title: Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA.
abstract: Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany. jens.mueller@ukb.uni-bonn.de
authors.forename: Jens
authors.initials: J
authors.lastname: Mueller
authors.forename: Matthias
authors.initials: M
authors.lastname: Gessner
authors.forename: Anja
authors.initials: A
authors.lastname: Remberg
authors.forename: Jochen
authors.initials: J
authors.lastname: Hoch
authors.forename: Gerold
authors.initials: G
authors.lastname: Zerlauth
authors.forename: Peter
authors.initials: P
authors.lastname: Hanfland
mesh_terms: D001483:Base Sequence; D001782:Blood Donors; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006510:Hepatitis B Antibodies / Q000097:blood; D006801:Humans; D008403:Mass Screening; D011786:Quality Control; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D006510:Hepatitis B Antibodies; D012367:RNA, Viral