Premium
Comparative study of a modified competitive RT‐PCR and amplicor HCV monitor assays for quantitation of hepatitis C virus RNA in serum
Author(s) -
Olmedo Eva,
Costa Josep,
LópezLabrador Fancesc X.,
Forns Xavier,
Ampurdanés, Sergi,
Maluenda Maria D.,
Guilera Magdalena,
SánchezTapias Jose M.,
Rodes Joan,
Jimenez de Anta Maria T.
Publication year - 1999
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/(sici)1096-9071(199905)58:1<35::aid-jmv5>3.0.co;2-v
Subject(s) - virology , hepatitis c virus , flaviviridae , virus , hepacivirus , rna , biology , real time polymerase chain reaction , polymerase chain reaction , microbiology and biotechnology , gene , genetics
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 × 10 3 to 6 × 10 7 copies/ml, whereas Amplicor was linear up to 1–2 × 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. J. Med. Virol. 58:35–43, 1999. © 1999 Wiley‐Liss, Inc.