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United States Department of Agriculture

Agricultural Research Service

Title: Establishment of a Real-Time Pcr Assay for Swine Influenza Viruses

Authors
item Richt, Juergen
item Clouser, Deborah
item Lager, Kelly
item Spackman, Erica
item Suarez, David

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 10, 2002
Publication Date: November 10, 2002

Technical Abstract: Swine influenza (SI) is an acute respiratory disease of swine caused by type A influenza viruses. Before 1998, mainly "classical" H1N1 SI viruses (SIV) were isolated from swine in the United States. Since then, antigenetically distinct reassortant H3- and H1-SIVs have been identified as causative agents of respiratory disease in pigs on U.S. farms. Improvement in SIV diagnostics is needed in the light of the recently observed rapid evolution of H1 and H3 swine influenza viruses and their potential threat to human health. To address this need, a real-time RT-PCR assay for H1- and H3-SIVs was developed. We established a M-gene based RT-PCR assay which is able to detect H1- and H3-subtypes of SIVs with a sensitivity of ~5 RNA copies of in vitro generated M-specific negative-sense RNA molecules and ~1 TCID50 in nasal swabs of experimentally SIV-infected pigs. This RT-PCR assay can be performed in less than 2 hours. In addition, we have designed H1, H3-, N1, and N2-specific primer and probe sets in order to differentiate between different SIVs subtypes. The H3-and N1 primer sets detect approx. 10**3 RNA copies and were found to be (i) specific for their respective viral gene, and (ii) able to distinguish between their respective SIV-subtypes. The H1- and N2- primer sets are being evaluated. The feasibility of these primer/probes sets for multiplex RT-PCR assays will be tested in future experiments.

Last Modified: 10/1/2014
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