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.
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 influenzaA/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 influenzaA/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.
Authors: Jürgen J Wenzel; Marcus Panning; Karen L Kaul; Kathy A Mangold; Paula A Revell; Ruth Ann Luna; Héctor Zepeda; Lizbeth Perea; Joel A Vazquez-Perez; Steve Young; Bojana Rodic-Polic; Markus Eickmann; Christian Drosten; Wolfgang Jilg; Udo Reischl Journal: J Clin Microbiol Date: 2010-07-07 Impact factor: 5.948
Authors: Nguyen thi Tham; Vu thi Ty Hang; Trong Huu Khanh; Do Chau Viet; Tran Tinh Hien; Jeremy Farrar; Nguyen van Vinh Chau; H Rogier van Doorn Journal: Diagn Microbiol Infect Dis Date: 2012-07-09 Impact factor: 2.803
Authors: Markus Bauswein; David Peterhoff; Annelie Plentz; Andreas Hiergeist; Ralf Wagner; André Gessner; Bernd Salzberger; Barbara Schmidt; Stilla Bauernfeind Journal: iScience Date: 2022-01-05