1、EN18057June2025EuropeanstandardNormeeuropeenneEuropaischenormICS67.120.10EnglishVersionFoodauthenticity-QuantitationofroedeerDNArelativetomammalianDNAinmeatandmeatproductsbyreal-timePCRAuthenticitedesaliments-QuantificationdeADNdechevreuilparrapportaADNdemammiferedanslaviandeetlesproduitscarriesparP
2、CRentempsreelLebensmittelauthentizitat-QuantifizierungvonReh-DNAimVerhaltniszuSaugetier-DNAinFleischundFleischproduktenmittelsReal-time-PCRThisEuropeanStandardwasapprovedbyCENon4May2025.CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEuropeanSta
3、ndardthestatusofanationalstandardwithoutanyalteration.Up-to-datelistsandbibliographicalreferencesconcerningsuchnationalstandardsmaybeobtainedonapplicationtotheCEN-CENELECManagementCentreortoanyCENmember.ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlangua
4、gemadebytranslationundertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCEN-CENELECManagementCentrehasthesamestatusastheofficialversions.CENmembersarethenationalstandardsbodiesofAustria,Belgium,Bulgaria,Croatia,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France,Germany,Greece,Hungary
5、Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal,RepublicofNorthMacedonia,Romania,Serbia,Slovakia,Slovenia,Spain,Sweden,Switzerland,TiirkiyeandUnitedKingdom.EUROPEANCOMMITTEEFORSTANDARDIZATIONCOMITEEUROPEENDENORMALISATIONEuropaischeskomiteefurnormungCEN-CEN
6、ELECManagementCentre:RuedelaScience23,B-1040Brussels2025CENAllrightsofexploitationinanyformandbyanymeansreservedRef.No.EN18057:2025EworldwideforCENnationalMembers.ContentsPageEuropeanforeword3Introduction41 Scope52 Normativereferences53 Termsanddefinitions54 Symbolsandabbreviations65 Principle66 Rea
7、gentsandmaterials67 Apparatus88 Procedure88.1 Preparationofthetestsampleandtestportion88.2 PreparationofDNAextracts88.3 Preparationofroedeerandmammaliancalibrationstandards88.4 PCRsetup98.4.1 Samplesandcontrols98.4.2 Reactionmixes98.4.3 Thermalcycling109 Accept/rejectcriteria109.1 General109.2 Dataa
8、nalysis1110 Validationstatusandperformancecriteria1110.1 General1110.2 Repeatability1410.3 Reproducibility1410.4 Recovery1410.5 LimitofDetection(LOD)1410.6 Specificity1511 Testreport15Bibliography16EuropeanforewordThisdocument(ENl8057:2025)hasbeenpreparedbyTechnicalCommitteeCEN/TC460Foodauthenticity
9、thesecretariatofwhichisheldbyDIN.ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextorbyendorsement,atthelatestbyDecember2025,andcon11ictingnationalstandardsshallbewithdrawnatthelatestbyDecember2025.Attentionisdrawntothepossibilitythatsomeoftheelements
10、ofthisdocumentmaybethesubjectofpatentrights.CENshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.Anyfeedbackandquestionsonthisdocumentshouldbedirectedtotheusersnationalstandardsbody.AcompletelistingofthesebodiescanbefoundontheCENwebsite.AccordingtotheCEN-CENELECInternalRegulations,then
11、ationalstandardsorganisationsofthefollowingcountriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,Bulgaria,Croatia,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal,Republico
12、fNorthMacedonia,Romania,Serbia,Slovakia,Slovenia7Spain,Sweden,Switzerland,TiirkiyeandtheUnitedKingdom.IntroductionFoodauthenticityandintegrityarekeyaspectsintermsofconsumerprotection.Sincethelate1980s,globalizationhastakenplaceinthetradeoffood.Duringthelastdecades,manyofmethodsapplyingPCR,particular
