Multiplex assay for simultaneous typing of influenzaA viruses and subtyping into H1N1 and H3N2, including novel 2009 swine origin influenzaA H1N1 using a microarray-based platform
Abstract number: P1103
Alemán A., Salazar O., Villahermosa M.L., Cospedal R.
Objectives: Recent outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) makes necessary the design of well-validated diagnostic tools for the rapid, sensitive and specific identification of the 2009 pandemic H1N1 influenza virus. We have developed and validated a multiplex PCR assay followed for a hybridization in a low-density microarray (CLART® FluAVir) for simultaneously typing and subtyping of Influenza A viruses including novel H1N1 swine origin influenza virus [S-OIV], human H1N1 and H3N2 influenza A and generic Influenza A viruses.
Methods: The assay targets the genes NP (nucleoprotein) to type the influenza A viruses and subtype human influenza A H1N1 and N3N2, and the gene HA (haemagglutinin) to subtype and S-OIV H1N1. 446 clinical isolates were typed and subtyped using CLART® FluAVir assay.
Results: The assay achieved an analytical sensitivity of 10 to 102 copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 446 clinical isolates, including 228 S-OIV H1N1, 15 A/H1N1 human, 60 A/H3N2 human, and 62 from other viruses and 113 negative samples. Testing of the analytical specificities revealed no-cross reactivity and demonstrated reproducibility of results. Clinical tests showed 98.3 of sensitivity for Influenza A H3N2 and 100% sensitivity and specificity for the rest of the types, including S-OIV H1N1. Comparisons of the CLART® FluAVir results with multiple validated in-house molecular assays, and CDC-validates FDA-approved assay demonstrates a 100% positive agreement for all the viruses.
Conclusions: CLART® FluAVir is a rapid, sensitive, specific and multiparametric assay for the typing and subtyping of influenza A viruses, which allow to process up to 96 samples simultaneously in a working day, being useful for de diagnosis and surveillance of 2009 pandemic (H1N1) influenza virus and necessary for clinical and public health decisions.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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