Simultaneous amplification and detection of specific hepatitis B virus and hepatitis C virus genomic sequences in serum samples.
A sensitive and specific two-stage polymerase chain reaction (PCR) technique was developed for the simultaneous amplification and detection of specific genomic sequences of hepatitis B virus (HBV) and hepatitis C virus (HCV) in serum samples. Initially, HCV-RNA was reverse transcribed to cDNA. This cDNA and DNA from HBV were then co-amplified using primer pairs derived from conserved regions of HBV and HCV nucleotide sequences. The specificity of PCR products was confirmed by liquid hybridization analysis using 32P end-labeled oligomer probes specific for the target HBV and HCV nucleotide sequences. Independent human serum samples, positive and negative by PCR for both HBV-DNA and HCV-RNA, were used as controls. We tested sera from nine donors, of which seven were reactive for HBsAg, anti-HBc, and anti-HCV (multiantigen test), one of whom was reactive for anti-HCV and anti-HBc, and one of whom was reactive for HBsAg and anti-HBc. The assay detected HBV- and HCV-specific genomic sequences in eight of eight sera reactive for both HBV and HCV serological markers and also in the serum that was reactive for HBV markers only.
Nedjar
S
S
Laboratory of Hepatitis, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland.
Mitchell
F
F
Biswas
R
R
D001483:Base Sequence; D017931:DNA Primers; D016679:Genome, Viral; D006508:Hepatitis Antibodies / Q000097:blood; D006509:Hepatitis B / Q000097:blood / Q000235:genetics*; D006526:Hepatitis C / Q000097:blood / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D006508:Hepatitis Antibodies; D015345:Oligonucleotide Probes; D012367:RNA, Viral