Abstract
The quantification of hepatitis C virus (HCV) is essential for the management of chronic hepatitis C therapy. We have developed a fully automated microfluidic RT-qPCR system for rapid quantitative detection of HCV RNA in human EDTA-plasma and serum, and the performance of the method was assessed. The platform for the assay, µTASWako g1 Fully Automated Genetic Analyzer, performs automated sample preparation and RNA extraction, followed by amplification and detection on an integrated RT-qPCR-CE (capillary electrophoresis (CE)) microfluidic chip. The total assay time from sample input to data output is less than 120 minutes. The HCV assay has a linear quantitative range of 15 to 107 IU/mL, with a limit of detection (LOD) of 10.65 IU/mL in EDTA-plasma and 12.43 IU/mL in serum. The assay has a reproducibility of SD ≤ 0.16 log10 IU/mL and an accuracy of ≤ 0.22 log10 IU/mL difference when compared to the assigned values. The main HCV genotypes 1 to 6 are detected with an accuracy of ± 0.3 log10 IU/mL. The assay is specific for HCV RNA and is free of interference from non-HCV pathogens, elevated levels of anti-viral and anti-bacterial drugs, and common endogenous interferents. In the linear quantitative range, the assay is highly correlated with the Roche cobas AmpliPrep/cobas TaqMan HCV Test, version 2.0 (r2 = 0.949). As the assay is highly sensitive, accurate and specific, and provides reliable quantification of HCV in plasma and serum, it can potentially be applicable for monitoring the therapy and management of HCV infection.
Highlights
Hepatitis C virus (HCV) is a single-stranded RNA virus that has been identified as the major etiologic agent responsible for acute or chronic hepatitis[1,2]
The graph depicts the growth of the amplicons of hepatitis C virus (HCV) RNA target and the non-competitive internal control (IC), MS2 non-competitive internal control (MS2 IC) as the thermal cycle progressed
A growth curve for each HCV concentration was generated by plotting the amplicon peak height (Relative Fluorescence Unit) against the PCR cycle number, and the Cq value was determined by using a threshold value of 5
Summary
Hepatitis C virus (HCV) is a single-stranded RNA virus that has been identified as the major etiologic agent responsible for acute or chronic hepatitis[1,2]. Two widely used commercial RT-qPCR assays for HCV RNA quantification are the Roche cobas AmpliPrep/cobas TaqMan HCV Test, version 2.0 and the Abbott RealTime HCV Assay[8,9,10] Both assays are performed on systems automated for sample preparation, followed by RT-qPCR amplification and detection using fluorescent probes, and they exhibit similar detection sensitivity We have developed the μTASWako g1 Fully Automated Genetic Analyzer for the quantitative detection of nucleic acids in human clinical samples. We assessed the analytical and clinical performances of the μTASWako g1 Fully Automated Genetic Analyzer for the quantitative detection of HCV RNA in human EDTA-plasma and serum. The assay requires minimal hands-on time and can be completed within 120 minutes
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