DEVELOPMENT OF A REAL-TIME RT-PCR ASSAY FOR TYPE A INFLUENZA AND THE AVIAN H5 AND H7 HEMAGGLUTININ SUBTYPES

Authors: Spackman, Erica; SENNE, DENNIS - NVSL - AMES,IA; MYERS, T - USDA-APHIS,RIVERDALE,MD; BULAGA, LESLIE - USDA-APHIS, NEW JERSEY; GARBER, P - FORT COLLINS, CO; PERDUE, MICHAEL - USDA-ARS, BELTSVILLE,MD; LOHMAN, KENTON - BROOKS AFB-SAN ANTONIO,TX; DAUM, LUKE - BROOKS AFB-SAN ANTONIO,TX; Suarez, David

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/18/2002
Publication Date: 9/17/2002
Citation: Spackman, E., Senne, D.S., Myers, T.J., Bulaga, L.L., Garber, P., Perdue, M., Lohman, K., Daum, L.T., Suarez, D.L. 2002. Development Of A Real-Time RT-PCR Assay For Type A Influenza And The Avian H5 And H7 Hemagglutinin subtypes. Journal of Clinical Microbiology.

Interpretive Summary: Avian influenza (AI) is a virus that can cause severe economic losses in poultry. Although the virus is considered to be exotic to the United States, it is currently circulating the live-bird market (LBM) system in the Northeast. In order to eliminate the threat that this disease may be transmitted to commercial poultry operations, it must be eradicated from the markets. Eradication of AI from the LBM's requires a rapid and sensitive diagnostic test. With current detection methods for AI, results may not be obtained for one to two weeks. We have developed a rapid method for the detection of AI and for the identification of strains that are associated with severe disease in poultry. With this novel test results may be obtained in as little as three hours. This method, real-time reverse-transcriptase-PCR, was compared directly with the current standard AI detection method for its ability to detect AI in clinical samples obtained from LBM's. The new, rapid method compared well with the current standard method and the results could be obtained as quickly as expected.

Technical Abstract: A real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) assay was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limi of 10fg or approximately 1000 copies of target RNA and can detect 0.1 EID50 of virus. The H5 and H7 specific probe sets each have a detection limit of 100fg of target RNA or approximately 1000-10,000 gene copies. The sensitivity and specificity of the real-time PCR assay was directly compared with the current standard for detection of influenza virus; virus isolation (VI) in embryonated chicken eggs and hemagglutinin (HA) subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets. Overall the sensitivity and specificity of the influenza, H7 and H5 specific RRT-PCR was similar to VI and HI.