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[学习]肺炎支原体巢式pcr的优化[学习]肺炎支原体巢式pcr的优化Mycoplasmapneumoniaeisafrequentcauseofcommunity-acquiredpneumoniafor10-40%inchildrenandadults.BecauseofthetreatmentofM.pneumoniainfectionwithβ-lactamantibioticsisineffectiveandtheclinicalmanifestationsofM.pneumoniaeinfectionarecomplicatedandnonspecific,soarapid,sensitiveandspecificlaboratorytestisvitalforearlydiagnosisofM.pneumoniaeinfection.ConventionaltestsfordetectingM.pneumoniaehavetheirlimitations.IntroductionMycoplasmapneumoniaeisafreIntroduction

SeveralPCR-relatedmethodsprovideenhancedsensitivityandhavebeensuccessfullyappliedforresearchpurposessuchasnestedPCR.TheP1adhesiongeneandthe16SrRNAgenehavebeenutilizedwidelyinPCRtechniquesasthetargetsfordetectionofM.pneumoniae.Inthisstudy,wesoughttoidentifythemoresensitiveandspecifictarget(P1or16SrRNA)inM.pneumoniaedetectionandtoevaluatetheuseofnestedPCRforthediagnosisofMPinfectionfrompatientsinwhomM.pneumoniaewassuspected.Introduction

SeveralPCR-relatMaterialsandmethodsStrainsandclinicalsamplesDNApreparationOrthogonalarraydesignOptimizationofsinglefactorconditionsNestedPCRsensitivitytestDetectionofclinicalsamplesMaterialsandmethodsStrainsaOrthogonalarraydesign

FactorsLevels123Primer(μΜ)Mg2+(mM)1.52.54Annealingtemperature(℃)58(54)60(56)62(58)DilutionmultipleNO50100Table1NestedPCRfactorsandtheirlevelsfororthogonalprojects(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets)Orthogonalarraydesign

Orthogonalarraydesign

FactorsReactionPrimer(μΜ)Mg2+(mM)AnnealingDilutionmultipletemperature(℃)10.11.558(54)NO20.12.560(56)5030.1462(58)10040.31.560(56)10050.32.562(58)NO60.3458(54)5070.51.562(58)5080.52.558(54)10090.5460(56)NOTable2OrthogonalarraydesignfornestedPCR(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets)

Orthogonalarraydesign

Figure1:ElectrophoresisanalysisofvariednestedPCRproductsofMycoplasmapneumoniaeFHwiththetargetoftheP1adhesion(16SrRNA)gene.M1234567

8

9100bp→150bp→←107bpM123456789144bp150bp→P1gene16SrRNAFigure1:ElectrophoresisanalSinglefactorexperiment

