title: Evaluation of commercially available and in-house reverse transcription-PCR assays for detection of hepatitis G virus or GB virus C.
abstract: Serum samples from 96 Spanish hemodialysis patients, as well as serial dilutions of RNA extracted from a reference strain of hepatitis G virus (HGV), were tested for HGV or GB virus C (GBV-C) RNA. Two different reverse transcription (RT)-PCR-based methods of detection were compared for the ability to detect RNA extracted from the samples: an RT-nested PCR assay with primers derived from the 5' noncoding region (5'NC) or nonstructural region 3 (NS3) sequences and a commercially available RT-PCR assay with primers derived from the 5'NC or NS5A sequences. When RT-nested PCR was performed on 10-fold serial dilutions of RNA from the HGV reference strain, the last positive dilution was 10(-7) to 10(-8). With the commercial RT-PCR assay, the last positive dilution was 10(-6) to 10(-7). When equal amounts of RNA extracted from serum samples from 96 hemodialysis patients were tested for HGV or GBV-C RNA, 25 patients (26%) were positive by the RT-nested PCR. However, only 21 (84%) of these 25 positive patients were positive for HGV or GBV-C by the commercial RT-PCR assay. Analysis of the 5'NC and NS3 sequences amplified by RT-nested PCR demonstrated that all but two positive patients had unique HGV or GBV-C sequences. In summary, RT-nested PCR and a commercially available RT-PCR assay for HGV or GBV-C gave concordant results for 96% of the patients tested.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0740, USA. xforns@atlas.niaid.nih.gov
authors.forename: X
authors.initials: X
authors.lastname: Forns
authors.forename: D
authors.initials: D
authors.lastname: Tan
authors.forename: H J
authors.initials: HJ
authors.lastname: Alter
authors.forename: R H
authors.initials: RH
authors.lastname: Purcell
authors.forename: J
authors.initials: J
authors.lastname: Bukh
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D019143:Evolution, Molecular; D018067:Flaviviridae / Q000145:classification / Q000235:genetics*; D006525:Hepatitis, Viral, Human / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D006435:Renal Dialysis / Q000009:adverse effects; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D011933:Reagent Kits, Diagnostic