwith peroxidesinPBSTbufferwaspreparedand100␮Lofthis

solu-tionwasaddedtoeachwell(includingcontrols)andafter1h in37◦_{Ctheplateswerewashedanddriedasdescribed}

ear-lier.100␮Lofsubstratesolution(2mgOPD,100␮Ldetection

buffer,5␮L30%hydrogenperoxide)wasaddedtowells.100␮L

of1MH2SO4wasaddedtostopthereaction.21Theoptical den-sitywasmeasuredat490nmusinganELISA reader(Dynex Technologies,Guornesey,ChannelIslandsandGreatBritain).

EvaluationPCRspecificityandsensitivity

Todeterminethespecificity,PCRwascarriedoutwithgenomic DNA extracted from Pseudomonas aeruginosa, Salmonella

typhimurium, Salmonella paratiphiC, Klebsiellapneumonia and

Vibriocholera.Productswereanalyzedon1%agarosegel.To

determine the minimum genomic DNA concentration that couldbedetectedbythemethod,seriallydilutedgenomicDNA inTEbuffer(pH=8)wasusedasPCRtemplateandtheproduct wasanalyzedon1%agarosegel.

SensitivityevaluationofPCR-ELISAtechniqueusing labeledPCRproductsofstx1andstx2

To determine the detection limit for stx gene, genomic DNA was extracted and tenfold serial dilutions containing 108ng/␮Lto0.108pg/␮Land156ng/␮Lto0.156pg/␮LofE.coli O157:H7and Shigelladysenteriae genomicDNA,respectively, werepreparedandallthestepswerecarriedoutasdescribed previously.22

PCR-ELISAspecificityevaluationusingbacterialstrains

ToevaluatespecificityofPCR-ELISA,E.coliO157:H7,Shigella

dysenteriae, Pseudomonas aeruginosa, Salmonella typhimurium,

Salmonella paratiphiC and Klebsiellapneumonia strains were

growninLBmedium.GenomicDNAswereextractedandafter evaluatingtheirconcentrationbyNanoDrop,10−2_folddilution

waspreparedastemplates.ThePCRwascarriedout accord-ingtomentionedprotocolsandtheproductswereanalyzed byagarosegelelectrophoresisandELISA.22

Clinicalsamplesanalysis

Inthisstudy63positivesamplesofShigelladysenteriaeand E.coliO157:H7obtainedfromstoolandurinecultureswere analyzed.SamplesweregatheredfromMazandaranhospitals ina3-monthperiodbetweenJanuaryandMarchof2013.Age groupsincludedchildrenbetween8monthsto10yearsold, adultsbetween20and30 andbetween50and 90yearsold (Table2).Samplesweretransferredtothelab,cultured,and aftergrowthwereidentifiedindifferentialandspecificmedia andverifiedbyspecificantiserums.FollowingDNAextraction byboilingmethod,PCR-ELISAwascarriedout.Dataanalysis wasperformedonSPSS.

Results

PCR

DNA from extracted genome of bacteria cultured in LB mediumwas availableinlargequantitiesand was ofgood quality. Thepurity ofthe DNA sampleswas confirmed by absorbance(A260/A280)ratio,whichwas1.8–2.0.PCRwas per-formedforeachstrainwithspecificprimers.EachPCRproduct wasobtainedasclearbandat275and490bpgeneratedbystx2 andstx1genesrespectively(Fig.1b).ThesizesofPCRproducts werethesameaspredicted.

1 2 3 2 1

490bp

275bp

500bp

300bp

a

b

Fig.1–AgarosegelelectrophoresisofextractedE.coli

O157:H7andShigelladysenteriaegenomicDNAandanalysis

Table2–Clinicalcharacteristicsofpatients.

PatientID Age(years) Sex Typeofspecimen Clinicalmanifestations Stxgenotype

Diarrhea Abdominalpain

2 30 F Bloodystool + + Stx1/Stx2

3 20 F Bloodystool + + Stx1/Stx2

8 8 M Bloodystool + + Stx1/Stx2

10 6 M Bloodystool + + Stx2

14 20 F Bloodystool + − Stx2

15 56 M Bloodystool + − Stx2

18 20 M Bloodystool + − Stx2

20 6 F Bloodystool + + Stx2

23 9 M Waterystool + − Stx2

27 8 M Bloodystool + + Stx1/Stx2

32 57 M Bloodystool + − Stx2

35 9 M Bloodystool + − Stx2

43 25 F Bloodystool + + Stx2

47 50 F Bloodystool + − Stx2

53 7 M Bloodystool + + Stx2

55 55 F Waterystool + − Stx2

59 8 M Bloodystool + − Stx2

a

b

275bp 490bp

7 6 5 4 3 2 1 1 2 3 4 5 6 7

Fig.2–SpecificityofPCRdetectionofE.coliO157:H7(a)andShigelladysenteriae(b).Lane1,positivecontrol;Lane2,100bp DNAladder;Lane3,Pseudomonasaeruginosa;Lane4,Klebsiellapneumonia;Lane5,Vibriocholera;Lane6,Salmonella typhimurium;Lane7,SalmonellaparatyphiC.

