Clinical Chemistry
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Clinical Chemistry 51: 1217-1222, 2005; 10.1373/clinchem.2005.048603
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2005;51:1217-1222.)
© 2005 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

High-Throughput Purification of Viral RNA Based on Novel Aqueous Chemistry for Nucleic Acid Isolation

Michael K. Hourfar1,a, Uwe Michelsen2, Michael Schmidt1, Annemarie Berger3, Erhard Seifried1 and W. Kurt Roth1

1 Institute of Transfusion Medicine and Immunohaematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany.
2 Merck KGaA, Darmstadt, Germany.
3 Institute for Medical Virology, Johann Wolfgang Goethe University Hospital, Frankfurt, Germany.

aAddress correspondence to this author at: Institute of Transfusion Medicine and Immunohaematology, German Red Cross, Johann Wolfgang Goethe University, Sandhofstrasse 1, 60528 Frankfurt am Main, Germany. Fax 49-696782-289; e-mail khourfar{at}bsdhessen.de.

Background: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead–based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.

Methods: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 µL plasma in 1 batch.

Results: The detection limit of the method was comparable to the spin-column–based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R2) was >0.99 for the viruses evaluated.

Conclusions: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.






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Copyright © 2005 by the American Association for Clinical Chemistry.