13、lyreal-timePCRprotocols,havebeenestablishedfortheidentificationofanimalspeciesusedforfoodconsumption(e.g.pig,cattle,sheep,horse,chickenandturkey).Gamemeatisparticularlysusceptibletofraudulentlabellingsinceitismorevaluablethanmeatfromdomesticanimals.Avarietyofgamemeatproducts,commonlycontainingreddee
14、rorroedeer,iscommerciallyavailable.Forverifyingthecorrectlabellingofcommercialgamemeatproducts,thedevelopmentofharmonizedandstandardizedprotocolsfortheauthenticationofmeatproductsisimportanttoestablishreliablemethodsforthedetectionofpotentialfoodfraud.1 ScopeThisdocumentspecifiesareal-timePCRprocedu
15、reforthequantitationoftheamountofroedeerDNArelativetototalmammalianDNAinmeatandmeatproducts.Resultsofthisroedeerassayareexpressedintermsofroedeer(CaPreOlUSCaPreOlUS)haploidgenomecopynumbersrelativetototalmammalianhaploidgenomecopynumbers.Thecontentofroedeercanalsobeexpressedasmassfractionin%usinggra
16、vimetricallypreparedcalibrationmaterialfrommeatmixturesormodelsamples.ThemethodhasbeenpreviouslyvalidatedinacollaborativestudyandappliedtoDNAextractedfromsamplesthatconsistofrawroedeermeatinarawpigmeatbackgroundaswellasrawandboiledsausages.ThelimitofdetectionoftheroedeerPCRhasbeendeterminedexperimen
17、tallytobeatleast5targetgenecopiesoratleast0,1%roedeer.Thecomplianceassessmentprocessisnotpartofthisdocument.2 NormativereferencesThefollowingdocumentsarereferredtointhetextinsuchawaythatsomeoralloftheircontentconstitutesrequirementsofthisdocument.Fordatedreferences,onlytheeditioncitedapplies.Forunda
18、tedreferences,thelatesteditionofthereferenceddocument(includinganyamendments)applies.ENISO1178LMolecularbiomarkeranalysis-Requirementsandguidanceforsingle-laboratoryvalidationOfqualitativereal-timepolymerasechainreaction(PCR)methods(ISO11781)ENISO20813,Molecularbiomarkeranalysis-Methodsofanalysisfor
19、thedetectionandidentificationofanimalspeciesinfoodsandfoodproducts(nucleicacid-basedmethods)-Generalrequirementsanddefinitions(ISO20813)ENISO215712005,AsimpactedbyENISO21571:2005/Al:2013.Foodstuffs-MethodsOfanalysisforthedetectionofgeneticallymodifiedorganismsandderivedproducts-Nucleicacidextraction
20、ISO21571:2005)ISO16577,Molecularbiomarkeranalysis-Vocabularyformolecularbiomarkeranalyticalmethodsinagricultureandfoodproduction3 TermsanddefinitionsForthepurposesofthisdocument,thetermsanddefinitionsgiveninISO16577apply.ISOandIECmaintainterminologydatabasesforuseinstandardizationatthefollowingaddr
21、esses: ISOOnlinebrowsingplatform:availableathttps:/www.iso.org/obp/ IECElectropedia:availableathttps:/www.electropedia.org/4 SymbolsandabbreviationsForthepurposesofthisdocument,thefollowingsymbolsandabbreviationsapply:dNTPdeoxyribonucleotidetriphosphateDNAdeoxyribonucleicacidPCRpolymerasechainreacti
22、onHGEhaploidgenomeequivalentsAadenineCcytosineGguanineRpurine(guanineoradenine)Tthymine5 PrincipleTestsamplescontainingroedeerDNAinabackgroundofDNAfromothermammalianspeciesareanalysedbyarelativequantitationapproachutilizingSingleplexreal-timePCRassaystargetingthepromoterregionofthelactoferringeneofr
