title: "Single step" PCR with a sensitivity similar to nested PCR for the detection of hepatitis C virus RNA.
abstract: OBJECTIVE
  Evaluation of the performance of different HCV PCR detection systems for HCV RNA: A nested PCR, considered the reference assay, was compared with two single-step methods (ss-PCR): the first is based on the detection of PCR products by liquid hybridization with a 32P end-labelled probe (isotopic ss-PCR), while the second assay is a colorimetric method (colorimetric ss-PCR) using microwell plate hybridization with a specific nucleic acid probe (Amplicor HCV PCR, Roche Diagnostics Systems).
  
  
  METHODS
  Sera from 56 patients with suspected hepatitis C infection based on reactive serology or altered liver parameters, and sera from 15 blood donors were tested for HCV RNA: After RNA extraction, the synthesized HCV cDNA was amplified in parallel using isotopic ss-PCR, colorimetric ss-PCR and nested PCR. The products were detected by autoradiography, color development and ethidium bromide fluorescence, respectively.
  
  
  RESULTS
  In order to assess the analytical sensitivity of ss-PCR versus that of nested of PCR, experiments included serial dilutions of positive control samples. Results showed that both methods had an extinction signal at the 1:512 dilution. A comparative analysis of 71 clinical sera samples was obtained using the three protocols and the results clearly documented 100% concordance.
  
  
  CONCLUSIONS
  Single step PCR methods for HCV RNA have a sensitivity equal to that of nested PCR and appear more suitable for diagnostic applications. Ss-PCR is safer than nested PCR in terms of both specificity and contamination problems. In particular, the Roche Amplicor HCV PCR assay minimizes sample exposure and management problems.
authors.affiliation: Laboratory of Virology, AIDS Center S. Luigi, Hospital S. Raffaele, Milan, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Farma
authors.forename: E
authors.initials: E
authors.lastname: Boeri
authors.forename: G
authors.initials: G
authors.lastname: Morsica
authors.forename: J
authors.initials: J
authors.lastname: McDermott
authors.forename: L
authors.initials: L
authors.lastname: Soldini
authors.forename: C M
authors.initials: CM
authors.lastname: Repetto
authors.forename: B
authors.initials: B
authors.lastname: Ferioli
authors.forename: G
authors.initials: G
authors.lastname: Pellizzari
authors.forename: F
authors.initials: F
authors.lastname: Molinari
authors.forename: A
authors.initials: A
authors.lastname: Molinelli
mesh_terms: D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral