AOACOfficialMethod996.10EnterohemorrhagicEscherichiacoli(EHEC).pdf

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1、17.4.01B(d)Microplatereader.Photometerwith405410nmfilter,forAOACOfficialMethod996.10readingmicrowellplates.Mayincludeoptionalprinter.EnterohemorrhagicEscherichiacoli(EHEC)O157:H7D.GeneralInstructionsDetectioninSelectedFoodsâStoreallreagentsat28°Cwhennotinuse.LetreagentswarmtoAssuranceP

2、olyclonalEnzymeImmunoassayMethodFirstAction1996roomtemperaturebeforeuse.IncludeduplicatepositiveandoneRevisedFirstAction1999blanktestwellswitheachrunoftestsamples.UseseparatepipetforFinalAction1999eachtestportionandreagenttoavoidcross-contamination.Usekitreagentsandcomponentsasanintegr

3、atedunitanddonotmixwith(ApplicabletodetectionofenterohemorrhagicE.coli[EHEC]O157componentsfromothermanufacturingbatchesorsources.Usededi-includingstrainH7indairyfoods,meats,poultryproducts,fruits,catedtroughorglasswareforeachreagenttoavoidnutmeats,seafood,pasta,andliquideggs.)cross-con

4、tamination.Donotusereagentsafterexpirationdate.Donotreusemicrowells.SeeTable996.10fortheresultsoftheinterlaboratorystudysup-portingtheacceptanceofthemethod.E.PreparationofTestSamples(a)Enrichment.Asepticallyweigh25gtestportionintoA.Principle225mLmTSB+n.Iflargertestportionsizesareanalyz

5、ed,propor-ProprietaryantibodieswithhighspecificitytoEHECantigensaretionatelyincreasevolumeofmTSB+ntomaintain1:9dilutionratio.boundtomicrowellplates.AppropriatelyenrichedtestsamplesandMixwell.Incubateovernight(1828h)at3537°C.positivecontrolsareaddedtoplates.AnyEHECantigenspresentwill(b)

6、PreparationofsampleforEIAanalysis.Followingincuba-bindtomicrowells,formingantibodyantigencomplex.Nonreactivetion,mixbottlecontentsthoroughly.Letparticulatemattersettle1materialiswashedaway.Alkalinephosphataseantibodyconjugateisminandtransfer1.0mLintotesttube.Retainthesampleandadded,and

7、,afterincubation,unboundconjugateiswashedaway.ThemTSB+nmixtureforconfirmationofpresumptivepositiveresults.substrate,p-nitrophenylphosphate,isaddedandabsorbanceofresult-ingcoloredproductisreadspectrophotometricallyat405410nm.Submerge1.0mLaliquotinboilingH2Obath20mintoinactivatemicroor