Atlast,wedeterminedthefinaloptimalnestedPCRreactionconditions:FortheP1gene,theoptimalcombinationofMg2+concentration3mM,primerconcentration0.3uM,annealingtemperature60℃,thefirstroundPCRproduct50-folddilution;Forthe16Sgene,themostexcellentcombinationofMg2+concentration3mM,primerconcentration0.3uM,annealingtemperature56℃,thefirstroundPCRproduct50-folddilution.P1gene16SrRNASinglefactorexperimentAtlaNestedPCRsensitivitytestsensitivitiesofnestedPCRforM.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.M123456789P1gene:1pg12345678912345678916SrRNA；0.1pgNestedPCRsensitivitytestsenDetectionofclinicalsamplesP1adhesiongene:(25/55;43.6%)DetectionofclinicalsamplesPDetectionofclinicalsamples16SrRNAgene(30/55;56.3%)Detectionofclinicalsamples1discussionWiththedevelopmentofmolecularbiologytechniques,PCRtechnologyhasbecomethemostvaluablemethodforrapiddiagnosisofMycoplasmapneumoniaeinfection.BothP1adhesiongeneand16SrRNAgenearewidelyusedastargetsfordetectionofM.pneumoniaebyPCR.However,itisstillinconclusiveforwhichtargetisbetterandoureffortistofindthemostsuitableone.Inthisstudy,wejointedorthogonalexperimentandsinglefactorteststooptimizeseveralcrucialconditionsofnestedPCRandfinallyconcludedtheoptimumreactionconditionsofthetwotargets.Thenwedetectedthesensitivityofthetwotargetsonthebasisoftheoptimalconditionsandtheresultsshowedthatthe16SrRNAgeneismoresensitivethantheP1adhesiongene.Wealsopresentedastudybyusingclinicalspecimensfromadultpatientsandfoundthat16SrRNAgenehasahigherpositiveratethantheP1adhesiongene.Sothe16SrRNAgeneisthemostexcellenttargetfordetectionofM.pneumoniaebynestedPCR.discussionWiththedevelopmentdiscussionInthisstudy,weadoptednestedPCRtocomparethetwotargets.ThesuperiorsensitivityisthemajoradvantageofnestedPCR.ThesensitivitycanbeincreasedbynestedPCRbecauseitinvolvesthereamplificationofaPCRproductwithasecondprimerset.NestedPCRmayleadtoa103-foldincreaseinsensitivitythansingle-stepPCR,asnestedPCRenablesthedetectionof1-100fgofDNA,andsingle-stepPCRassayscanonlydetect10-100pgofDNA.Abele-Hornetal.candetect30-100fgofM.pneumoniaeDNAbynestedPCR,andthesensitivityis103-foldbetterthanthatforsingle-stepPCRandexceedsthatforantigencaptureenzymeimmunoassayandcultureby104-to105-fold.discussionInthisstudy,weaddiscussionAlthoughnestedPCRisarapidandsensitivemethodforearlydiagnosisofM.pneumoniaeinfection,theimpactofnestedPCRreactionconditionsisnumerousanditistime-consumingtofindtheoptimalcondition.Whatismore,onlyonthebasisoftheoptimumconditionscanobtainamoreaccurateandobjectiveresults.SoweadoptedtheorthogonalarraydesigntooptimizeseveralcrucialfactorsaffectingthenestedPCRusing

thestandardstrainofM.pneumoniae,sinceorthogonaltestdesigncangreatlyshortenthetestnumberandcanquicklyarriveatamoreappropriatereactioncondition.Thenwealsoutilized

acompletelysinglefactortestdesignbasedontheresultsoftheorthogonaldesign.Atlast,thefinaloptimalreactionconditionsofnestedPCRaredeterminedbyintegratedtheresultsofthemethodsabove,sothecomparisonoftheP1adhesiongeneandthe16SrRNAgeneprimersunderthisoptimalconditioncanbemoreobjectivethanthatofotherlaboratories.discussionAlthoughnestedPCRdiscussionInourstudy,OrthogonalarraydesignwasadoptedtooptimizefourcommonfactorsaffectingthenestedPCR,whichweretheconcentrationofprimersandMg2+,dilutionmultipleofthefirstroundPCRproduct,annealingtemperature.AllofthemarethecriticalelementintheperformanceofnestedPCR.Firstly,throughthetestwefoundthatexcessivelylowprimerconcentrationcanreducePCRyieldandexcessivelyhighprimerconcentrationincreasetheprobabilityofmisprimingandgenerationsofnon-specificPCRproducts.Secondly,formourexperiments,IfMg2+concentrationwastoolow,theyieldofPCRproductcouldbereduced,sinceTapDNApolymerasesareMg2+-dependentenzymeanditissensitivetotheconcentrationofMg2+.Thirdly,

ourresultsshowsthatpoorspecificityofamplifiedbandappearsatlowannealingtemperature,andweakenedamplifiedbandsathighannealingtemperature.ForthespecificityofPCRmainlydependsonannealingtemperatureandimprovetheannealingtemperaturewithinacertainrangecanincreasethespecificityofthePCRreaction.Lastly,wealsofoundthatalotofnon-specificbandsappearifnotdilution,thatwasprobablybecausethetemplateconcentrationofsecondroundofPCRisexcessivelyhigh.discussionInourstudy,OrthogdiscussionThesensitivityofnestedPCRwastestedbyusingserialdilutions(1:10)ofM.pneumoniaeDNA,ourresultssuggestedthatthe16SrRNAgeneprimersweremoresensitivethantheP1adhesiongeneprimers,asthe16SrRNAgeneprimerscandetectupto0.1pgofM.pneumoniaeDNAandtheP1geneprimerscandetect1pgofM.pneumoniaeDNAatmost.

