Quantitative assay of hepatitis C virus RNA using an automated extraction system for specific capture with probes and paramagnetic particle separation.
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.
Miyachi
Hayato
H
Department of Laboratory Medicine, Tokai University School of Medicine,Tokyo, Japan. miyachi@is.icc.u-tokai.ac.jp
Masukawa
Atsuko
A
Asai
Satomi
S
Miura
Toshiaki
T
Tamatsukuri
Shigeru
S
Hirose
Toru
T
Ando
Yasuhiko
Y
D001331:Automation; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods*; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results
D015347:RNA Probes; D012367:RNA, Viral