New quantitative assay of hepatitis B and C viruses by competitive PCR using alternative internal sequences.
A competitive PCR was developed for quantitation of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA, alternatively, using only two constructions containing both priming sites. DNAs corresponding to the HBV-S gene and the HCV-5' non-coding region were introduced into distinct plasmids. HBV plasmid was used as a standard for HBV-DNA quantitation, in competition with the HCV plasmid as internal control. HBV and HCV plasmids also served as template for transcription of HBV-RNA, and HCV-RNA, which was used as internal control and standard, respectively, in competition for HCV-RNA quantitation. The analyzed samples for HBV and HCV quantitation were processed in the same way in competition with the internal controls and to the respective calibration curves obtained by serial dilutions of the mimic standard. This method showed very good specificity and sensitivity, allowing absolute quantitation in a large linear range from 5 viral genomic copies per assay up to 10(6) copies, in sera of chronically HBV and HCV infected patients, as well as in supernatants of cell cultures inoculated with these viruses.
Payan
C
C
Unit d'Immunologie Microbienne, Institut Pasteur, Paris, France.
Véal
N
N
Crescenzo-Chaigne
B
B
Bélec
L
L
Pillot
J
J
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics; D006526:Hepatitis C / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D016014:Linear Models; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014709:Vero Cells