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United States Department of Agriculture

Agricultural Research Service

Research Project: APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS 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: April 16, 2010
Publication Date: April 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.

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.

Last Modified: 11/28/2015
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