SpecificityofPCR

AftervalidationofPCRproducts,specificity ofthereaction was examined using genomes of Pseudomonas aeruginosa,

Salmonellatyphimurium,SalmonellaparatiphiC,Klebsiella pneu-moniaandVibriocholera.AsshowninFig.2,nocross-reaction

was found between the individual primers and non-target pathogensinthePCR.

SensitivityofPCR

After preparing a serial dilution from E. coli O157:H7 and

Shigella dysenteriae ata primary concentration of108ng/␮L

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

a

b

275bp

490bp

490bp

275bp

1 2 3 4 5

Fig.4–RepresentativeelectrophoreticgelsofPCRproducts labeledwithdigoxigenin.Lane1,100bpDNAladder;Lane 2,PCRproductofstx1fromShigelladysenteriaegenomic DNA;Lane3,PCRproductofstx1fromShigelladysenteriae

genomicDNAlabelingwithdNTPDIGmix;Lane4,PCR

productofstx2fromE.coliO157:H7genomicDNA;Lane5,

PCRproductofstx2fromE.coliO157:H7genomicDNA

labelingwithdNTPDIGmix.

and 156ng/␮L,the PCRreactions were performedon these dilutionsandtheresultsaredisplayedinFig.3.Regarding con-centrationsoftheprimarysample(108ng/␮Land156ng/␮L), sensitivityofthereactionwas calculatedas1.08pg/␮Land 1.56pg/␮L,respectively.

SpecificPCRwithdNTPDIGlabelingmix

APCRreactionwasperformedwithdigoxigeninlabelingmix andtheresultswereanalyzedon1%agarosegel(Fig.4).

PCR-ELISAspecificityandsensitivityassay

ThespecificityofthePCR-ELISAwasanalyzedusinggenomes

ofPseudomonasaeruginosa,Salmonellatyphimurium,Salmonella

paratiphiC,KlebsiellapneumoniaandVibriocholera.

Todetermine the minimumdetectable concentration of genomic DNA of Shigella dysenteriae and E. coli O157:H7, serialdilutionswere subjectedtothePCR-ELISAtechnique. Theresults(Fig. 5)demonstratethe possibilityofdetecting 1.08pg/␮Land1.56pg/␮LE.coliO157:H7andShigella dysente-riae,respectively.

Clinicalresults

Atotalof63clinicalsampleswerecollectedandscreenedfor thepresenceofE.coliO157:H7andShigelladysenteriaestrains.

Seventeen(26.98%)samplesweredetectedasstxpositive. Agedistributionofthepatientsrangedfrom<1to90years. E.coliO157:H7andShigelladysenteriaeinfectedpatientshad an average age of 23.17 years, and 47.05% were less than 10 years old (Table 2). Themost common symptoms were bloodystools(88.23%)andabdominalpain(47.05%).Sex dis-tribution ofE. coli O157:H7 and Shigella dysenteriaeinfected patientswere7females(41.18%),and10males(58.82%). Bac-teriologicalcultureresultswerecomparedtothoseobtained byPCR-ELISA.ResultsobtainedfromPCR-ELISAassay(Fig.6) andfromselectiveculturewerecompared.Therewasa sig-nificantassociationbetweenPCR-ELISAandcultureresultsfor detectingandscreeningE.coliO157:H7andShigelladysenteriae. (p<0.001).

Discussion

Shigatoxinproducingbacteriainfectionisamajorhealth con-cernevenindevelopedcountriesallovertheworld.9,23,24_With increasingreports ofE.coliO157:H7 and Shigelladysenteriae infections,greatattentionhasbeengiventothedevelopment ofmethodsfordetectingthesepathogensandapproachesfor preventionandtreatmentoftheseinfections.Thisstudywas intendedtoreachpartoftheseaims.

Inrecentyears,ELISAtesthasbeendesignedtodetectshiga toxinsdirectlyinstoolsamples.Thetestisrapidandhasa goodpotentialforshigatoxindetectionsinceitcandetectthe presenceofshigatoxin-producingE.coli(STEC)orothershiga toxin-producingbacteria.Sinceshigatoxintype differentia-tionrequireshighcostmonoclonalandpolyclonalantibodies it isnotwidelyused.Hybridization methodisaneffective, highly sensitive and specificmolecular method forprecise detectionofshigatoxins,andusesnon-radioactivesubstances suchasdigoxigeninandbiotin.However,sinceitisnot suit-ableforusinginlargenumberofclinicalsamples,thismethod isnotusedinmanyclinicallabs.25

In contrast toserological and microbiologicaltests, PCR providesarapidandsensitivealternative.Thistechnique,first developedbyKarchandMeyer,includesaprimerpairfroma

1.4

1.2

1

0.8

0.6

0.4

0.2

0

1 2 3 4 5 6 7 8

DNA concentration (ng-pg)

E. coli O157:H7 (stx 2)

Shigella dysentriae (stx 1)

0.5 1.5

1

0 2 2.5

OD:

492nm

Bacterial samples

E.coli O1157:H7 Shigella...

