Development of a TaqMan assay for the six major genotypes of hepatitis C virus: comparison with commercial assays

Abstract

A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and <2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.

Fig. 1

Sequence alignment of target region for TaqMan primers and probe. The highly conserved target area on the HCV 5′ UTR that was used for constructing the TaqMan primers and probe is shown. This alignment shows consensus sequences for each of the six major genotypes and subtypes. The number of sequences represented per genotype in the alignment is shown in parenthesis. The 5′ UTR of 186 full-length confirmed (as defined by the LANL HCV database) sequences were queried. Lower case letters represent mismatches in reference to the consensus sequence. Mismatches at positions 261, 267, 276, 280, 284, 309, and 312 are present in no more than two isolates per subtype and were not accounted for in the probe or primer sequences. In contrast, single nucleotide differences at positions 262 (T/C), 270 (C/T) and 302 (T/C) for genotypes 2a, 2b, 3a, and 6a were accounted for by the use of degenerate bases at those positions. Arrows and boxes highlight the selected primers and probe.

Fig. 2

Titration of a prototype HCV H77 strain using three NAT assays for quantifying HCV RNA. The HCV H strain was tested by TaqMan (open circles), Versant HCV RNA 3.0 b-DNA (diamonds) and the HCV Monitor 2.0 (solid circles). Based on linear regression and titration values, the calculated quantity for the HCV H strain was 10^7.4, 10^7.5, and 10^7.7 IU/ml respectively by b-DNA, TaqMan and Amplicor Monitor, which correlated with the infectious titer (10^6.5 CID50/ml) of this strain as described by Feinstone et al. [1981] and Purcell (unpublished work). The slopes for b-DNA, Monitor and TaqMan (−0.975, −0.968, and −0.974) respectively, were not significantly different (P = 0.25).

Fig. 3

Comparison of three NAT assays for quantifying HCV RNA. Weekly samples from chimpanzees experimentally infected with HCV genotypes 1a (TN), 1b (Con1 strain), or 6a (HK strain) were tested with the Versant HCV RNA 3.0 b-DNA assay (solid diamonds), HCV Monitor 2.0 (solid circles) and TaqMan (open circles). Results are presented as HCV RNA log₁₀ IU/ml. The Roche Monitor assay generated values approximately 0.3 log₁₀ IU/ml higher than those of the Versant b-DNA assay throughout the dynamic range of each test.

Fig. 4

Production of HCV in Huh7.5 cells. Samples from cultures inoculated with 500 ffu of JFH1 into 1 million Huh7.5 cells (m.o.i. = 0.0005) and maintained for 21 days were assayed with TaqMan (open circles) and HCV Monitor (solid circles). Pairwise comparison of the parallel data points showed no significant difference (P = 0.5).