OurfindingsarewellconfirmedtothestudybyMohamedNouretal,whofoundthatthefragmentintensityaftervisualinspectionofgelswasalwayshigherwith16SrDNAprimersthanwiththosedirectedtoP1adhesiongene,thisshowedthattheamplificationofthe16SrRNAgenebynestedPCRweremoresensitiveforthedetectionofM.pneumoniae.Thiswasmainlybecausethepresenceofapproximately103copiesof16SrRNApermycoplasmacellandthehighdegreeofconservationoftherRNAgenesallowingahighlyfixationofprimersonthetargetandleadtoahigherPCRyield.Whatismore,duetotheRNAisdestroyedmorerapidlythantheDNAafterthedeathofthemycoplasmacell,detectionofRNAprovidesfurtherevidenceofviablemycoplasmasinthespecimen.

K.Loens

etal.thoughtthattheP1adhesiongeneprimerswerefoundtobemoresensitivethanthe16SrRNAones.However,theycometothisconclusionmerelybyspeculation,nottocomparetheboth.discussionThesensitivityofn[学习]肺炎支原体巢式pcr的优化[学习]肺炎支原体巢式pcr的优化Mycoplasmapneumoniaeisafrequentcauseofcommunity-acquiredpneumoniafor10-40%inchildrenandadults.BecauseofthetreatmentofM.pneumoniainfectionwithβ-lactamantibioticsisineffectiveandtheclinicalmanifestationsofM.pneumoniaeinfectionarecomplicatedandnonspecific,soarapid,sensitiveandspecificlaboratorytestisvitalforearlydiagnosisofM.pneumoniaeinfection.ConventionaltestsfordetectingM.pneumoniaehavetheirlimitations.IntroductionMycoplasmapneumoniaeisafreIntroduction

SeveralPCR-relatedmethodsprovideenhancedsensitivityandhavebeensuccessfullyappliedforresearchpurposessuchasnestedPCR.TheP1adhesiongeneandthe16SrRNAgenehavebeenutilizedwidelyinPCRtechniquesasthetargetsfordetectionofM.pneumoniae.Inthisstudy,wesoughttoidentifythemoresensitiveandspecifictarget(P1or16SrRNA)inM.pneumoniaedetectionandtoevaluatetheuseofnestedPCRforthediagnosisofMPinfectionfrompatientsinwhomM.pneumoniaewassuspected.Introduction

SeveralPCR-relatMaterialsandmethodsStrainsandclinicalsamplesDNApreparationOrthogonalarraydesignOptimizationofsinglefactorconditionsNestedPCRsensitivitytestDetectionofclinicalsamplesMaterialsandmethodsStrainsaOrthogonalarraydesign

FactorsLevels123Primer(μΜ)Mg2+(mM)1.52.54Annealingtemperature(℃)58(54)60(56)62(58)DilutionmultipleNO50100Table1NestedPCRfactorsandtheirlevelsfororthogonalprojects(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets)Orthogonalarraydesign

Orthogonalarraydesign

FactorsReactionPrimer(μΜ)Mg2+(mM)AnnealingDilutionmultipletemperature(℃)10.11.558(54)NO20.12.560(56)5030.1462(58)10040.31.560(56)10050.32.562(58)NO60.3458(54)5070.51.562(58)5080.52.558(54)10090.5460(56)NOTable2OrthogonalarraydesignfornestedPCR(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets)

Orthogonalarraydesign

Figure1:ElectrophoresisanalysisofvariednestedPCRproductsofMycoplasmapneumoniaeFHwiththetargetoftheP1adhesion(16SrRNA)gene.M1234567

8

9100bp→150bp→←107bpM123456789144bp150bp→P1gene16SrRNAFigure1:ElectrophoresisanalSinglefactorexperiment