NEG

Vibr io cho

lerae 01 Salmonella...

Pseudomonas ...

Enterobacter Klebcillae

OD:

492 nm

0

2 3 8 10 14 15 18 20 23 27 32 35 43 47 53 55 59 Neg

Clinical samples

0.2 0.4 0.6 0.8 1 1.2 1.4

OD: 492 nm

E. coli 0157 :H7 (stx 2) Shigella dysentriae (stx 1)

Fig.6–PCR-ELISAanalysisofclinicalsamples,E.coliO157:H7(stx2)andShigelladysenteriae.(stx1).Negativecontrolsare alsoindicated.Assaycut-offwascalculatedbyreplicateanalysisofnegativesamples(OD:0.125).

conservedregionofstx1andstx2inhomologousgeneswhose maindefectswere lowTm and ineffectivenessindifferent typesofshigatoxins.15_{Inthatregard,todetectdifferenttypes} ofshigatoxinsitisnecessarytodesignamultiplexPCRwithat leasttwopairsofprimerstodetectshigatoxingene.Thefirst studyonmultiplexPCRdetectionshigatoxinswasbyCebula etal.whodesignedspecificprimersandusedafragmentas positiveinternalcontrol.Nonetheless,primerswereonlyable todetectstx2.26_{SubsequentstudieswerecarriedoutbyPaton} etal.,Passetal.,Philotetal.,andBelangeretal.Theirmain dis-advantagewasthatallfragmentswerelow-sizeandwerehard todissolveinagarosegel.14,27,28_{Theprimersthatwedesigned} forthisstudy,aftercomparisonwithavailabledatafromgene banks,using softwareand experimenton clinicalsamples, provedtolacktheabovementioneddisadvantagesandtobe ableofamplifydifferentshigatoxingenes.Inaddition,these amplifiedfragmentswereeasilydissolvedinagarosegeland bothstx1andstx2genescouldbesimultaneouslydetectedin clinicalsamples.OnepotentialproblemofPCRisthepresence ofinhibitors,whichmaycausefalseresultsofthetest.InGram negativebacteriadifferentlipids,carbohydrates,andproteins presentincellwallcanactasinhibitorsandreducethe sensi-tivityofthereaction.29_{Toovercometheselimitationsandto} increasesensitivity,bacterialgenomemustbepurifiedbefore carryingoutthereaction.Franketal.in1998usedthe boil-ingmethodtoextractgenomicDNA;however,inthismethod inhibitorscouldnotbeeliminated.30_{Wangetal.in2002used} SDS,lysozyme,tris,glucoseandEDTAforextractionwhich increasedsensitivityto100pmol/␮L.31_{In2011,Marzonyetal.} removedinhibitorsandextractedgenomicDNAbyusingCTAB and5MNaClandreportedasensitivityof2.1pg/␮L.32 Com-paringPCR-ELISAwithVerocellassays,Fachetal.33_showed thatresultswithPCRwereobtainedwithin24hwhereaswith Vero cell assaysresultswere available onlyafterfivedays. Furthermore,Beilei et al.developedPCR-ELISA methods to

detectEscherichiacoliO157:H7andotherShigatoxin-producing E. coli (STEC)in food, where105 _{CFU ofSTEC per gram of}

groundbeefdetectedwithoutanycultureenrichmentby PCR-ELISA.34

In this study, inhibitors were effectively eliminated by using TE buffer, lysozyme, 20% SDS, proteinase K, RNase A,3MSodiumAcetateand coldisopropanol.Consequently, sensitivity of the test improved to 1.08pg/␮L. PCR-ELISA was successfully carried out with genomic DNA extracted fromclinicalsamples;fromShigelladysenteriae,a490bp frag-mentwasamplifiedasmentionedearlier.ForE.coliO157:H7 both490and 275bpfragmentswereamplified demonstrat-ing that it can produce both toxins. PCR-ELISA correctly confirmed the specific stx1 and stx 2 genes. Non-positive results for other strains indicate that the tests are highly specific. Moreover, we have compared the performanceof PCR-ELISA with standard agarose gel electrophoresis. PCR-ELISAassaywasmoresensitivethanthegelelectrophoresis. Our data indicate that PCR-ELISA is highly specific, and hashighersensitivitythanconventionalgelelectrophoresis. By offering shorter turnaround time and high sensitiv-ity, PCR-ELISA has the potential to serve as a powerful detectiontoolinmedicineandinfoodandagricultural indus-tries.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

submittedto AppliedMicrobiology Research Center, Baqiy-atallahUniversityofMedicalSciences,Tehran,Iraninpartial fulfillmentoftherequirementsfortheMScinmicrobiology.

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