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Clin Chem. 2009 Dec;55(12):2218-22. doi: 10.1373/clinchem.2009.136192. Epub 2009 Oct 1.

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.

Author information

1
Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany.

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.

PMID:
19797710
DOI:
10.1373/clinchem.2009.136192
[Indexed for MEDLINE]
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