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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.

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.

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Wenzel JJ,Walch H,Bollwein M,Niller HH,Ankenbauer W,Mauritz R,Höltke HJ,Zepeda HM,Wolf H,Jilg W,Reischl U

doi

10.1373/clinchem.2009.136192

subject

Has Abstract

pub_date

2009-12-01 00:00:00

pages

2218-22

issue

eissn

0009-9147

issn

1530-8561

pii

clinchem.2009.136192

journal_volume

pub_type

杂志文章

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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.