Clinical Chemistry
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Clinical Chemistry 55: 2218-2222, 2009. First published October 1, 2009; 10.1373/clinchem.2009.136192
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(Clinical Chemistry. 2009;55:2218-2222.)
© 2009 American Association for Clinical Chemistry, Inc.

Brief Communications

Library of Prefabricated Locked Nucleic Acid Hydrolysis Probes Facilitates Rapid Development of Reverse-Transcription Quantitative Real-Time PCR Assays for Detection of Novel Influenza A/H1N1/09 Virus

Jürgen J. Wenzel1,a, Heiko Walch2, Markus Bollwein1, Hans Helmut Niller1, Waltraud Ankenbauer2, Ralf Mauritz2, Hans-Joachim Höltke2, Héctor Manuel Zepeda3, Hans Wolf1, Wolfgang Jilg1 and Udo Reischl1

1 Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany; 2 Roche Diagnostics GmbH, Penzberg, Germany; 3 Laboratorio Medicina de Conservatión, Escuela Superior de Medicina, Instituto Politécnico Nacional, Colonia de Santo Tomás, México;

aaddress correspondence to this author at: Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Fax +49-941-944-6402; e-mail juergen.wenzel{at}klinik.uni-regensburg.de.


Abstract

Background: The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods.

Methods: We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)—a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes—specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin.

Results: Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100–1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent.

Conclusions: Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.






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Copyright © 2009 by the American Association for Clinical Chemistry.