C Drosten1, M Weber, E Seifried, W K Roth. 1. Institute of Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service of Hesse, Frankfurt am Main, Germany.
Abstract
BACKGROUND: High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing. STUDY DESIGN AND METHODS: Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template-derived competitive internal control sequence designed through site-directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single-donor and single-patient testing and by spiking sample mini-pools. Three seroconversion panels were tested. RESULTS: Sensitivity is high, reaching 300 HBV genomes per mL of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors. CONCLUSION: The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.
BACKGROUND: High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing. STUDY DESIGN AND METHODS: Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template-derived competitive internal control sequence designed through site-directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single-donor and single-patient testing and by spiking sample mini-pools. Three seroconversion panels were tested. RESULTS: Sensitivity is high, reaching 300 HBV genomes per mL of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors. CONCLUSION: The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.
Authors: Roman Wölfel; Janusz T Paweska; Nadine Petersen; Antoinette A Grobbelaar; Patricia A Leman; Roger Hewson; Marie-Claude Georges-Courbot; Anna Papa; Volker Heiser; Marcus Panning; Stephan Günther; Christian Drosten Journal: J Clin Microbiol Date: 2009-02-18 Impact factor: 5.948
Authors: Stefanie Kramme; Le Van An; Nguyen Dinh Khoa; Le Van Trin; Egbert Tannich; Jan Rybniker; Bernhard Fleischer; Christian Drosten; Marcus Panning Journal: J Clin Microbiol Date: 2009-01-14 Impact factor: 5.948
Authors: Christian Drosten; Stephan Göttig; Stefan Schilling; Marcel Asper; Marcus Panning; Herbert Schmitz; Stephan Günther Journal: J Clin Microbiol Date: 2002-07 Impact factor: 5.948