Accurate Real-Time PCR Strategy for Monitoring Bloodstream Parasitic Loads in Chagas Disease Patients 英文参考文献.docVIP

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Accurate Real-Time PCR Strategy for Monitoring Bloodstream Parasitic Loads in Chagas Disease Patients 英文参考文献.doc

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Accurate Real-Time PCR Strategy for Monitoring Bloodstream Parasitic Loads in Chagas Disease Patients 英文参考文献

AccurateReal-TimePCRStrategyforMonitoring BloodstreamParasiticLoadsinChagasDiseasePatients TomasDuffy1,MargaritaBisio1,JaimeAltcheh2,JuanMiguelBurgos1,MirtaDiez3,MarianoJorgeLevin1, RobertoReneFavaloro3,HectorFreilij2,AlejandroGabrielSchijman1* 1LaboratoriodeBiolog?′aMoleculardelaEnfermedaddeChagas,InstitutodeInvestigacionesenIngenier?′aGene′ticayBiolog?′aMolecular(INGEBI-CONICET),BuenosAires, Argentina, 2Parasitology Unit of the ‘‘Ricardo Gutierrez’’ Children’s Hospital, Buenos Aires, Argentina, 3Transplant Unit of the Instituto de Cardiolog?′a y Cirug?′a Cardiovascular,Fundacio′n‘‘Rene′ Favaloro’’,BuenosAires,Argentina Abstract Background: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. Methodology/PrincipalFindings:TheQ-PCRhasadetectionlimitof0.1and0.01parasites/mL,withadynamicrangeof106 and107forSilvioX10cl1(T.cruziI)andClBrenerstocks(T.cruziIIe),respectively,anefficiencyof99%,andacoefficientof determination(R2)of0.998.Inordertoexpressaccuratelytheparasiticloads:(1)weadaptedacommercialkitbasedon silica-membrane technologyto enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimizePCRinhibition;(2)resultswerenormalizedincorporatingalinearizedplasmidasaninternalstandardofthewhole procedure;and(3)acorrectionfactoraccordingtotherepresentativityofsatellitesequencesineachparasitelineagegroup wasdeterminedusingamodifiedreal-timePCRprotocol(Lg-PCR).TheQ-PCRstrategywasapplied(1)toestimatebasal parasiteloadsin43pediatricChagasdiseasepatients,(2)tofollow-up38ofthemreceivingtreatmentwithbenznidazole, and(3)tomonitorthreechronicChagasheartdiseasepatientswhounderwentheart-transplantationanddisplayedevents ofclinicalreactivationduetoimmunosupression. Conclusion/Significance:Alltogether,thehighanalyticalsensitivityoftheQ-PCRstrategy,thelowlevelsofintra-andinter-