23、oedeer1andthemyostatingene2presentinmammalsandpoultry.DNAtemplateconcentrationisquantifiedpriortothereal-timePCRtonormalizetestinputlevels.DNAfrommodelmeatmixturesderivedfromauthenticatedroedeermeatinthemeatbackgroundofothermammalianspeciesisextracted,orsolutionsofgenomicroedeerDNAandDNAofothermamma
24、lianspeciesareusedanddilutedtogenerateseparatecalibrationcurvesforboththeroedeer-specifictargetandthemammaliantarget.TestsamplesextractedinthesamemannerasthecalibrationstandardsareevaluatedinthesamePCRrunusingtheroedeer-specificanduniversalmammalianreal-timePCRassays.DNAconcentrationsofroedeerandmam
25、malianDNAaredeterminedforthetestsamplesusingtheroedeerandmammaliancalibrationcurves.ThepercentageofroedeerDNAcontentinthetestsampleisexpressedasaratiooftheamountofroedeerDNArelativetothetotalmammalianDNApresentinthesample.6 ReagentsandmaterialsDuringtheanalysis,unlessotherwisestated,useonlyreagentso
26、frecognizedmolecularbiologygradeanddistilledordemineralizedwaterorwaterofequivalentpurity,accordingtoENISO20813.Regardinglaboratoryorganization,seeENISO20813.Althoughitisnotobligatorytousethereagents,instrumentationorconditionsspecifiedinthisdocumentWhenalaboratorychoosesnottodoso,thenitistherespons
27、ibilityofthatlaboratorytoensurethattheresultsobtainedarefitforpurpose.6.1 PCRmastermixPcRmastermixcontainsthermostableDNApolymerase,pHbuffer,KCbMgC127andthefourdNTPs(dATP,dCTP,dGTPanddTTP)anddUTPasadilutableconcentrate,whichisready-to-useandsuitableforthemakeandmodelofthermocycleremployed23.6.2 Olig
28、onucleotidesThequalityoftheoligonucleotidesshallbesufficientfortheiruseasprimersandprobes,seeTable1andTable2.Table1Oligonucleotidesforamplificationoftheroedeer-specificgeneregion1NameDNAsequenceofoligonucleotideFinalconcentrationinPCRRoedeerpromotorregionofthelactoferringene,GenBank4accessionnumberA
29、Y122040aroedeer2aFW(Forwardprimer)5-TGGCTGCTGCGTGCAGAA-30,2molLroedeer2aRV(Reverseprimer)5,-TCTAAAATGCTTGGGAACCAGATAT-3,0,2molLroedeer2aPr(Probe)5,-FAM-GAAGGGTCTCCGTCTGC-NFQ-MGB-3b0,1molLaPCRproduct=212-TGGCTGCTGCGTGCAGAATGAAGGGTCTCCGTCTGCCATATCTGGTTCCCAAGCATTTTAGA-273bFAM:6-Carboxyfluorescein,NFQ-M
30、GB:Non-AuorescentquencherminorgroovebinderTable2Oligonucleotidesforamplificationofthemammaliangeneregion2NameDNAsequenceofoligonucleotideFinalconcentrationinPCRMyostatingene,GenBank4accessionnumberAF320998aMYw-f(Forwardprimer)5,-TTGTGCARATCCTGAGACTCAT-30,2molLMY-r(Reverseprimer)5-ATACCAGTGCCTGGGTTCA
31、T-3,0,2molLMY-Probe(Probe)5-FAM-CCCATGAAAGACGGTACAAGRTATACTG-BHQl-3b0,1molLaPCRproduct=2500-TTGTGCAAATCCTGAGACTCATCAAACCCATGAAAGACGGTACAAGGTATACTGGAATCCgatctctgaaacttgacatgaacccaggcactggtat-2596bFAM:6-Carboxyfluorescein,BHQl:blackholequencher12Duringthecollaborativestudy,QuantiTect(B)MultiplexPCRKit
32、Qiagen)wasused.ThisinformationisgivenfortheconvenienceofusersofthisdocumentanddoesnotconstituteanendorsementbyCENoftheproductsnamed.Equivalentproductsmaybeusediftheycanbeshowntoleadtothesameresults.3TheformulationofthePCRmastermixemployedmustbesuitableforusewiththemakeandmodelofthethermocycleremplo
33、yed.Equivalentproductstothosespecifiedmaybeusediftheycanbedemonstratedtoleadtothesameresults.4GenBankisatrademarkofaproductsuppliedbytheNationalCenterforBiotechnologyInformation(NCBI).ThisinformationisgivenfortheconvenienceofusersofthisdocumentanddoesnotconstituteanendorsementbyCENoftheseproducts.6.
