The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholars Program

GVSP Summer 2006 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Joel Landrum University of Georgia Class of 2009	Dr. David Suarez

Development of RRT-PCR for Detection and Identification of Avian Influenza Virus H13 Subtype

Joel Landrum*, Erica Spackman
College of Veterinary Medicine, The University of Georgia, Athens, Ga. and Southeast Poultry Research Laboratory, USDA Agricultural Research Service, Athens, Ga.

Abstract: Avian influenza virus (AIV) is a major concern of the US poultry industry and of increasing importance as it relates to public health. AIV can cause disease in chickens and turkeys and adversely affect not only poultry production, but also impact international trade of poultry and associated products. Considering that US poultry is a multi-billion dollar industry, outbreaks can potentially disrupt regional economies and employment. With this in mind, development of rapid and efficient detection tests for all AIV hemagglutinin (HA) subtypes has been undertaken by the USDA Southeast Poultry Research Lab (SEPRL). The HA protein, a surface protein of the virus, is known to be one of the main determinants of pathogenicity in chickens and turkeys. With 16 HA subtypes of AIV, diagnostic protocol with optimal specificity for each subtype is important for distinguishing known potentially HPAI subtypes, presently only H5 and H7, from other HA subtypes. A real-time reverse transcriptase polymerase chain reaction (RRT-PCR) protocol was developed for detection and identification of the H13 subtype of AIV. The RRT-PCR assay utilized subtype specific primers and fluorogenic hydrolysis probe selected based on the nucleotide sequence of known North American H13 virus isolates. Two primer sets were designed and compared for sensitivity. The most sensitive set was selected and optimized for conditions such as primer ratios, annealing temperatures, probe concentration, salt (MgCl2) concentration and PCR cycle times. The optimal primer set and specific assay conditions were chosen based on sensitivity. The optimized primer and protocol was successful in achieving positive detection for the H13 subtype at a greater sensitivity than that of virus isolation (VI), the reference standard method of detection. A specificity panel with H1 through H15 AIV subtypes was tested against the optimized primers using the RRT-PCR protocol. The H13 primers and protocol did not cross-react with any other AIV subtype samples.