title: A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus replication.
abstract: The detection of negative-strand hepatitis C virus (HCV) RNA is a hallmark of replication. A highly sensitive and specific method is required to quantify the very low level of replication inherent to in vitro infection systems. Based on reverse transcription with a tagged primer in the 5' non-coding region of the HCV genome, followed by a nested PCR with a second round of real-time PCR, a novel method is described with improved sensitivity for negative-strand HCV RNA quantification. The lower detection level was 25 copies per reaction of negative-strand HCV RNA, even in the presence of 1 x 10(5) copies of positive-strand HCV RNA. This protocol was applied to the detection of negative HCV strand RNA in the liver of HCV-infected patients as well as in primary human hepatocytes infected in vitro. In both models, and particularly in each of three, independent in vitro infection experiments, this assay permitted the quantitation of HCV replication.
authors.affiliation: UPRESS 1833, Faculté de Médecine Paris 5, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Carrière
authors.forename: Véronique
authors.initials: V
authors.lastname: Pène
authors.forename: Adrien
authors.initials: A
authors.lastname: Breiman
authors.forename: Filoména
authors.initials: F
authors.lastname: Conti
authors.forename: Sandrine
authors.initials: S
authors.lastname: Chouzenoux
authors.forename: Eliane
authors.initials: E
authors.lastname: Meurs
authors.forename: Muriel
authors.initials: M
authors.lastname: Andrieu
authors.forename: Patrick
authors.initials: P
authors.lastname: Jaffray
authors.forename: Lilia
authors.initials: L
authors.lastname: Grira
authors.forename: Olivier
authors.initials: O
authors.lastname: Soubrane
authors.forename: Philippe
authors.initials: P
authors.lastname: Sogni
authors.forename: Yvon
authors.initials: Y
authors.lastname: Calmus
authors.forename: Stanislas
authors.initials: S
authors.lastname: Chaussade
authors.forename: Arielle R
authors.initials: AR
authors.lastname: Rosenberg
authors.forename: Philippe
authors.initials: P
authors.lastname: Podevin
mesh_terms: D002478:Cells, Cultured; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D019698:Hepatitis C, Chronic / Q000821:virology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008099:Liver / Q000166:cytology / Q000821:virology; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014779:Virus Replication
chemical_list: D012367:RNA, Viral