High-Speed Detection of Blood-borne Hepatitis C Virus RNA by Single-Tube Real-Time Fluorescence Reverse Transcription-PCR with the LightCycler

Hepatitis C virus (HCV) has been identified as the agent responsible for the vast majority of cases of posttransfusion non-A, non-B hepatitis. Although generally asymptomatic, ∼85% of the infections become chronic with a wide spectrum of outcomes (1). Current assays developed to detect antibodies against HCV proteins are successful in detecting most cases of chronic HCV infection. Antibody tests may be negative, however, in cases of acute HCV infection during the window that precedes seroconversion. No immunoassay for direct detection of HCV antigen is available at the present time. With nucleic acid amplification tests, it is possible to detect HCV viremia an average of 59 days before immunological seroconversion (2)(3). Nucleic acid amplification tests for detection of HCV sequences in blood products became compulsory in Germany on April 1, 1999 (4)(5). Because HCV, with its extremely heterogeneous genome, circulates in the blood in concentrations that range from undetectable (1000 IU/mL), with both our standard HCV nucleic acid amplification tests giving a concordance rate of 100%. Moreover, the real-time RT-PCR showed similar sensitivities for the genotypes 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, and 5a in a genotype panel (data not shown). The real-time RT-PCR is well suited for quantification, using CT analysis on the basis of an RNA calibration curve. From amplification plots using a series of 10-fold dilutions of a genotype 2a sample, we obtained a linear correlation between the CT and the template at concentrations of 103–106 IU/mL HCV RNA (r = 0.96). In clinical samples, the calibration curve could be extrapolated up to 6.3 × 107 IU/mL HCV RNA (data not shown). The results from 20 HCV RNA-positive patients with HCV RNA titers between 2.1 × 103 and 6.3 × 107 IU/mL HCV RNA were then compared with those of the Cobas Amplicor HCV Monitor assay. The correlation was statistically significant (r = 0.92). The mean values of the results obtained with our RT-PCR protocol and the Amplicor assay were comparable. Single-tube real-time quantification of HCV RNA using TaqMan technology (Roche) and the ABI PRISM 7700 system (Perkin-Elmer) has been described recently (11)(12). Compared with our test, these real-time tests showed comparable reproducibility with lower (11) and higher sensitivity (12), but sequence variation among HCV targets presented problems (12). The Light Cycler test has the advantage of a running time of only 70 min compared with 150 min with the PRISM 7700, but the PRISM 7700 has a higher throughput and is better suited for automation. Our method has been approved by PEI for HCV virus testing in blood samples and is ideally suited for rapid analysis of smaller sample numbers. This convenient approach is very economic without the risk of carryover contamination. Fluorescence vs cycle number in serially diluted PEI reference preparations and in a negative control (A), and logarithmic plot of all fluorescence data vs cycle number (B). (A), serially diluted PEI reference preparations contained 5000 (run control) to 156 IU/mL HCV RNA. The noise band was set to 0.04, which corresponds to the final fluorescence intensity of the negative sample. Background subtraction was used, and the fluorescence was set to channel 2/channel 1. (B), the cutoff value was adjusted to 0.12, which was the highest final fluorescence intensity in 149 HCV RNA-negative samples. Only samples with sufficient HCV RNA content to generate fluorescence >0.12 (∼600 IU/mL in this serial dilution experiment) are seen on the screen. ▵, 5000 IU/mL; □, 2500 IU/mL; ✲, 1250 IU/mL; ◊, 625 IU/mL; ○, 312 IU/mL; •, 156 IU/mL; dotted line, negative control. This work was supported by the Robert Bosch Foundation.