肺炎支原体巢式pcr的优化.ppt

ComparisonofP1and16SrRNAgenesfordetectionofMycoplasmapneumoniaebyoptimizednestedPCRinadultpatientsinZhejiang,China,汇报人：周子博,Mycoplasmapneumoniaeisafrequentcauseofcommunity-acquiredpneumoniafor10-40%inchildrenandadults.BecauseofthetreatmentofM.pneumoniainfectionwith-lactamantibioticsisineffectiveandtheclinicalmanifestationsofM.pneumoniaeinfectionarecomplicatedandnonspecific,soarapid,sensitiveandspecificlaboratorytestisvitalforearlydiagnosisofM.pneumoniaeinfection.ConventionaltestsfordetectingM.pneumoniaehavetheirlimitations.,Introduction,Introduction,SeveralPCR-relatedmethodsprovideenhancedsensitivityandhavebeensuccessfullyappliedforresearchpurposessuchasnestedPCR.TheP1adhesiongeneandthe16SrRNAgenehavebeenutilizedwidelyinPCRtechniquesasthetargetsfordetectionofM.pneumoniae.Inthisstudy,wesoughttoidentifythemoresensitiveandspecifictarget(P1or16SrRNA)inM.pneumoniaedetectionandtoevaluatetheuseofnestedPCRforthediagnosisofMPinfectionfrompatientsinwhomM.pneumoniaewassuspected.,Materialsandmethods,StrainsandclinicalsamplesDNApreparationOrthogonalarraydesignOptimizationofsinglefactorconditionsNestedPCRsensitivitytestDetectionofclinicalsamples,Orthogonalarraydesign,Table1NestedPCRfactorsandtheirlevelsfororthogonalprojects(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets),Orthogonalarraydesign,Table2OrthogonalarraydesignfornestedPCR(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets),Figure1:ElectrophoresisanalysisofvariednestedPCRproductsofMycoplasmapneumoniaeFHwiththetargetoftheP1adhesion(16SrRNA)gene.,M123456789,100bp,150bp,107bp,M123456789,144bp,150bp,P1gene,16SrRNA,Singlefactorexperiment,Atlast,wedeterminedthefinaloptimalnestedPCRreactionconditions:FortheP1gene,theoptimalcombinationofMg2+concentration3mM,primerconcentration0.3uM,annealingtemperature60,thefirstroundPCRproduct50-folddilution;Forthe16Sgene,themostexcellentcombinationofMg2+concentration3mM,primerconcentration0.3uM,annealingtemperature56,thefirstroundPCRproduct50-folddilution.,P1gene,16SrRNA,NestedPCRsensitivitytest,sensitivitiesofnestedPCRforM.pneumoniae:LaneM:DNAmarker.Lane1:20ngofM.pneumoniaeFHstrain;Lane2:10ng;Lane3:10-1ng;Lane4:10-2ng;Lane5:10-3ng;Lane6:10-4ng;Lane7:10-5ng;Lane8:10-6ng;Lane9:negativecontrol.,M123456789,P1gene:1pg,123456789,123456789,16SrRNA；0.1pg,Detectionofclinicalsamples,P1adhesiongene:(25/55;43.6%),Detectionofclinicalsamples,16SrRNAgene(30/55;56.3%),discussion,Withthedevelopmentofmolecularbiologytechniques,PCRtechnologyhasbecomethemostvaluablemethodforrapiddiagnosisofMycoplasmapneumoniaeinfection.BothP1adhesiongeneand16SrRNAgenearewidelyusedastargetsfordetectionofM.pneumoniaebyPCR.However,itisstillinconclusiveforwhichtargetisbetterandoureffortistofindthemostsuitableone.Inthisstudy,wejointedorthogonalexperimentandsinglefactorteststooptimizeseveralcrucialconditionsofnestedPCRandfinallyconcludedtheoptimumreactionconditionsofthetwotargets.Thenwedetectedthesensitivityofthetwotargetsonthebasisoftheoptimalconditionsandtheresultsshowedthatthe16SrRNAgeneismoresensitivethantheP1adhesiongene.Wealsopresentedastudybyusingclinicalspecimensfromadultpatientsandfoundthat16SrRNAgenehasahigherpositiveratethantheP1adhesiongene.Sothe16SrRNAgeneisthemostexcellenttargetfordetectionofM.pneumoniaebynestedPCR.,discussion,Inthisstudy,weadoptednestedPCRtocomparethetwotargets.ThesuperiorsensitivityisthemajoradvantageofnestedPCR.ThesensitivitycanbeincreasedbynestedPCRbecauseitinvolvesthereamplificationofaPCRproductwithasecondprimerset.NestedPCRmayleadtoa103-foldincreaseinsensitivitythansingle-stepPCR,asnestedPCRenablesthedetectionof1-100fgofDNA,andsingle-stepPCRassayscanonlydetect10-100pgofDNA.Abele-Hornetal.candetect30-100fgofM.pneumoniaeDNAbynestedPCR,andthesensitivityis103-foldbetterthanthatforsingle-stepPCRandexceedsthatforantigencaptureenzymeimmunoassayandcultureby104-to105-fold.,discussion,AlthoughnestedPCRisarapidandsensitivemethodforearlydiagnosisofM.pneumoniaeinfection,theimpactofnestedPCRreactionconditionsisnumerousanditistime-consumingtofindtheoptimalcondition.Whatismore,onlyonthebasisoftheoptimumconditionscanobtainamoreaccurateandobjectiveresults.SoweadoptedtheorthogonalarraydesigntooptimizeseveralcrucialfactorsaffectingthenestedPCRusingthestandardstrainofM.pneumoniae,sinceorthogonaltestdesigncangreatlyshortenthetestnumberandcanquicklyarriveatamoreappropriatereactioncondition.Thenwealsoutilizedacompletelysinglefactortestdesignbasedontheresultsoftheorthogonaldesign.Atlast,thefinaloptimalreactionconditionsofnestedPCRaredeterminedbyintegratedtheresultsofthemethodsabove,sothecomparisonoftheP1adhesiongeneandthe16SrRNAgeneprimersunderthisoptimalconditioncanbemoreobjectivethanthatofotherlaboratories.,discussion,Inourstudy,OrthogonalarraydesignwasadoptedtooptimizefourcommonfactorsaffectingthenestedPCR,whichweretheconcentrationofprimersandMg2+,dilutionmultipleofthefirstroundPCRproduct,annealingtemperature.AllofthemarethecriticalelementintheperformanceofnestedPCR.Firstly,throughthetestwefoundthatexcessivelylowprimerconcentrationcanreducePCRyieldandexcessivelyhighprimerconcentrationincreasetheprobabilityofmisprimingandgenerationsofnon-specificPCRproducts.Secondly,formourexperiments,IfMg2+concentrationwastoolow,theyieldofPCRproductcouldbereduced,sinceTapDNApolymerasesareMg2+-dependentenzymeanditissensitivetotheconcentrationofMg2+.Thirdly,ourresultsshowsthatpoorspecificityofamplifiedbandappearsatlowannealingtemperature,andweakenedamplifiedbandsathighannealingtemperature.ForthespecificityofPCRmainlydependsonannealingtemperatureandimprovetheannealingtemperaturewithinacertainrangecanincreasethespecificityofthePCRreaction.Lastly,wealsofoundthatalotofnon-specificbandsappearifnotdilution,thatwasprobablybecausethetemplateconcentrationofsecondroundofPCRisexcessivelyhigh.,discussion,ThesensitivityofnestedPCRwastestedbyusingserialdilutions(1:10)ofM.pneumoniaeDNA,ourresultssuggestedthatthe16SrRNAgeneprimersweremoresensitivethantheP1adhesiongeneprimers,asthe16SrRNAgeneprimerscandetectupto0.1pgofM.pneumoniaeDNAandtheP1geneprimerscandetect1pgofM.pneumoniaeDNAatmost.OurfindingsarewellconfirmedtothestudybyMohamedNouretal,whofoundthatthefragmentintensityaftervisualinspectionofgelswasalwayshigherwith16SrDNAprimersthanwiththosedirectedtoP1adhesiongene,thisshowedthattheamplificationofthe16SrRNAgenebynestedPCRweremoresensitiveforthedetectionofM.pneumoniae.Thiswasmainlybecausethepresenceofapproximately103copiesof16SrRNApermycoplasmacellandthehighdegreeofconservationoftherRNAgenesallowingahighlyfixationofprimersonthetargetandleadtoahigherPCRyield.Whatismore,duetotheRNAisdestroyedmorerapidlythantheDNAafterthedeathofthemycoplasmacell,detectionofRNAprovidesfurtherevidenceofviablemycoplasmasinthespecimen.K.Loensetal.thoughtthattheP1adhesiongeneprimerswerefoundtobemoresensitivethanthe16SrRNAones.However,theycometothisconclusionmerelybyspeculation,nottocomparetheboth.,discussion,Accordingtoourresultsfromclinicaltest,16SrRNAgeneprovedclearlytobethebesttargetforthispurpose,yieldingapositivePCRresultin56.3%ofcases,whilethepositiveratewas43