In this research we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays including manual HCV RNA extraction protocols. assay and standard methods was observed. The introduced amount of IC was recognized in 45 of E-7050 45 serum samples 41 of 45 EDTA tube whole-blood samples and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory. Molecular techniques have been shown to be effective tools for direct detection of hepatitis C disease (HCV). Such assays for detection of pathogens basically consist of several steps: extraction of HCV RNA (also called sample preparation) reverse transcription (RT) amplification of cDNA hybridization of amplified products and detection of nucleic acid hybrids. Molecular techniques are labor-intensive and time-consuming when manual home brew methods are used. To meet the needs of the routine diagnostic laboratory PCR amplification and detection of amplified products have recently been automated with the COBAS AMPLICOR (Roche Molecular Diagnostics Pleasanton Cal.) analyzer (1 4 5 14 Sample preparation is currently considered the major weakness in molecular detection of HCV RNA. Conventional sample preparation protocols are usually time-consuming labor-intensive and susceptible to contamination. It has been E-7050 demonstrated that the probability of obtaining false-positive results because of contamination increases in relation to the number of manipulations involved in sample processing (3 E-7050 11 To save time and labor more rapid and automated nucleic acid extraction protocols with fewer manipulation steps have largely replaced conventional protocols. Several ready-to-use sample preparation kits available either separately or as part of the entire molecular kit have been brought to market and found suitable for inclusion in molecular assays for detection of RNA viruses (6 9 15 Recently a new automated specimen preparation instrument the MagNA Pure LC (Roche Applied Science Mannheim Germany) was developed to automate sample preparation (7). Previous studies have shown that HCV can infect peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (2 17 19 The presence of HCV in PBMCs may have implications for the response to antiviral therapy (10 13 16 It has been demonstrated recently that detection of HCV RNA in PBMCs may be an additional tool to demonstrate the persistence of HCV RNA. Reappearance of HCV RNA is detected earlier in EDTA tube whole-blood samples than in sera (6). Therefore recovery of total HCV RNA (i.e. intracellular RNA as well as plasma RNA) appears to be of major importance to detect low-level viremia. Until recently the sustained virological response rate to anti-HCV therapy was rather low. Introduction of novel drugs such as e.g. Mouse monoclonal to CD59(PE). pegylated interferon α-2b has been shown to significantly increase the rate of patients with nondetectable serum HCV RNA (12 18 These new therapeutic approaches have led to a strong increase in the number of EDTA tube whole-blood samples to be tested with HCV E-7050 RNA. The aims of the present study were to establish a fully automated molecular assay for qualitative detection of HCV RNA in serum and whole-blood samples which included automated RNA extraction with the MagNA Pure LC device and to evaluate it with regular molecular assays such as manual HCV RNA removal protocols. Blood examples were gathered from individuals with persistent hepatitis C (non-responders and the ones with suffered virological response) and from healthful bloodstream donors. For qualitative recognition of serum HCV RNA bloodstream was gathered in 9-ml pipes (Vacuette; Greiner Bio-one GmbH Kremsmünster Austria). Furthermore whole bloodstream was gathered in 3.0-ml Vacuette EDTA tubes (Greiner) and in 3.5-ml Vacuette Nucleic Acid solution Stabilization Tubes (NASTs; Greiner) that have a liquid nucleic acidity stabilizer. The scholarly study protocol was approved by the neighborhood Ethics Committee and everything patients gave informed consent. Within 2 h of bloodstream being attracted 9 tubes had been centrifuged at 1 500 × for 20 min at space temperature. After centrifugation serum aliquots had been ready that have been freezing at instantly ?70°C until tests. Whole-blood collection pipes were freezing at ?70°C without prior planning. Inside our ISO.