Determination of HCV RNA concentration by direct quantitation of the products from a single RT-PCR.
A novel method for the estimation of HCV RNA levels in vivo was developed, based on competitive RT-PCR. The use of the Tth DNA polymerase and 5' 32P-labeled antisense primer respectively reduced cross-contamination and permitted the direct quantification of viral loads by the analysis of the radioactivity of PCR products derived from a clinical sample and a competitive deleted template, separated previously on a polyacrilamide gel. A HCV fragment (H) and a competitive (deltaH) RNA templates were synthesized for optimizing the method. The minimal starting RNA detectable by RT-PCR was 40 copies. RT-PCR performed with ratios deltaH/H ranging from 1/1 to 1/20 revealed different relative percentages of both H and deltaH products, changing from 90% of deltaH product when the ratio was 1/1 to 5%, when it was 1/20. Regression analysis was adjusted to a linear model and served to further estimate HCV RNA loads from clinical samples. HCV RNA quantitation was carried out in 19 patients. Higher viral loads were related to type 1b infection and persistence of HCV RNA after interferon therapy. This method is simple, reproducible and useful for rapid estimation of HCV RNA load in vivo.
Pérez-Ruiz
M
M
Instituto de Parasitología y Biomedicina López Neyra CSIC, Hospital Clínico Universitario, Granada, Spain.
Torres
C
C
García-López
P A
PA
Ruiz-Extremera
A
A
Salmerón
J
J
Berzal-Herranz
A
A
D000998:Antiviral Agents / Q000627:therapeutic use; D001483:Base Sequence; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D007372:Interferons / Q000627:therapeutic use; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood*; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic; D019562:Viral Load; D014766:Viremia
D000998:Antiviral Agents; D012367:RNA, Viral; D007372:Interferons