Performance characteristics of a quantitative, homogeneous TaqMan RT-PCR test for HCV RNA

Abstract

We developed a homogeneous format reverse transcription-polymerase chain reaction assay for quantitating hepatitis C virus (HCV) RNA based on the TaqMan principle, in which signal is generated by cleaving a target-specific probe during amplification. The test uses two probes, one specific for HCV and one specific for an internal control, containing fluorophores with different emission spectra. Titers are calculated in international units (IU)/ml by comparing the HCV signal generated by test samples to that generated by a set of external standards. Endpoint titration experiments demonstrated that samples containing 28 IU/ml give positive results 95% of the time. Based on these data, the limit of detection was set conservatively at 40 IU/ml. All HCV genotypes were amplified with equal efficiency and accurately quantitated: when equal quantities of RNA were tested, each genotype produced virtually identical fluorescent signals. The test exhibited a linear range extending from 64 to 4,180,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 21.6 to 30.4%, which implies that titers that differ by a factor of twofold (0.3 log10) are statistically significant (P = 0.005). The test did not react with other organisms likely to co-infect patients with hepatitis C and exhibited a specificity of 99% when evaluated on a set of samples from HCV seronegative blood donors. In interferon-treated patients, the patterns of viral load changes revealed by the TaqMan HCV quantitative test distinguished responders from nonresponders and responder-relapsers. These data indicate that the TaqMan quantitative HCV test provides an attractive alternative for measuring HCV viral load and should prove useful for prognosis and for monitoring the efficacy of antiviral treatments.

Figure 1.

Efficiency of amplification of different HCV genotypes. HCV transcripts representing genotypes 1a, 1b, 2a, 2b, 3a, 4, and 5 were diluted to 100,000 copies/ml and tested with the TaqMan HCV quantitative Test. The C_T for four replicates of each genotype were averaged to determine the mean C_T and SD.

Figure 2.

Linear range for the TaqMan HCV test. Two independent dilution series were prepared by diluting an external HCV RNA quantitation standard in HCV-negative plasma. Each dilution in each series was tested in duplicate. At each input, RNA concentration, the mean of the log₁₀ calculated RNA concentrations (circles and squares), and the SD were determined. Linear regression analysis was performed on the mean log₁₀ values for each dilution series (solid and dashed lines and associated equations).

Figure 3.

Quantitation of HCV RNA (left axis) and serum ALT (right axis) levels in a responder-relapser (A), responder (B), and nonresponder (C) to interferon therapy. Baseline HCV RNA and ALT levels (first data point) were measured 12 (A), 4 (B), and 65 (C) weeks before the start of interferon therapy (day 0, vertical dashed line). RNA and ALT levels were periodically monitored starting at 1 week after initiating therapy (second data point). The posttreatment RNA levels (last data point) were measured at 25 (A), 4 (B) or 38 (C) weeks after completion of interferon therapy at week 48 (vertical dashed line). The dotted-dashed line indicates both the limit of detection for the HCV RNA test and the upper limit of the normal ALT range. Samples that tested negative for HCV RNA were assigned an arbitrary value (10 IU/ml) below the limit of detection.