Single-step reverse transcription-polymerase chain reaction for hepatitis C virus RNA with DNA enzyme immunoassay hybridization.
A single reverse transcription-polymerase chain reaction (SRT-PCR) for HCV RNA detection, confirmed by hybridization of amplified products with biotinylated probes using DNA enzyme immunoassay (DEIA), was compared to nested-PCR (N-PCR) on 20 sera from patients with chronic (n = 18) or acute (n = 2) hepatitis. Results obtained by SRT-PCR confirmed by DEIA and by N-PCR identical. All but one of the patients with chronic hepatitis and positive HCV serology as well as the patients with acute hepatitis had detectable HCV RNA in serum; all patients with chronic hepatitis and indeterminate HCV serology and all controls (n = 5) were negative by the two PCR methods. Both SRT-PCR and N-PCR remained positive after 7 x 10(-2) and 5 x 10(-4) dilutions of two HCV RNA-positive sera. The threshold of detection for SRT-PCR was 15 RNA copies per assay, as assessed by testing serial dilutions of an in vitro synthesized 5'-NCR HCV RNA transcript. SRT-PCR confirmed by DEIA for HCV RNA appears to be as sensitive and specific as N-PCR. Furthermore, it is easier to perform, with less of contamination, is less time-consuming, requires fewer enzymes, and it permits semi-quantification of PCR products.
Payan
C
C
Laboratoire de Virologie, Hôpital Broussais, Paris, France.
Bloch
F
F
Gaultier
I
I
Bélec
L
L
D000208:Acute Disease; D000914:Antibodies, Viral / Q000097:blood; D001483:Base Sequence; D002908:Chronic Disease; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006505:Hepatitis / Q000097:blood / Q000821:virology*; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D000914:Antibodies, Viral; D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral