A quantitative HCV-PCR test for routine diagnostics.
The aim of this study was to develop a reliable and simple method for hepatitis C virus (HCV)-PCR using standard, automated laboratory equipment. HCV-RNA was extracted from serum and amplified in a single PCR with an internal standard. The PCR product was detected using fluoroimmunoassay. Quantification was based on external and internal standards. Linearity was observed over a wide range (10-10(7) geq). Mean inter and intra serial coefficients of variation were 35% and 23%, respectively. The limit of quantification was 1000 geq/ml based on intra and inter serial variations, while levels of 110 geq/ml were always detectable. Lower concentrations were intermittently positive. The ability to separate HCV-signals in healthy and infected persons was good, based on the distribution of HCV-signals from 353 random blood donors and 191 patient samples. To illustrate the applicability of the test, HCV-RNA quantification was performed in 11 patients during treatment with interferon alpha-2b. Ten of 11 patients showed a decline in HCV-RNA within the first few weeks of treatment. After four weeks most patients were still HCV-RNA positive but below the limit of quantification. The present method for quantification of HCV-RNA was shown to have sensitivity at the level of nested PCR techniques. Until now HCV-PCR has been complicated, time-consuming and costly, and therefore not suitable for routine diagnostics. The PCR method described here is easy to perform, fast and cost-effective.
Krarup
H B
HB
Department of Clinical Chemistry, Aalborg Hospital, Denmark.
Drewes
A M
AM
Madsen
P H
PH
D001483:Base Sequence; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D011994:Recombinant Proteins
D017931:DNA Primers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins