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 850,000 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.

The Role of Human Metapneumovirus in Upper Respiratory Tract Infections in Children: A 20‐Year Experience

“…In the present study, the RNA was extracted by automated methods. Some studies have suggested that automated nucleic acid-extraction methods are less efficient than manual methods [21][22][23], whereas others have reported similar sensitivities between the 2 methods [24,25]. Although the URI specimens tested with the LRI specimens in our previous pilot-scale study were selected randomly (using computer methods) from all URI specimens, there were more specimens from recent years in that subset (data not shown), and this factor may have affected RNA quality as well.…”

Kinetics of hepatitis C viral RNA and HCV‐antigen during dialysis sessions: Evidence for differential viral load reduction on dialysis

“…The assay is an automated real-time PCR-based system that amplifies a 244-base target sequence located in a highly conserved 5 0 -untranslated region of the HCV genome for detection and quantitation of HCV [Lee et al, 2000]. Heparin does not affect viral load detection [Miyachi et al, 2003].…”

Comparative evaluation of the Cobas Amplicor HIV-1 Monitor™ Ultrasensitive Test, the new Cobas AmpliPrep/Cobas Amplicor HIV-1 Monitor™ Ultrasensitive Test and the Versant HIV RNA 3.0 assays for quantitation of HIV-1 RNA in plasma samples

“…The sample preparation is currently considered as the most labor-intensive part of the PCR testing process, accounting for most of the handson time (Klapper et al, 1998;Verhofstede et al, 1996). A new automated specimen preparation system, the Cobas AmpliPrep TM , was developed to automate this last part of the PCR procedure (Jungkind, 2001;Miyachi et al, 2003;Stelzl et al, 2002).…”