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Journal of Clinical Microbiology, September 2002, p. 3256-3260, Vol. 40, No. 9
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.9.3256-3260.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes

Erica Spackman,1 Dennis A. Senne,2 T. J. Myers,3 Leslie L. Bulaga,4 Lindsey P. Garber,5 Michael L. Perdue,6 Kenton Lohman,7 Luke T. Daum,7 and David L. Suarez1*

Southeast Poultry Research Laboratory, USDA Agricultural Research Service, Athens, Georgia 30605,1 National Veterinary Services Laboratories, USDA Animal and Plant Health Inspection Service, Ames, Iowa 50010,2 USDA Animal and Plant Health Inspection Service, Riverdale, Maryland,3 USDA Animal and Plant Health Inspection Service, Robbinsville, New Jersey 08691,4 Centers for Epidemiology and Animal Health, National Animal Health Monitoring System, Fort Collins, