title: Detection of hepatitis C virus RNA by a combined reverse transcription PCR assay: comparison with nested amplification and antibody testing.
abstract: Many of the current reverse transcription (RT)-PCR assays for the detection of hepatitis C virus (HCV) RNA are multistep processes which use multiple enzymes and buffers. The assays are also often suboptimal, requiring nested amplification to achieve the desired levels of sensitivity. As a result, these assays are cumbersome and prone to false-positive results. The susceptibility to contamination is further aggravated by the lack of carryover controls. We have previously reported the development of a combined RT-PCR assay for HCV RNA detection which is sensitive and simple to perform. We have since successfully integrated dUTP-uracil-N-glycosylase carryover prevention into the combined assay. Restriction of as much as 0.5 microliter of deoxyuridine-containing amplification products has been achieved. The performance of the improved combined assay was compared directly with conventional nested RT-PCR and antibody detection. The combined assay was found to have sensitivity similar to that of nested RT-PCR in detecting HCV RNA from HCV antibody-positive specimens. In an analysis of hepatitis B virus antibody-positive specimens, nested amplification had false-positive rates ranging from 8 to 31%, while no false-positive results were seen with the combined assay. In comparison with serological methods, the combined assay had specificity and sensitivity of 100 and 95%, respectively.
authors.affiliation: Department of Infectious Diseases, Roche Molecular Systems, Inc., Alameda, California 94501, USA.
authors.forename: K K
authors.initials: KK
authors.lastname: Young
authors.forename: J J
authors.initials: JJ
authors.lastname: Archer
authors.forename: O
authors.initials: O
authors.lastname: Yokosuka
authors.forename: M
authors.initials: M
authors.lastname: Omata
authors.forename: R M
authors.initials: RM
authors.lastname: Resnick
mesh_terms: D001483:Base Sequence; D005189:False Positive Reactions; D005784:Gene Amplification; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006508:Hepatitis Antibodies / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
chemical_list: D006508:Hepatitis Antibodies; D012367:RNA, Viral