A novel TaqMan RT-PCR for rapid HCV RNA screening of blood donations

H. Hennig*, J. Luhm, H. Klüter, H. Kirchner

*Corresponding author for this work


Purpose: The objective of our work was to develop a novel automated and highly sensitive RT-PCR method which is suitable for reliable and rapid HCV RNA screening of blood donations according to the criteria released by the Paul-Ehrlich-Institute (PEI) for routine HCV NAT. Methods: RNA was prepared from pools of up to 20 single blood donations using the NucliSens™ Extractor (Organon Teknika, Boxtel, The Netherlands). For reverse transcription, amplification and simultaneous detection of PCR products we developed a novel approach based on the TaqMan® EZ RT-PCR Kit on the ABI Prism 7700 SDS (PE Applied Biosystems, Weiterstadt, Germany). Primers and fluorogenic TaqMan probe for HCV RNA detection are highly homologous to 165 sequences containing the 5′non-coding region of HCV genome which were available in Genbank via Internet. An RNA sequence which exists ubiquitously in human plasma was coamplified in each reaction as internal positive control to avoid false negative results due to PCR inhibition. Results: The HCV genotypes and subtypes 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4 and 5 were detected with comparable amplification efficiency. The 95% detection limit related to the WHO HCV RNA standard preparation was calculated by Probit analysis and amounted to between 400 and 500 IU/ml plasma of the single blood donation. Complete validation results according to the validation guidelines of the PEI have been demonstrated. By disposing of closed PCR tubes, cross-over contamination by PCR products was practically excluded. The performance of screening of up to 180 blood donations took 5 hours, the blood products as a rule could be released on the day of donation. Conclusions: The TaqMan HCV RT-PCR is a semi-automated, highly sensitive and rapid method which is reliable for HCV RNA screening of blood donations. It allows a closed-tube HCV RNA detection with simultaneous coamplification of an internal positive control and without risk for contamination by PCR products.

Original languageEnglish
JournalInfusionstherapie und Transfusionsmedizin
Issue numberSUPPL. 1
Pages (from-to)23
Number of pages1
Publication statusPublished - 1999


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