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

摘要

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 (50 copies/mL) up to 109 copies/mL (6), a detection limit of 5000 IU/mL (according to WHO, 1 IU corresponds to 2–5 genome copies, depending on the HCV-RNA method) in a single blood specimen is acceptable by the criteria of the Paul-Ehrlich-Institut (PEI, Langen, Germany).In blood bank settings, the Cobas Amplicor Hepatitis C Virus Test, Ver. 2.0 (Roche Molecular Biochemicals) (7), is the most frequently used assay. Testing of only a few samples with this assay is quite expensive. Furthermore, turnaround time of PCR and detection exceeds 4 h. For selected single donations of thrombocytes collected by thrombocytapheresis, e.g., for severely ill patients with HLA antibodies, the time period between preparation and release of the result often is too long.The recent development of real-time quantitative PCR based on the LightCycler (Roche Molecular Biochemicals) (8) offers the opportunity for detection of HCV RNA in up …