Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces 英文参考文献.docVIP

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Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces 英文参考文献.doc

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Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces 英文参考文献

DevelopmentandEvaluationofaSensitivePCR-ELISA SystemforDetectionofSchistosomaInfectioninFeces LucianaIna′ciaGomes1,Let?′ciaHelenadosSantosMarques1,MartinJohannesEnk2,MariaCla′udia deOliveira1,PauloMarcosZechCoelho2,AnaRabello1* 1Laborato′rio de Pesquisas Cl?′nicas, Centro de Pesquisas Rene′ Rachou, Fundac?a?o Oswaldo Cruz (Fiocruz), Belo Horizonte, Minas Gerais, Brazil, 2Laborato′rio de Esquistossomose,CentrodePesquisasRene′ Rachou,Fundac?a?o OswaldoCruz(Fiocruz),BeloHorizonte,MinasGerais,Brazil Abstract Background:APCR-enzyme-linkedimmunosorbentassay(PCR-ELISA)wasdevelopedtoovercometheneedforsensitive techniquesfortheefficientdiagnosisofSchistosomainfectioninendemicsettingswithlowparasiticburden. Methodology/Principal Findings: This system amplifies a 121-base pair tandem repeat DNA sequence, immobilizes the resultant 59 biotinylated product on streptavidin-coated strip-well microplates and uses anti-fluorescein antibodies conjugated to horseradish peroxidase to detect the hybridized fluorescein-labeled oligonucleotide probe. The detection limitoftheSchistosomaPCR-ELISAsystemwasdeterminedtobe1.3fgofS.mansonigenomicDNA(lessthantheamount foundinasinglecell)andestimatedtobe0.15S.mansonieggspergramoffeces(fractionsofanegg).Thesystemshowed goodprecision andgenusspecificitysincetheDNAtargetwasfoundin seven SchistosomaDNAsamples: S.mansoni,S. haematobium,S.bovis,S.intercalatum,S.japonicum,S.magrebowieiandS.rhodaini.Byevaluating206patientslivinginan endemicareainBrazil,theprevalenceofS.mansoniinfectionwasdeterminedtobe18%byexamining12Kato-Katzslides (41.7mg/smear,500mgtotal)ofasinglefecalsamplefromeachperson,whiletheSchistosomaPCR-ELISAidentifieda30% rate ofinfection using500-mg of thesamefecalsample. Whenconsidering theKato-Katz methodas thereference test, artificial sensitivity and specificity rates of the PCR-ELISA system were 97.4% and 85.1%, respectively. The potential for estimatingparasiticloadbyDNAdetectioninfeceswasassessedbycomparingabsorbancevaluesand