Atlast,wedeterminedthefinaloptimalnestedPCRreactionconditions:FortheP1gene,theoptimalcombinationofMg2+concentration3mM,primerconcentration0.3uM,annealingtemperature60℃,thefirstroundPCRproduct50-folddilution;Forthe16Sgene,themostexcellentcombinationofMg2+concentration3mM,primerconcentration0.3uM,annealingtemperature56℃,thefirstroundPCRproduct50-folddilution.P1gene16SrRNASinglefactorexperimentAtlaNestedPCRsensitivitytestsensitivitiesofnestedPCRforM.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.M123456789P1gene:1pg12345678912345678916SrRNA；0.1pgNestedPCRsensitivitytestsenDetectionofclinicalsamplesP1adhesiongene:(25/55;43.6%)DetectionofclinicalsamplesPDetectionofclinicalsamples16SrRNAgene(30/55;56.3%)Detectionofclinicalsamples1discussionWiththedevelopmentofmolecularbiologytechniques,PCRtechnologyhasbecomethemostvaluablemethodforrapiddiagnosisofMycoplasmapneumoniaeinfection.BothP1adhesiongeneand16SrRNAgenearewidelyusedastargetsfordetectionofM.pneumoniaebyPCR.However,itisstillinconclusiveforwhichtargetisbetterandoureffortistofindthemostsuitableone.Inthisstudy,wejointedorthogonalexperimentandsinglefactorteststooptimizeseveralcrucialconditionsofnestedPCRandfinallyconcludedtheoptimumreactionconditionsofthetwotargets.Thenwedetectedthesensitivityofthetwotargetsonthebasisoftheoptimalconditionsandtheresultsshowedthatthe16SrRNAgeneismoresensitivethantheP1adhesiongene.Wealsopresentedastudybyusingclinicalspecimensfromadultpatientsandfoundthat16SrRNAgenehasahigherpositiveratethantheP1adhesiongene.Sothe16SrRNAgeneisthemostexcellenttargetfordetectionofM.pneumoniaebynestedPCR.discussionWiththedevelopmentdiscussionInthisstudy,weadoptednestedPCRtocomparethetwotargets.ThesuperiorsensitivityisthemajoradvantageofnestedPCR.ThesensitivitycanbeincreasedbynestedPCRbecauseitinvolvesthereamplificationofaPCRproductwithasecondprimerset.NestedPCRmayleadtoa103-foldincreaseinsensitivitythansingle-stepPCR,asnestedPCRenablesthedetectionof1-100fgofDNA,andsingle-stepPCRassayscanonlydetect10-100pgofDNA.Abele-Hornetal.candetect30-100fgofM.pneumoniaeDNAbynestedPCR,andthesensitivityis103-foldbetterthanthatforsingle-stepPCRandexceedsthatforantigencaptureenzymeimmunoassayandcultureby104-to105-fold.discussionInthisstudy,weaddiscussionAlthoughnestedPCRisarapidandsensitivemethodforearlydiagnosisofM.pneumoniaeinfection,theimpactofnestedPCRreactionconditionsisnumerousanditistime-consumingtofindtheoptimalcondition.Whatismore,onlyonthebasisoftheoptimumconditionscanobtainamoreaccurateandobjectiveresults.SoweadoptedtheorthogonalarraydesigntooptimizeseveralcrucialfactorsaffectingthenestedPCRusing

thestandardstrainofM.pneumoniae,sinceorthogonaltestdesigncangreatlyshortenthetestnumberandcanquicklyarriveatamoreappropriatereactioncondition.Thenwealsoutilized

acompletelysinglefactortestdesignbasedontheresultsoftheorthogonaldesign.Atlast,thefinaloptimalreactionconditionsofnestedPCRaredeterminedbyintegratedtheresultsofthemethodsabove,sothecomparisonoftheP1adhesiongeneandthe16SrRNAgeneprimersunderthisoptimalconditioncanbemoreobjectivethanthatofotherlaboratories.discussionAlthoughnestedPCRdiscussionInourstudy,OrthogonalarraydesignwasadoptedtooptimizefourcommonfactorsaffectingthenestedPCR,whichweretheconcentrationofprimersandMg2+,dilutionmultipleofthefirstroundPCRproduct,annealingtemperature.AllofthemarethecriticalelementintheperformanceofnestedPCR.Firstly,throughthetestwefoundthatexcessivelylowprimerconcentrationcanreducePCRyieldandexcessivelyhighprimerconcentrationincreasetheprobabilityofmisprimingandgenerationsofnon-specificPCRproducts.Secondly,formourexperiments,IfMg2+concentrationwastoolow,theyieldofPCRproductcouldbereduced,sinceTapDNApolymerasesareMg2+-dependentenzymeanditissensitivetotheconcentrationofMg2+.Thirdly,

our

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