34、3 AuthenticroedeerDNAsampleThesampleshallconsistofrawmuscletissuetrimmedfreeofsurfaceinter-muscularfatandconnectivetissue.Thesourcematerialshouldbederivedfromanappropriatereferencematerial,ifavailable,orinhousematerialauthenticatedthroughappropriatemeatverificationtests,e.g.DNAsequencing-basedanalys
35、esorverificationbythecompetentauthority.PurifiedandquantitatedgenomicDNAderivedfromtheauthenticatedroedeermuscletissuewithamassfractionof100%shalladheretorecommendedqualitycharacteristics3(i.e.spectrophotometricmeasurementoftheA260A280l,8andA260A2301,8to2,2).7 ApparatusRequirementsconcerningapparatu
36、sandmaterialsshallfollowENISO20813.Thefollowingequipmentisrequired.7.1 Real-timePCRthermocyclerinstrumentAdevicethatamplifiesDNAinvitroandperformstemperature-timecyclesshallbeusedforPCR.Additionally,thedeviceshallbecapableofexcitingHuorescencemoleculesatspecificwavelengthsandsufficientlydetectingthe
37、emittedfluorescentlightoftheusedHuorophoretoperformreal-timePCRassaysusinghydrolysisprobes.8 Procedure8.1 PreparationofthetestsampleandtestportionThetestsampleusedforDNAextractionshallberepresentativeofthelaboratorysampleandhomogeneous,e.g.bygrindingorhomogenizingthelaboratorysampletoafinemixture.Te
38、stsampleandtestportionpreparationshallfollowthegeneralrequirementsandspecificmethodsspecifiedinENISO20813.8.2 PreparationofDNAextractsTheextraction,purification,andquantitationofDNAfromthetestportionshallfollowthegeneralrequirementsandmethodsspecifiedinENISO21571:2005.DNAextractionmethodsdescribedin
39、ENISO21571:2005,AnnexAarerecommended.ItistheresponsibilityoftheanalyticallaboratorytoensurethattheextractedDNAisfitforpurpose.Asindicatedin6.3,thepurifiedgenomicDNAshalladheretotherecommendedqualitycharacteristics3.8.3 PreparationofroedeerandmammaliancalibrationstandardsAnappropriatenumberofcalibrat
40、ionpointsandreplicatescoveringtherangeofquantitationshallbeapplied.AnexampleofthepreparationofcalibrationstandardsisshowninTable3.Preparea5-pointdilutionseries(Sl-S5)rangingfromapproximately29600to115roedeerhaploidgenomeequivalentsperPCRusingaroedeermodelmeatmixtureDuringthecollaborativestudy,amodel
41、meatmixturecontainingamassfractionof24,8%roedeermeatinpigmeatwasused.oramixtureofgenomicDNAsasthecalibrant.Table3Preparationofaroedeer5-pointcalibrationserieswithafourfolddilutionstepStandardDNAapgperPCRHGEperPCRabVolumeofstandardLVolumeofwaterLSl10000029600-0S225000740020(Sl)60S36250185020(S2)60S41
42、56246220(S3)60S539011520(S4)60aValueroundeddowntothenearestwholenumber.bGenomeequivalentsofthecalibrationstandardSlweredeterminedbydigitalPCR(1C-valueroedeer3,38pg)8.4 PCRsetup8.4.1 SamplesandcontrolsTheDNAextractedfromeachtestportionshallbeanalysedusingreplicatesandPCRcontrolsshallbeusedasspecified
43、inENISO20813.8.4.2 ReactionmixesThemethodisdevelopedforatotalvolumeof25LfortheroedeerPCRandthesameforthemammalianPCR.ThereactionsetupisgiveninTable4andshallbeperformedasfollows:一Reagentsshallbecompletelythawed. Eachreagentshallbecarefullymixedandbrie11ycentrifugedtoensurecollectionbeforepipetting. A
44、PCRreagentmixtureispreparedtocontainallcomponentsexceptforthesampleDNA.TherequiredtotalamountofthePCRreagentmixtureprepareddependsonthenumberofreactionstobeperformed,includingatleastoneadditionalreactionasapipettingreserve.SetupthePCRtestsasfollows: MixthePCRreagentmixture,centrifugebrieflytoensuret
45、hecollectionoftheliquidatthebottomofthemicrotubeandpipette20LintotheappropriatewellsofaPCRplateorPCRvials.一Add5LofeachsampleDNA(5ngL)orcalibrationstandards(SltoS5)orpositiveDNAtargetcontrolorextractionblankcontrolorwatertotherespectivewellsofaPCRplateorPCRvialsandseal. MixandcentrifugethePCRplateorP
46、CRvialsbrie11ytoremoveairbubblesandensureliquidcollectionatthebottomoftheplateorvials.Table4ReactionsetupfortheamplificationReagentcomponentRoedeerassay1Mammalianassay2PCRmastermix11Forwardprimer0,2molL0,2molLReverseprimer0,2molL0,2molLProbe0,1molL0,1molLWaterMakeupto20LMakeupto20LSample5Lvolume5Lvo
47、lume8.4.3ThermalcyclingTransferthepreparedreactionsintothethermocyclerandstartthetemperature-timeprogram.Thetemperature-timeprogramoutlinedinTable5wasusedinthecollaborativestudy6.Theuseofdifferentreactionconditionsandreal-timePCRcyclesshallbeverified.Thetimeforinitialdenaturationdependsonthemastermixused.Table5Temperature-timeprogramfortheroedeerandmammalianPCRsStepParameterTemperatureTimeCycles1Initialdenaturation/activationofthehot-startpolymerase95600s12AmplificationDenaturation9460s45Annealingandelongation6060s9 Accept/rejectcriter
