Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA.docVIP

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Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA.doc

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Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA

Paraskevisetal.VirologyJournal2010,7:57 /content/7/1/57 METHODOLOGY OpenAccess Developmentofanewultrasensitivereal-time PCRassay(ultrasensitiveRTQ-PCR)forthe quantificationofHBV-DNA DimitriosParaskevis ,ApostolosBeloukas,CatherineHaida,AntigoniKatsoulidou,ZisisMoschidis1, 1* 1 1 1 HelenHatzitheodorou ,AgoritsaVaraklioti ,VanaSypsa,AngelosHatzakis1 1 2 1 Abstract Background:ImprovedsensitivityofHBV-DNAtestsisofcriticalimportanceforthemanagementofHBVinfection. Ouraimwastodevelopandassessanewultrasensitivein-housereal-timePCRassayforHBV-DNAquantification (ultrasensitiveRTQ-PCR). st Results:PreviouslyusedHBV-DNAstandardswerecalibratedagainsttheWHO1 InternationalStandardforHBV- DNA(OptiQuant?HBV-DNAQuantificationPanel,AccrometrixEuropeB.V.).The95%and50%HBV-DNAdetection end-pointoftheassaywere22.2and8.4IU/mL.Accordingtothecalibrationresults,1IU/mLequals2.8copies/mL. Importantlytheclinicalperformanceoftheultrasensitivereal-timePCRwastestedsimilar(67%)totheProcleix UltriodiscriminatoryHBVtest(dHBV)(70%)inlow-titersamplesfrompatientswithoccultHepatitisB.Finally,inthe comparisonofultrasensitiveRTQ-PCRwiththecommerciallyavailableCOBASTaqManHBVTest,thein-house assayidentified94.7%ofthe94specimensaspositiveversus90.4%identifiedbyTaqMan,whilethequantitative resultsthatwerepositivebybothassaywerestronglycorrelated(r=0.979). Conclusions:WereportanewultrasensitiverealtimePCRmolecularbeaconbasedassaywithremarkable st analyticalandclinicalsensitivity,calibratedagainsttheWHO1 Internationalstandard. Background quantitativeassaysforHBV-DNAhavebeendeveloped, Chronic hepatitis B virus (HBV) infection can be basedmostlyonreal-timePCRmethodology,showinga assessedbyevaluatingclinicalfeaturesandbiochemical, remarkablesensitivityandawidelinearrangeforquan- virologic, and histologic parameters. Knowledge of tification[6,7,11-14]. HBV-DNA viral load is useful for predicting disease Importantly,improvedsensitivityofHBV-DNAtestsis prognosis,determininginfectivity,evaluatin