Comparative study of a modified competitive RT-PCR and amplicor HCV monitor assays for quantitation of hepatitis C virus RNA in serum

✍ Scribed by Olmedo, Eva; Costa, Josep; L�pez-Labrador, Fancesc X.; Forns, Xavier; Ampurdan�s,, Sergi; Maluenda, Maria D.; Guilera, Magdalena; S�nchez-Tapias, Jose M.; Rodes, Joan; Jimenez de Anta, Maria T.

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 169 KB
• Volume: 58
• Category: Article
• ISSN: 0146-6615
• DOI: 10.1002/(sici)1096-9071(199905)58:1<35::aid-jmv5>3.0.co;2-v

✦ Synopsis

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 genotyperelated 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.