title: Single-step PCR in molecular diagnosis of hepatitis C virus infection.
abstract: The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection.
authors.affiliation: Laboratory of Virology, S. Luigi AIDS Centers, S. Raffaele Hospital, Milan, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Farma
authors.forename: E
authors.initials: E
authors.lastname: Boeri
authors.forename: P
authors.initials: P
authors.lastname: Bettini
authors.forename: C M
authors.initials: CM
authors.lastname: Repetto
authors.forename: J
authors.initials: J
authors.lastname: McDermott
authors.forename: F B
authors.initials: FB
authors.lastname: Lillo
authors.forename: O E
authors.initials: OE
authors.lastname: Varnier
mesh_terms: D001483:Base Sequence; D003124:Colorimetry; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology*; D006801:Humans; D015336:Molecular Probe Techniques; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
chemical_list: D017931:DNA Primers; D012367:RNA, Viral