Comparative study of a modified competitive RT-PCR and Amplicor HCV monitor assays for quantitation of hepatitis C virus RNA in serum.
A modified competitive RT-PCR (mcRT-PCR) to measure HCV RNA in serum and the Amplicor HCV Monitor assay were compared. For mcRT-PCR, the RNA extracted was retrotranscribed and coamplified in one step with a known amount of a DNA internal control (IC). Digoxigenin-labeled amplified products were hybridized to specific HCV DNA and IC-DNA probes and quantified by colorimetry. HCV RNA concentration was calculated by plotting the ratio of HCV/IC ODs against a calibration curve. Multiple samples were analyzed in the same round and tedious titration of each sample with a competitor was unnecessary. The mcRT-PCR assay was linear from 6 x 10(3) to 6 x 10(7) copies/ml, whereas Amplicor was linear up to 1-2 x 10(6) copies/ml. HCV RNA was measured in samples from 75 carriers. There was agreement between both methods in type 1 infections but not in type 2 or type 3 infections, in which the values measured by Amplicor were, on average, 15 times lower than those measured by the mcRT-PCR. HCV RNA measured by Amplicor was higher in type 1 infections than in type 2 or 3 infections, but no differences were found when viral load was assessed by mcRT-PCR. The binding efficiency of the Amplicor-probe was greater for type 1 than for types 2 or 3, suggesting Amplicor underestimates the viral load in the latter types. In contrast, the mcRT-PCR is not affected by genotype-related variation of HCV. This study suggests that mcRT-PCR assay is reliable for sensitive and accurate measurement of HCV RNA over a broad range of values independently of the HCV genotype.
Olmedo
E
E
Liver Unit, Hospital Clínic i Provincial, University of Barcelona, Catalonia, Spain.
Costa
J
J
López-Labrador
F X
FX
Forns
X
X
Ampurdanés
S
S
Maluenda
M D
MD
Guilera
M
M
Sánchez-Tapias
J M
JM
Rodes
J
J
Jimenez de Anta
M T
MT
D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000097:blood / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D007372:Interferons / Q000627:therapeutic use; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012367:RNA, Viral; D007372:Interferons