H Hennig1, J Luhm, D Hartwig, H Klüter, H Kirchner. 1. Institute of Immunology and Transfusion Medicine, University of Lübeck School of Medicine, Lübeck, Germany. hennig@immu.mu-luebeck.de
Abstract
BACKGROUND: The objective of this work was to develop a novel and highly sensitive RT-PCR method that is suitable for HCV RNA screening of blood donations according to the criteria released by the Paul Ehrlich Institute, the federal licensing agency of Germany, for routine HCV NAT. STUDY DESIGN AND METHODS: RNA was prepared from plasma pools of up to 20 single blood donations using an automated nucleic acid isolation system (NucliSens Extractor, Organon Teknika). For reverse transcription, amplification, and simultaneous detection of PCR products, a novel approach based on the TaqMan technology was developed. Glyceraldehyde-3-phosphate dehydrogenase messenger RNA, which is detectable in human plasma, was coamplified in each reaction as an internal positive control. RESULTS: The HCV genotypes and subtypes 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, and 5a were detected in parallel with comparable amplification efficiency. The 95-percent detection limit related to the WHO HCV RNA standard preparation was calculated to be 389 IU per mL of plasma of the single blood donation. Total CVs (%) were <4. The screening of up to 180 blood donations took 5 hours; as a rule, the blood components could be released on the day of donation. CONCLUSION: The TaqMan HCV RT-PCR is an almost completely automated, highly sensitive, specific, and rapid method that is reliable for HCV RNA screening of blood donations. It allows a closed-tube HCV RNA detection without risk of contamination by PCR products.
BACKGROUND: The objective of this work was to develop a novel and highly sensitive RT-PCR method that is suitable for HCV RNA screening of blood donations according to the criteria released by the Paul Ehrlich Institute, the federal licensing agency of Germany, for routine HCV NAT. STUDY DESIGN AND METHODS: RNA was prepared from plasma pools of up to 20 single blood donations using an automated nucleic acid isolation system (NucliSens Extractor, Organon Teknika). For reverse transcription, amplification, and simultaneous detection of PCR products, a novel approach based on the TaqMan technology was developed. Glyceraldehyde-3-phosphate dehydrogenase messenger RNA, which is detectable in human plasma, was coamplified in each reaction as an internal positive control. RESULTS: The HCV genotypes and subtypes 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, and 5a were detected in parallel with comparable amplification efficiency. The 95-percent detection limit related to the WHO HCV RNA standard preparation was calculated to be 389 IU per mL of plasma of the single blood donation. Total CVs (%) were <4. The screening of up to 180 blood donations took 5 hours; as a rule, the blood components could be released on the day of donation. CONCLUSION: The TaqMan HCV RT-PCR is an almost completely automated, highly sensitive, specific, and rapid method that is reliable for HCV RNA screening of blood donations. It allows a closed-tube HCV RNA detection without risk of contamination by PCR products.