简介：Objective:ToobtainrecombinantTreponemapallidumsubsp,pallidum(TP17KD)lipoproteininlargequantitiesbyamplificationandtofurtherpurifyantigensforlaboratorydiagnosisofsyphilisanddevelopmentofasyphilisvaccine.Method:TheTppl7lipoproteingenewasamplifiedfromtheTP(strainNichols),andthenitwasrecombinatedintoaplasmidpMAL-2candclonedwithinE.coli12-TB1.ThehostbacteriacontainingrecombinantplasmidswereinducedwithIPTG.TheTpp17KDlipoproteingenewasamplifiedbyus-ingPCRandpositivecloneswerescreenedwithdoubledigestionandPCR.RecombinantplasmidsweretransformedintoE.coliandtheE.colicarryingrecombinantplasmidswereinduced.TheexpressionofTP17KDwasdetectedbysodiumdedecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)andimmunoblot.Results:GelstainingwithCoomassieblueG-250showedthattheinducedE.colicarryingrecombinantplasmidcouldproduce60KDfusionproteinathighlevels.Gelscanningshowedthat17KDproteinexpressioninE.coliaccountedfor10%oftotalcellularprotein.Therecombinantproteinantigenreactedwiththeseraofsyphilispatients.Conclusion:Ourstudylaysacornerstonefordevelopingnewtechniquesoflaboratorydiagnosisforsyphilisandnewvaccines.Preliminaryclinicalapplicationshowedthatthefusionproteincouldbeusedforthediagnosisofsyphilis.

简介：Objective:Todevelopasensitive,specificandsimplemethodfordetectionofextremelylownumbersofT.palliduminclinicalspecimens,asasignificantadditiontotheserologictestsforsyphilisdiagnosis.Methods:Double-tubenestedPCR(DN-PCR)andsingle-tubenestedPCR(SN-PCR)assayswereperformedtoamplifyspecificfragmentsoftheDNApolymeraseIgene(polA)ofT.pallidum.SensitivityandspecificityofthetwoPCRassaysweretested.EightysixwholebloodspecimensfrompersonswithsuspectedsyphilisweredetectedbythetwonestedPCRmethods.TheTPPAtestwasusedasacomparisonfordetectingsyphilisinserafromcorrespondingpatients.Results:OnlyspecificampliconscouldbeobtainedduringamplificationoftheT.pallidumpolAgeneandthedetectionlimitwasapproximately1organismwhenanalyzedongelbythetwoPCRmethods.Of86clinicalspecimens,62werepositivebyTPPA.Ofthese,54and51werepositivebytheDN-PCRandSN-PCR,respectively,whichdoesnotrepresentastatisticallysignificantdifferencebetweenthetwoPCRtests.Of24TPPA-negativespecimens,5werepositivebybothDN-PCRassayandSN-PCRassay.Conclusion:TheSN-polAPCRmethodisextremelysensitive,specificandeasytoperformfordetectinglownumbersofT.palliduminclinicalbloodspecimensasacomplementarytoserologyforsyphilisdiagnosis.

简介：Objectives:TodevelopamethodofsimultaneousPCRdetectionofHaemophilusducreyi,Treponemapallidum,andHerpesSimplexVirusTypes1and2fromgenitalulcersamongpatientsattendingSTDclinicsinGuangzhou,China;andevaluatetheclinicalapplicationofmultiplexPCR(M-PCR)assayfordiagnosingtheetiologyofgenitalulcerdiseases(GUD).Methods:244patientswithagenitalulcerwereevaluated.ClinicaletiologyofGUDwasbasedonphysicalappearanceandmicrobiologicevaluationsthatincludeddarkfieldmicroscopyexamination(D-F)andserologytestforsyphilis(STS).SwabsofeachgenitalulcerweretestedforHSVantigenbyenzymeimmunoassay(EIA)andprocessedinanM-PCRassayforsimultaneousdetectionofT.pallidum,HSVandH.ducreyi.Results:ThestandardstrainsofT.pallidum,HSVandH.ducreyiwereamplifiedbyM-PCR,producingamplifiedproductsof260bp,432bp,170bp,respectively.ThesensitivityofM-PCRis102pgDNA.M-PCRassayforT.pallidum,HSVandH.ducreyishowedgoodagreementwhencomparedwithD-FdetectionforT.pallidum,STS,H.ducreyicultureandEIAforHSVantigen(Kappascoresare0.774,0.704,0.793,0.756,respectively).Conclusions:TheM-PCRisaconvenient,accurateandreliableassayforthedetectionofT.pallidum,HSVandH.ducreyifromgenitalulcers,andcanbeusedasamethodofdiagnosingtheetiologyofGUD.