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Title: Development and validation of a real-time RT-PCR assay for the 2009 pandemic H1N1 virus in veterinary specimens

item Spackman, Erica
item Suarez, David
item Killian, Mary
item Koster, Leo
item Swenson, Sabrina
item Pedersen, Jan
item Vincent, Amy
item Lager, Kelly
item Jenkins-moore, Melinda
item Schmitt, Beverly

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/16/2010
Publication Date: 4/18/2010
Citation: Spackman, E., Suarez, D.L., Killian, M.L., Koster, L., Swenson, S., Pedersen, J., Vincent, A.L., Lager, K.M., Jenkins-Moore, M., Schmitt, B. 2010. Development and validation of a real-time RT-PCR assay for the 2009 pandemic H1N1 virus in veterinary specimens [abstract]. Swine Origin H1N1: The First Pandemic of the 21st Century meeting, April 18-20, 2010, Atlanta, Georgia. p. 30.

Interpretive Summary:

Technical Abstract: Due to the transmissibility of the pH1N1 among different species including swine and turkeys, development of a differential diagnostic test for animal specimens was crucial for establishing surveillance and control programs. The current diagnostic paradigm for veterinary specimens is to test for type A influenza, then to confirm positive samples with a lineage specific test. To accomplish this the USDA type A influenza real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) test, which targets the M gene, was updated to match the sequence of the pH1N1 M gene, and a new test which targets the neuraminidase (NA) gene was developed to specifically identify the 2009 pH1N1 lineage. The modified M gene test achieved the highest sensitivity for the pH1N1 lineage although it can detect other type A influenza viruses. The N1 test was successfully evaluated for specificity with a panel of 50 influenza viruses which included viruses of all 9 NA subtypes (18 isolates), 4 pH1N1 lineage viruses, 2 seasonal H1N1 lineage viruses, 14 North American H1N1 swine influenza virus (SIV) lineage viruses and 2 European Lineage H1N1 SIVs and 10 avian N1 subtype viruses. The limit of detection was one 50% egg infectious doses per reaction. Virus was detected in nasal swabs from experimentally infected pigs 1-5 days post exposure with the N1 test. This assay has been implemented as an official test by the National Animal Health Lab Network.