Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region 英文参考文献.docVIP

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Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region 英文参考文献.doc

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Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region 英文参考文献

DevelopmentofHepatitisCVirusGenotypingbyReal- TimePCRBasedontheNS5BRegion SueliM.Nakatani1,7,CarlosA.Santos2,IrinaN.Riediger1,8,MarcoA.Krieger3,CesarA.B.Duarte3, MarcoA.Lacerda4,AlexanderW.Biondo5,6,FlairJ.Carilho7,SuzaneK.Ono-Nita7* 1Laborato′rioCentraldoEstado(LACEN-PR),Sa?oJose′dosPinhais,Parana′,Brazil,2CentrodeGenomas,Sa?oPaulo,Sa?oPaulo,Brazil,3InstitutoCarlosChagas–Fundac?a?o OswaldoCruz(ICC-FioCruz),Curitiba,Parana′, Brazil,4DivisionofGastroenterologyandHepatology,IndianaUniversity,Indianapolis,Indiana,UnitedStatesofAmerica, 5DepartmentofVeterinaryMedicine,FederalUniversityofParana′, Curitiba,Brazil,6DepartmentofPathobiology,UniversityofIllinois,Urbana,Illinois,UnitedStatesof America,7DepartmentofGastroenterology,SchoolofMedicine,UniversityofSa?oPaulo(USP),Sa?oPaulo,Brazil,8DepartamentodeBiologiaCelular,UniversidadeFederal doParana′, Curitiba,Brazil Abstract Background:HepatitisCvirus(HCV)genotypingisthemostsignificantpredictoroftheresponsetoantiviraltherapy.The aimofthisstudywastodevelopandevaluateanovelreal-timePCRmethodforHCVgenotypingbasedontheNS5Bregion. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and anothertodetectgenotypes2a, 2b,and2c. Thisapproach hadanoverallsensitivityof 97.0%,detecting295ofthe304 testedsamples.Allsamplesgenotypedbyreal-timePCRhadthesametypethatwasassignedusingLiPAversion1(Linein ProbeAssay).AlthoughLiPAv.1wasnotabletosubtype68ofthe295samples(23.0%)andrendereddifferentsubtype resultsfromthoseassignedbyreal-timePCRfor12/295samples(4.0%),NS5Bsequencingandreal-timePCRresultsagreed inall146testedcases.Analyticalsensitivityofthereal-timePCRassaywasdeterminedbyend-pointdilutionofthe5000IU/ mlmemberoftheOptiQuantHCVRNApanel.Thelowerlimitofdetectionwasestimatedtobe125IU/mlforgenotype3a, 250IU/mlforgenotypes1band2b,and500IU/mlforgenotype1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours require