肺炎支原体巢式PCR的优化ppt课件

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1、ComparisonofP1and16SrRNAgenesfordetectionofMycoplasmapneumoniaebyoptimizednestedPCRinadultpatientsinZhejiang,China汇报人：周子博Mycoplasmapneumoniaeisafrequentcauseofcommunity-acquiredpneumoniafor10-40%inchildrenandadults.BecauseofthetreatmentofM.pneumoniainfectionwithβ-lactamantibioticsisineffectiv

2、eandtheclinicalmanifestationsofM.pneumoniaeinfectionarecomplicatedandnonspecific,soarapid,sensitiveandspecificlaboratorytestisvitalforearlydiagnosisofM.pneumoniaeinfection.ConventionaltestsfordetectingM.pneumoniaehavetheirlimitations.IntroductionIntroductionSeveralPCR-relatedmethodsprovideenh

3、ancedsensitivityandhavebeensuccessfullyappliedforresearchpurposessuchasnestedPCR.TheP1adhesiongeneandthe16SrRNAgenehavebeenutilizedwidelyinPCRtechniquesasthetargetsfordetectionofM.pneumoniae.Inthisstudy,wesoughttoidentifythemoresensitiveandspecifictarget(P1or16SrRNA)inM.pneumoniaedetectionand

4、toevaluatetheuseofnestedPCRforthediagnosisofMPinfectionfrompatientsinwhomM.pneumoniaewassuspected.MaterialsandmethodsStrainsandclinicalsamplesDNApreparationOrthogonalarraydesignOptimizationofsinglefactorconditionsNestedPCRsensitivitytestDetectionofclinicalsamplesOrthogonalarraydesignFactorsLe

5、vels123Primer(μΜ)0.10.30.5Mg2+(mM)1.52.54Annealingtemperature(℃)58(54)60(56)62(58)DilutionmultipleNO50100Table1NestedPCRfactorsandtheirlevelsfororthogonalprojects(Theannealingtemperatureof16SrRNAgeneisexpressedinthebrackets)OrthogonalarraydesignFactorsReactionPrimer(μΜ)Mg2+(mM)AnnealingDiluti

6、onmultipletemperature(℃)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)Figure1:Electrophoresisanalysisofvar

7、iednestedPCRproductsofMycoplasmapneumoniaeFHwiththetargetoftheP1adhesion(16SrRNA)gene.M123456789100bp→150bp→←107bpM123456789144bp150bp→P1gene16SrRNASinglefactorexperimentAtlast,wedeterminedthefinaloptimalnestedPCRreactionconditions:FortheP1ge