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17.9.24AOACOfficialMethod998.09SalmonellainFoodsColorimetricPolyclonalEnzymeImmunoassayScreeningMethodwithRappaport-Vassiliadis(R10)BrothandTetrathionateBroth(TECRASalmonellaVisualImmunoassay)FirstAction1998RevisedFirstAction1999FinalAction2001(MethodisscreeningprocedureforpresenceofSalmonellainallfoods;itisnotaconfirmatorytestbecausepolyclonalantibodiesusedintestmaycross-reactwithsmallpercentageofnon-Salmo-nellaorganisms.Testspositivebytheimmunoassaymustbecon-firmedbybiologicalandserologicalprocedures.)SeeTables998.09A–Cfortheresultsoftheinterlaboratorystudysupportingtheacceptanceofthemethod.A.PrincipleDetectionofSalmonellaantigensisbasedonenzymeimmunoassayusinghighlypurifiedantibodiespreparedfromanti-gensuniquetoSalmonella.PolyclonalantibodiestoSalmonellaan-tigensareadsorbedontointernalsurfaceof96-wellmicrotitertray.Brothtobetestedisplacedintoawellofatray.IfSalmonellaanti-gensarepresent,theywillattachtospecificantibodyadsorbedonwell.Allothermaterialiswashedaway.ConjugateisaddedandwillbindtoSalmonellaantigensiftheyareattachedtoadsorbedantibodyonsurfaceofwell.Wellsarewashedtoremoveunboundconjugate,andenzymesubstrateisadded.Darkblue-greencolorindicatespres-enceofSalmonellaantigen.©2002AOACINTERNATIONALTable998.09AStatisticalanalysesofcollaborativestudyresultsforTECRAassayusing2selectiveenrichmentbrothsFood(No.labs)MPNa(cfu/g)Methodagreement,%bc2cIncidenceoffalsenegativesamongtotalpositivesamples,%dSensitivityrate,%eIncidenceoffalsepositivesamongtotalnegativesamples,%fSpecificityrate,%gELISACultureELISACultureELISACultureELISACultureMilkchocolate(14)0.003100—h————1.48.699910.009100—0.00.01001000.02.3100980.0931000.00.00.01001000.02.110098Driedwholeegg(13)0.003100—————4.60.0951000.00998.40.00.02.8100971.10.0991000.430100—0.00.01001000.00.0100100Nonfatdrymilk(18)0.003100—————0.00.01001000.2498.80.01.30.0991000.0(1.2r)0.0100(99r)1002.4100—0.00.01001000.00.0100100Blackpepper(15)0.003100—————0.04.0100960.014100—0.00.01001000.00.9100990.088100—0.00.0100(97r)i1000.01.210099Soyflour(15)0.003100—————0.01.3100990.2498.7——0.01001001.21.299992.4100——0.01001000.00.0100100Rawturkey(12)0.003100—————5.0559545(BAM,7thEd.)0.04393.30.01.85.398.294.73.27.996.892.10.4695.01.30.05.110094.90.04.910095.1aMostprobablenumberofcolonyformingunitspergramoffood.bRatereflectsthenumberofconfirmeddeterminationsthatwereequivalentbetweentheTECRAandculturemethods.cc2isdefinedbyMcNemaras(|a–b|–1)2/(a+b)wherea=samplespositivebyTECRAandnegativebyculturemethodandb=samplesnegativebyTECRAandpos-itivebyculturemethod.Ac2valuegreaterthan3.84indicatessignificanceatP0.05.dIncidenceoffalsenegativesis100–sensitivityrate.eSensitivityrateisdefinedas100timesthetotalnumberofanalyzedpositivetestportionsamong“known”positivetestportionsdividedbytotalnumberof“known”testportions,where“known”positiveisdefinedassamplesconfirmedpositivebythereferencemethod.fIncidenceoffalsepositivesis100–specificityrate.gSpecificityrateisdefinedas100timesthetotalnumberofanalyzednegativetestportionsamong“known”negativetestportionsdividedbythetotalnumberof“known”negativetestportions,where“known”negativeisdefinedassamplesconfirmednegativebythereferencemethodandnegativecontrols.h—=Statisticalanalysisnotapplicable.ir=Readeronly.B.ReagentsItems(a)–(m)areavailableasTECRASalmonellaVisualImmunoassayfromTECRAInternationalPtyLtd.,POBox788,Wil-loughby,NSW,Australia,andInternationalBioProducts,Inc.,POBox0746,Bothell,WA98041,USA.Substitutionsmustbepretestedforequivalency.(a)Antibodyadsorbedstrips.—Removawell®Strips(DynatechTechnology,14340SullyfieldCircle,Chantilly,VA20151-1683,USA).PolyclonalantibodiestoSalmonella96wells.Storewellsat2–8 Cwhennotinuse.(b)Tray.—Sufficienttosecureindividualwellsorstrips.(c)Controlantigen.—Positivecontrol(lyophilized).PurifiedSalmonellaantigen,whichreactswithantibodiestoSalmonella,1vial.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 10H2O,0.12gSigmaDex-tranD9260producedbyLeuconostocmesenteroidesstrainB-512,0.06ghydrolyzedgelatin,0.0024gCaCl2 2H2O,and120ngthimerosal.Reconstitutedconjugateisstablefor2monthswhenstoredat2–8EC.(f)Conjugatediluent.—Twovials(13.5mL/vial).Contains0.42gNa2B4O7 10H2O,0.193gCaCl2 2H2O,0.22ghydrolyzedgel-atin,and0.0022gthimerosalinH2O.©2002AOACINTERNATIONALTable998.09BStatisticalanalysesofresultsforTECRAassaywithselectiveenrichmentinRappaport-Vassiliadis(R10)brothonlycomparedtoBAM,7thEd.Food(No.labs)MPNa(cfu/g)Methodagreement,%bc2cIncidenceoffalsenegativesamongtotalpositivesamples,%dSensitivityrate,%eIncidenceoffalsepositivesamongtotalnegativesamples,%fSpecificityrate,%gELISACultureELISACultureELISACultureELISACultureMilkchocolate(14)0.003100—h————08.6100910.009100—0.00.0100
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