AOAC989_14

17.9.13AOACOfficialMethod989.14SalmonellainFoodsColorimetricPolyclonalImmunoassayScreeningMethodwithSeleniteCystineBrothandTetrathionateBroth(TECRASalmonellaVisualImmunoassay)FirstAction1989FinalAction1991Modification1997FinalAction1999[MethodisscreeningprocedureforpresenceofSalmonellainallfoods;itisnotaconfirmatorytestbecausepolyclonalantibodiesusedintestmaycross-reactwithsmallpercentageofnon-Salmo-nella.EnrichmentbrothsandMbrothsfromtestportionspositivebyenzymeimmunoassay(EIA)methodmustbestreakedonselectivemediaasin967.26B(see17.9.02)andtypicalorsuspiciouscoloniesmustbeidentifiedasin967.26C(see17.9.02),967.27(see17.9.03),and967.28(see17.9.07).]Determinationofpositiveresultmaybeperformed(1)visuallybyaidofcolorcomparatorcardwherepositiveresultisvalidwhenneg-ativeandpositivecontrolsmatchthosedescribedoncard,or(2)in-strumentally,usingfilterphotometer,wherepositiveresultisvalidonlywhennegativeandpositivecontrolspossessacceptableopticaldensityreadings.SeeTables989.14A–Cfortheresultsoftheinterlaboratorystudysupportingtheacceptanceofthemethod.A.PrincipleDetectionofSalmonellaantigensisbasedonEIAusinghighlypurifiedantibodiespreparedfromantigensuniquetoSalmonella.PolyclonalantibodiestoSalmonellaantigenareabsorbedontointer-nalsurfaceof96-wellmicrotitertray.Testportiontobeassayedisplacedintoawellofatray.IfSalmonellaantigensarepresentintestportion,theywillattachtospecificantibodyabsorbedonwell.Allothermaterialintestportionsiswashedaway.ConjugateisaddedandwillbindtoSalmonellaantigensiftheyareattachedtoabsorbedanti-bodyonsurfaceofwell.Wellsarewashedtoremoveunboundconju-gate,andenzymesubstrateisadded.Darkblue-greencolorindicatespresenceofSalmonellaantigenintestportion.B.ReagentsItems(a)–(m)areavailableasTECRASalmonellaVisualImmunoassayfromTECRAInternationalPtyLtd.,POBox788,Wil-loughby,NSW,Australia,andInternationalBioProducts,Inc.,POBox0746,Bothell,WA98041,USA.Substitutionsmustbepretestedforequivalency.(a)Antibodyabsorbedstrips.—Removawell®(DynatechLabo-ratories,Inc.)strips.PolyclonalantibodiestoSalmonella96wells.Storewellsat2–8°Cwhennotinuse.(b)Tray.—Sufficienttosecureindividualwellsorstrips.(c)Controlantigen.—Positivecontrol(lyophilized).PurifiedSal-monellaantigen,whichreactswithantibodiestoSalmonella,onevial.Reconstitutedcontrolantigenisstable2monthswhenstoredat2–8°C.(d)Controlsdiluent.—Onevial(6mL/vial).Contains0.006gTris[tris(hydroxymethyl)aminomethane],0.044gNaCl,0.0025gTween20(polyoxyethylene20sorbitanmonolaurate),and0.005gthimerosalinH2O.Controlsdiluentalsoservesasnegativecontrol.(e)Conjugate.—Twovials(lyophilized).Containsca150nganti-Salmonellaantibodies(fromsheep)conjugatedtohorseradishperoxidase,0.00686gNa2B4O7,0.12gDextranT10,0.06ghydro-lyzedgelatin,0.0024gCaCl2,and120ngthimerosal.Reconstitutedconjugateisstablefor28dayswhenstoredat2–8°C.(f)Conjugatediluent.—Twovials(13.5mL/vial).Contains0.42gNa2B4O7,0.193gNaCl,0.22ghydrolyzedgelatin,and0.0022gthimerosalinH2O.(g)Substrate.—Onevial(lyophilized).Contains0.011g2,2¢-azino-di(3-ethylbenzthiazolinesulfonate)and0.123gNaH2PO4.Reconstitutedsubstrateisstable2monthswhenstoredat2–8°C.ª2000AOACINTERNATIONALTable989.14AInterlaboratorystudyresultsfordeterminationofSalmonellainallfoodsbyTECRASalmo-nellaVisualImmunoassay(basedoninterlaboratorystudyresultsoftheoriginalmethod)ResultsPercent95%Confidencerange(approximately)Agreementa96.895.4–98.2Falsenegative(BAM)b1.60.5–2.7Falsenegative(EIA)c1.40.4–2.4aThisratereflectsnumberoftestportionsreadidenticallybetweenAOAC/BAM[BacteriologicalAnalyticalManual,currentedition,AOACIN-TERNATIONAL,Gaithersburg,MD,USA]culturemethodandEIA.bThisratereflectsnumberoftestportionsfoundtobepositivebyEIAbutnegativebyAOAC/BAMculturemethod.cThisratereflectsnumberoftestportionsfoundtobepositivebyAOAC/BAMculturemethodbutnegativebyEIA.Table989.14BInterlaboratorystudyresultsfordeterminationofSalmonellainallfoodsbyTECRASalmonellaVisualImmunoassay(basedoninterlaboratorystudyresultsoftheoriginalmethod)ProductTestportionanalyzedaTestportionspositiveBAM/AOACEIAc2bEIAconfirmedc2Pepper9664630.00630.00Soyisolate1089090—c90—Driedwholeegg120100100—100—Chocolate9679800.0079—Nonfatdrymilk10879780.00780.00Turkey11873820.75770.31aDoesnotincludetestportionseliminatedbecauseofoutlierdataormethoddeviations.bMcNemartestc2value3.84indicatessignificance(P0.05).cStatisticalanalysisnotapplicable.Methodsgaveidenticalresults.食品伙伴网(basedonin-housevalidationofthemodi-fiedmethod)FoodSerovarLevelofSalmonellainoculationaMPN/gbModified989.14PositivesIncidenceoffalsepositivesamongtotalnegativetestportionsc,%Incidenceoffalsenegativesamongtotalpositivetestportionsd,%SuspectConfirmedPepperS.agonaUninoculated0000Low0.036141400High0.073141400SoyflourS.heidelbergUninoculated0000Low0.036871030High0.230131300NonfatdrymilkS.bovismorbificansUninoculated0000Low0.03121200High0.052161600RawgroundpoultryS.ramgatanandS.sofiaUninoculated3(6Ve)015(30