Quantitative HCV Assays

Currently there are several commercially available and proprietary laboratory-developed methods used to quanti-tate HCV RNA levels in patients. The Versant HCV RNA Assay v3.0 (Bayer Corp.),72 which is FDA approved, and the Amplicor HCV Monitor Test v2.0 (Roche Diagnostics)65 are the most widely used commercially available assays. The HCV Superquant is a proprietary RT-PCR assay developed by National Genetics Institute (Los Angeles, CA), which has been used by many investigators in clinical trials of treatment for HCV.

The Amplicor HCV Monitor test v2.0 is a quantitative RT-PCR assay that amplifies the same target region as the qualitative Amplicor HCV test. The assay uses an internal quantitation standard to calculate the amount of HCV RNA in a sample. The assay has a sensitivity of 600 IU/ml and a 2.9 logi0 dynamic range. Specimens with values greater than the upper limit of quantitation (500,000 IU/ml) can be diluted 100-fold and retested as recommended by the manufacturer. The Amplicor HCV Monitor test is available in both manual microwell plate and semiautomated COBAS instrument formats.

The Versant HCV RNA Assay v3.0 is based on bDNA technology. The sensitivity of the Versant HCV RNA Assay v3.0 is 615IU/ml,with a 4.1 log10 dynamic range.A number of changes to the design of the v3.0 test improved both the sensitivity and specificity over the v2.0 test. These include an increased number of capture probes, improved probe design, the use of nonnatural synthetic nucleotides in detection probes, and redesigned label extenders. The System 340 bDNA analyzer automates all incubations, wash steps, readings, and data analysis. The instrument can process two 96-microwell plates per run.

Overall, the results of the Versant HCV RNA Assay v3.0 and the Amplicor HCV Monitor Test v2.0 have been found to be in substantial agreement.72,73 The results of the Amplicor Monitor test are reported as IU/ml and those of the Versant assay are reported as either copies/ml or IU/ml. Both tests are free of significant HCV genotype bias and have similar analytical sensitivities. The Versant results tend to be more precise than the Amplicor Monitor results, but the Amplicor Monitor test is better able to discriminate low-positive from negative specimens. The Versant assay has a much greater dynamic range than the Amplicor Monitor test; however, a pretest sample dilution permits quantification of high viral load specimens by the Amplicor Monitor test. The throughput and level of automation are much greater with the Versant assay than with the Amplicor Monitor test.

A number of TaqMan RT-PCR assays for detection and quantitation for HCV RNA have been described.74 These tests have sensitivities comparable to qualitative tests, have a broad dynamic range, and provide precise quantitation of viral load. These tests also generate results more rapidly than conventional RT-PCR assays and are not prone to amplicon carryover contamination since the amplification and detection steps are combined in a single closed tube. With the development of real-time RT-PCR, clinical laboratories will no longer need to have separate qualitative and quantitative assay formats for HCV RNA to achieve both high sensitivity and broad dynamic range. Roche and Abbott manufacture TaqMan HCV analyte-specific reagents that contain the necessary HCV-specific primers, dual-labeled fluorescent HCV and HCV quantitation standard specific probes, enzyme, and dNTPs for the detection and quantitation of HCV RNA.75,76

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