Quantitative assay of hepatitis C virus RNA using an automated extraction system for specific capture with probes and paramagnetic particle separation

Abstract

A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log(10) (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R(2) = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.

FIG. 1.

Assay linearity in serial dilutions of a serum sample for HCV RNA levels as determined by the AMPLICOR HCV MONITOR Test using RNA extracted by the AmpliCap/GT-12 system. For linearity study, serial dilutions of a serum panel were subjected to HCV RNA extraction by the AmpliCap/GT-12, and measured for HCV RNA in the COBAS AMPLICOR HCV MONITOR Test, version 2.0. Data are means of duplicate assays.

FIG. 2.

HCV RNA levels for 86 clinical specimens as determined by the AMPLICOR HCV MONITOR Test using RNA extracted by the manual method or the AmpliCap/GT-12 system. A total of 90 clinical samples were subjected to RNA extraction by either the manual method or AmpliCap/GT-12, and measured in singlet using the version 2.0 AMPLICOR HCV MONITOR Test. Of the samples, 86 had levels of HCV RNA that were quantifiable by both assays.