Evaluation of the Abbott RealTime HCV assay for quantitative detection of hepatitis C virus RNA
Introduction
Molecular techniques are extensively used for both confirmation of reactive anti-hepatitis C virus (HCV) antibody assays and monitoring of patients with anti-HCV therapy. Maximum automation of assays for quantitative detection of HCV RNA is desirable. To meet the needs of the high-throughput diagnostic laboratory, extraction of HCV RNA, PCR amplification, and detection of amplified products have recently been automated. However, molecular assays based on conventional PCR show a limited linear range with the need to dilute for quantification above the upper range (Stelzl et al., 2002, Gourlain et al., 2005). This limitation has been resolved by introduction of molecular assays based on real-time PCR (Stelzl et al., 2004, Beuselnick et al., 2005).
Recently, Abbott RealTime assays (Abbott Molecular Inc., Des Plaines, IL) for quantitative detection of HCV RNA and HIV-1 RNA have been introduced. The previous versions included the m1000 instrument for HCV RNA extraction (Beuselnick et al., 2005, Garcia-Diaz et al., 2006). Recently, the m2000sp instrument has been introduced (Halfon et al., 2006, Swanson et al., 2006).
The aim of this study was to evaluate performance of the Abbott RealTime HCV assay based on automated sample preparation on the m2000sp instrument followed by real-time PCR and detection on the m2000rt instrument. Accuracy was tested with a reference material; linearity was analyzed by a dilution series of a high-titer sample. Both interassay and intra-assay variations were determined. The clinical performance of the new assay in the routine high-throughput laboratory was evaluated with routine clinical samples, and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test (Roche Molecular Systems, Inc., Branchburg, NJ).
Section snippets
Molecular assays
The Abbott RealTime HCV assay, which includes an automated sample preparation on the m2000sp instrument followed by real-time PCR and detection on the m2000rt instrument, and the COBAS AmpliPrep/COBAS TaqMan HCV test, which includes an automated sample preparation on the COBAS AmpliPrep instrument followed by real-time PCR and detection on the COBAS TaqMan instrument, were performed according to the manufacturer's package insert instructions. The lower limit of detection is 12 IU/ml for the
Results
When seven samples of the QCMD 2005 HCV proficiency panel were tested with the Abbott RealTime HCV assay, five were found within ±0.5 log10 unit of the expected panel results. Two samples showed differences of −0.55 and −0.67 log10 unit, respectively (Table 1). The HCV-negative sample tested negative. Linearity was tested with a dilution series of a high-titer routine clinical sample. A quasilinear curve was observed up to the original concentration (1.0 × 106 IU/ml; Fig. 1). HCV RNA was
Discussion
For quantification of HCV RNA in a routine high-throughput laboratory, maximum automation is desirable. Both the COBAS AmpliPrep/COBAS TaqMan HCV test (Roche) and the new Abbott RealTime HCV assay are based on automated sample preparation and real-time PCR. While Roche provides a largely closed platform, the Abbott instruments may easily be used as an open platform for in-house assays (Niesters and Scutten, 2006). In this study, the Abbott RealTime HCV assay was evaluated and compared with the
Acknowledgments
The authors gratefully acknowledge Sven Schaffer and Sven Thamm for stimulating discussions and Erich Hauptmann and Anette Niemann for technical assistance. This study was supported in part by Abbott Molecular.
References (10)
- et al.
Performance of the automated Abbott RealTime™ HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1.5, and LCx® HIV RNA Quantitative assays
J Virol Methods
(2006) - et al.
Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative real-time PCR
J Clin Microbiol
(2005) - et al.
Comparative evaluation of the performance of the Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for measurement of HIV-1 plasma viral load following automated specimen preparation
J Clin Microbiol
(2006) Dynamic range of hepatitis C virus RNA quantification with the Cobas Ampliprep-Cobas Amplicor HCV Monitor v2.0 assay
J Clin Microbiol
(2005)- et al.
Real-time assays for hepatitis C virus (HCV) RNA quantitation are adequate for clinical management of patients with chronic HCV infection
J Clin Microiol
(2006)
Cited by (49)
Molecular microbiology
2018, Principles and Applications of Molecular DiagnosticsDiagnostics in hepatitis C: The end of response-guided therapy?
2016, Journal of HepatologyPerformance characteristics of the VERSANT hepatitis C virus RNA 1.0 (kPCR) assay
2015, International Journal of Medical MicrobiologyCitation Excerpt :During the synthesis of the complementary DNA strand, the probe is uncoiled and the fluorophor is thus spatially separated from the quencher allowing fluorescence detection without cleavage of the probe. The generated fluorescence is registered during each PCR cycle, and the HCV cDNA is thus detected in real time (Halfon et al., 2006; Michelin et al., 2007; Schutten et al., 2007; Vermehren et al., 2011). In contrast to the two previously mentioned procedures, the VERSANT HCV RNA 3.0 Assay (bDNA) is not based on an amplification of a target sequence, but rather detects the HCV RNA by means of signal amplification, which results from hybridization with different oligonucleotides and in particular the “branched DNA complex”.
HCV RNA measurement in samples with diverse genotypes using versions 1 and 2 of the Roche COBAS<sup>®</sup> AmpliPrep/COBAS<sup>®</sup> TaqMan<sup>®</sup> HCV test
2015, Journal of Clinical VirologyCitation Excerpt :Genotypes 1a and 1b are most common in the United States, but accurate HCV RNA measurements are crucial for all genotypes. Several laboratory assays have been FDA-approved to measure HCV RNA, including the Versant HCV RNA 3.0 assay (bDNA, Siemens Healthcare Diagnostics Inc., Tarrytown, NY) [1,2], RealTime HCV (Abbott Molecular Inc., Des Plaines, IL) [3], and COBAS® AmpliPrep/COBAS® TaqMan® HCV Tests (Roche Molecular Systems, Inc., Branchburg, NJ) [4,5]. These assays have been designed for equivalent detection of all six genotypes of HCV.
Abbott RealTime PCR assay is useful for evaluating virological response to antiviral treatment for chronic hepatitis C
2011, Journal of Infection and ChemotherapyMulti-center evaluation of the Abbott RealTime HCV Assay for monitoring patients undergoing antiviral therapy for chronic hepatitis C
2011, Journal of Clinical VirologyCitation Excerpt :For the measurement of HCV RNA, a number of qualitative and quantitative assays are commercially available.16 Recently, real-time PCR-based assays have been introduced into clinical practice offering very low limits of detection and linear quantification over a broad dynamic range.17–26 In the present multi-center study, we evaluated the Abbott RealTime HCV assay (RealTime HCV; Abbott Molecular, Des Plaines IL, USA) for its clinical utility in monitoring patients infected with HCV GTs 1, 2, and 3 during treatment with PEG-IFN/RBV.