美国FDA细菌学分析手册第八版(BAM)产单核李斯特菌的血清学鉴定

BAM:SerodiagnosisofListeriamonocytogenesJanuary2001BacteriologicalAnalyticalManualChapter11SerodiagnosisofListeriamonocytogenesAuthors:ReginaldW.BennettandRobertE.WeaverAlthoughserologicalconfirmationisnotnecessaryforregulatoryidentificationofListeriamonocytogenes,itisusefulfordeterminingtheprevalenceofspecificserotypesinepidemiologicalstudiesandforenvironmentalrecontaminationtracking.EarlyattemptstoconfirmL.monocytogenesserologicallyweregenerallynotsuccessfulbecauseofcross-reactionswithotherorganisms(1).Serology,therefore,shouldalwaysfollowculturalandbiochemicalidentification(seeChapter10).SerologicalassaysofListeria,reviewedbyGrayandKillinger(4),includeagglutination,precipitation,andcomplementfixationassaysaswellasautomatedfluorescentantibodytechniques,usingflowcytometry(3)andrapidmethodELISAkits,whicharebasedonmonoclonalandpolyclonalantibodies(5).However,theseserologicalsystemsweredevelopedtodetectallListeriaspp.Theyhavebeenusedsuccessfullytoscreenfoodsforthegenus(seeChapter10),buttheydonotdifferentiateL.monocytogenesfromotherListeriaspecies.ThischapterpresentsproceduresfortheserologicalidentificationofL.monocytogenesbyflagellarandsomaticantigenicprofiles(2),usingagglutinationastheserologicaltool.1.A.Equipmentandmaterials1.Centrifuge2.Refrigerator(4-6°C)3.Steamer4.Inoculatingneedle5.Tubes,6x50mm6.Testtuberack(for6x50mmtubes)7.Dispenser,25,50,100µl,orcomparable8.Waterbath(48°C)9.Microscopeslides1.B.Media1andreagents21.EBmotilitymedium(M48)2.Tryptosephosphatebroth(TPB)(M168)3.Formaldehyde,37%solution4.Formalsaline,0.5%5.Physiologicalsaline,0.85%(R63)6.McFarlandNo.3turbidimetricstandard(R42)7.Somatic(O)antisera8.Flagellar(H)antisera1.C.Preparationofmaterialsandmedia1.EBmotilitymedium.Prepareasspecified.Distribute10m1amountsofmediumin18x125mmorcomparablesizescrew-captubes.Refrigeratemediumuntilused.2.Tryptosephosphatebroth(TPB).Prepareasdirected.Distribute8.0mlamountsin16x125mmorcomparablesizescrew-captubes.3.Formalsaline(0.5%).Addformaldehydeinaconcentrationof0.5%to0.85%physiologicalsaline.4.No.3McFarlandstandard.PrepareturbiditystandardNo.3oftheMcFarlandnephelometerscale.Mix3.0ml1%BaCl2solutionwith97.0ml1%H2SO4solution.5.HandOantisera.Diluteaccordingtomanufacturer'sdirections.6.Cells(antigenstobetested).PrepareL.monocytogenescellsasspecifiedunderprocedures.1.D.ProceduresTheuniformapproachtoserotypingisfirsttodeterminetheflagellar(H)serotype,andthentodeterminethesomatic(O)serotypeand/orsubserovar,dependingontherefinementoftheantisera.Specificsomatictypesareassociatedwithspecificflagellarserogroups.TherelationshipofsomaticandflagellarantigenicfactorsforL.monocytogenesisshowninTable1.Table1.Diagnosticschemeshowingrelationshipofsomatic(O)andflagellar(H)antigenicfactorsofL.monocytogenesHfactorsOfactorsA1a(1/2a)3a[1a(1);1a(1,2);3a(4)](b)C1b(1/2b)a;3b[4a(7,9);4b(5,6);4b(6);4d(8)](b)D2(1/2c)(a)3CaSeeligerandDonker-Voetdesignations.bBracketsindicatethatantiseratosomaticantigensareavailable.AntiseratoHantigensarealsoavailable.SchemeforroutineserodiagnosisofL.monocytogenes,basedonHandOantigenicfactors.2.RoutineserologicaltypingforflagellarantigensRemovegrowthfromagarslantwithstraightinoculatingneedleandstabtubeofEMmotilityagarifbothflagellarandsomaticantigensarerequiredforserodiagnosis.Seeschemeforgrowthandtreatmentevents.Incubateinoculatedmotilityagarat25°Cfor24-48h.PickcoloniesfromouteredgeofmotilegrowthonEBmotilityagar(Fig.1)andinoculatetubeofTPBorsimilaragar.IncubateinoculatedTPBfor18-24hat25°C.Fig.1.Typicalumbrella-likegrowthofL.monocytogenesonmotilityagar.Addformaldehydesolution(37%)forfinalvolumeof0.5%tobrothculture(0.04mlofformaldehydeto8.0mlofbrothculture).Allowformaldehyde-treatedbrothtoincubate4hat25°Cortreatasliveculture.Collectcellsbycentrifugation(1600xg,30min);thenresuspendcellsin0.5%formalsalinetoturbidityequaltoMcFarlandNo.3standard.Thebrothcellsuspensionworksequallyaswellasthewashedstandardizedcellsfortheagglutinationtest.In6x50mmtubes,mixcells(antigen)tobetestedwithequalvolumes(100l)ofpredetermineddilutionsofHantiserawithserologicalfactors,A,C,andD.Topreparenegativecontrol,place100loftestcellsinequalvolumeofsaline.Incubatetubescontainingbacterialcellsandantiseraaswellasnegativecontrolinwaterbathpresetat48°C.Observetubesforagglutinationafter1hincubation.Ifagglutinationoccurs,asedimentwillformandthesupernatantwillbeasclearasthenegativecontrol(orclearer).Agitatetubesslightly(executegentlywithfinger)toresuspendsediment.TypicaltubeagglutinationreactionisshowninFig.2.Fig.2.Comparativetubeagglutinationtestshowingpositiveagglutinationreaction(tubeonleftwithgranularappearance)andtypicalnegativereaction(tubeonrightwithsmooth,homogenousappearanceintheserodiagnosisofL.monocytogenes.2.RoutineserologicaltypingforsomaticantigensRemovegrowthfromagarslantorcomparablemediawithinoculatingneedleandinoculatetube(s)ofTPBifflagellarserodiagnosisisnotbeingdone.Ifbothflagellarandsomaticantigenprofilesarebeingdetermined,seeschemeforroutineserodiagnosisofL.monocytogenesbasedonHandOfactors.Collectcells(antigen)bycentrifugatio