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Title: DEVELOPMENT OF REAL-TIME RT-PCR FOR THE DETECTION OF AVIAN INFLUENZA VIRUS

Author
item Spackman, Erica
item SENNE, DENNIS - NVSL-USDA-APHIS, AMES,IA
item BULAGA, LESLIE - USDA-APHIS, NEW JERSEY
item MYERS, T - USDA-APHIS, RIVERDALE,MD
item Perdue, Michael
item GARBER, LINDSEY - NAT'L ANIMAL HEALTH, CO
item LOHMAN, KENTON - BROOKS AIR FORCE BASE,TX
item DAUM, LUKE - BROOKS AIR FORCE BASE,TX
item Suarez, David

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/30/2002
Publication Date: 9/1/2003
Citation: Spackman, E., Senne, D.A., Bulaga, L.L., Myers, T.J., Perdue, M.L., Garber, L.P., Lohman, K., Daum, L.T., Suarez, D.L. 2003. Development Of Real-Time RT-PCR For The Detection Of Avian Influenza Virus. Avian Diseases 47:1079-1082, 2003.

Interpretive Summary: Avian influenza is a virus that causes an economically important disease in commercial poultry. Currently, in the United States, the virus is not circulating in commercial poultry, however it is circulating in the urban live bird market (LBM) system in the Northeast, including LBM's in New York and New Jersey. With the persistence of the virus in the LBM system there exists the potential for the virus to be transmitted commercial poultry operations, possibly leading to severe economic consequences. In order to eliminate the threat of virus spread, the virus needs to be eradicated from the LBM system. An effective eradication program requires a fast and sensitive virus detection test, however current methods of virus detection take one to three weeks to obtain results. In this study we have developed real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) as a rapid and sensitive method for influenza virus detection and for identification of the virus subtypes associated with virulent virus in poultry. A direct comparison of the current standard virus detection test and the new RRT-PCR assay was performed and results indicated a similar level of sensitivity and specificity between the tests for detection of the virus in individual live bird markets.

Technical Abstract: A real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay was developed utilizing hydrolysis probes for the detection of avian influenza virus (AIV) and the H5 and H7 subtypes. The AIV specific primers and probes were directed to regions of the AIV matrix gene which are conserved among most type A influenza viruses. The H5 and H7 primers and probes are directed to H5 and H7 hemagglutinin gene regions which are conserved among North American avian influenza viruses. The sensitivity and specificity of this RRT-PCR assay was compared to virus isolation (VI) in chicken embryos with 1550 clinical swab samples from 109 live bird markets (LBM's) in New York and New Jersey. RRT-PCR detected influenza in samples from 61 of 65 (93.8%) of the LBM's which were the sources of VI positive samples. Of the 58 markets which were positive for H7 influenza by hemagglutination inhibition assay, RRT-PCR detected H7 influenza in 56 markets (96.5%). Too few H5 positive samples were obtained to validate the H5 RRT-PCR assay in this study. Although RRT-PCR was less sensitive than VI on an individual sample basis, this study demonstrated that the AIV and H7 RRT-PCR assays are good tools for the rapid screening of flocks and live bird markets.