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.

Wenzel, Jürgen J ; Walch, Heiko ; Bollwein, Markus ; Niller, Hans Helmut ; Ankenbauer, Waltraud ; Mauritz, Ralf ; Höltke, Hans-Joachim ; Zepeda, Héctor Manuel ; Wolf, Hans ; Jilg, Wolfgang ; Reischl, Udo

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Date of publication of this fulltext: 15 Dec 2009 07:34

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Details

Item type:	Article
Date:	27 November 2009
Institutions:	Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene
Identification Number:	Value Type 19797710 PubMed ID 10.1373/clinchem.2009.136192 DOI
Keywords:	Influenza A/H1N1/09; Locked Nucleic Acid Hydrolysis Probes; Molecular Detection ; Real-Time RT-PCR; Universal ProbeLibrary
Dewey Decimal Classification:	500 Science > 570 Life sciences 500 Science > 500 Natural sciences & mathematics 600 Technology > 610 Medical sciences Medicine
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	11456

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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 ...

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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Metadata last modified: 03 Jun 2018 22:47

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