title: A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping.
abstract: BACKGROUND
  Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5' untranslated region (5'UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples.
  
  
  METHODS
  Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples.
  
  
  RESULTS
  The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method.
  
  
  CONCLUSIONS
  A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.
authors: Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil.
authors: Yara Silva
authors: YS
authors: Casanova
authors: Cachoeirinha, Cachoeirinha, RS, Brasil.
authors: Thais da Rocha
authors: Tda R
authors: Boeira
authors: Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil.
authors: Elisa
authors: E
authors: Sisti
authors: Cachoeirinha, Cachoeirinha, RS, Brasil.
authors: Álvaro
authors: Á
authors: Celmer
authors: Cachoeirinha, Cachoeirinha, RS, Brasil.
authors: André Salvador Kazantzi
authors: AS
authors: Fonseca
authors: Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil.
authors: Nilo
authors: N
authors: Ikuta
authors: Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil.
authors: Daniel
authors: D
authors: Simon
authors: Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil.
authors: Vagner Ricardo
authors: VR
authors: Lunge
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D017931:DNA Primers; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000175:diagnosis*; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D020121:5' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral