Evaluation of a new kinetic amplification based assay for quantification of hepatitis C virus (HCV) RNA

Abstract number: 1134_01_293

Battersby T.R., Abraham M.L., Ahle J.D., Brooks E.J., Friesenhahn M.J., Grewal L., Jiang J., Sherman D., Wang C.

Objectives:  

The authors are developing an automated kinetic amplification assay targeting the 5'-UTR for quantification of HCV RNA. Analytical performance characteristics of this assay were evaluated, including: sensitivity, linearity, the ability to detect genotypes 1–6, and specificity.

Methods:  

Viral RNA was extracted from panels and donor samples using an automated method with magnetic silica beads. Reverse transcription, amplification, and detection were subsequently performed. Analytical performance of the assay, currently in development, was evaluated to determine sensitivity, dynamic range, specificity, and equivalence of genotype quantification. Linearity and limit of detection were evaluated with HCV 1a viral panels. Additionally, HCV 1a transcript quantified by phosphate analysis and A260 was used to evaluate linearity over an extended range. Transcripts representing HCV subtypes were similarly quantified and used to evaluate genotype detection. Specificity was evaluated using serum from HCV antibody negative donors.

Results:  

The assay was linear over the range tested (25–5 × 10⁸ copies/reaction) with subtype 1a transcripts as target. HCV 1a virus panels were detected >95% of the time at 20 IU/mL with a 0.5 mL sample volume. Preliminary experiments with transcripts representing genotypes 1–6 demonstrated equivalent quantification. Assay specificity was >99% using negative donor serum samples.

Conclusions:  

The new automated HCV viral load assay under development has been demonstrated to detect HCV RNA at very low concentration. The assay displays a wide dynamic range with excellent specificity.