title: Real-time multiplex PCR assay to quantify hepatitis C virus RNA in peripheral blood mononuclear cells.
abstract: Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ Detection System (Bio-Rad) coupled with TaqMan chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and C(T) values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R(2) > or = 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV < or= 3.94%) and reproducibility (CV < or = 2.20%). Quantitation of HCV RNA levels ranging from 10(3) to 10(7) IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor HCV Monitor test, v2.0 (Roche) (R(2) = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.
authors.affiliation: Division of Clinical Pathology, University Hospital, Geneva, Switzerland. pugnalp8@etu.unige.ch
authors.forename: Paolo
authors.initials: P
authors.lastname: Pugnale
authors.forename: Patrizia
authors.initials: P
authors.lastname: Latorre
authors.forename: Christine
authors.initials: C
authors.lastname: Rossi
authors.forename: Katia
authors.initials: K
authors.lastname: Crovatto
authors.forename: Valerio
authors.initials: V
authors.lastname: Pazienza
authors.forename: Andrea De
authors.initials: AD
authors.lastname: Gottardi
authors.forename: Francesco
authors.initials: F
authors.lastname: Negro
mesh_terms: D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D016022:Case-Control Studies; D005069:Evaluation Studies as Topic; D005814:Genes, Viral; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000097:blood*; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D019914:Taq Polymerase
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase