title: Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
abstract: BACKGROUND
  Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region.
  
  
  RESULTS
  Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a.
  
  
  CONCLUSIONS
  The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.
authors.affiliation: Laboratório Central do Estado (LACEN-PR), São José dos Pinhais, Paraná, Brazil.
authors.forename: Sueli M
authors.initials: SM
authors.lastname: Nakatani
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Santos
authors.forename: Irina N
authors.initials: IN
authors.lastname: Riediger
authors.forename: Marco A
authors.initials: MA
authors.lastname: Krieger
authors.forename: Cesar A B
authors.initials: CA
authors.lastname: Duarte
authors.forename: Marco A
authors.initials: MA
authors.lastname: Lacerda
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Biondo
authors.forename: Flair J
authors.initials: FJ
authors.lastname: Carrilho
authors.forename: Flair J
authors.initials: FJ
authors.lastname: Carilho
authors.forename: Suzane K
authors.initials: SK
authors.lastname: Ono-Nita
mesh_terms: D004279:DNA, Viral / Q000235:genetics; D016903:Drug Monitoring / Q000191:economics / Q000379:methods; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D004279:DNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
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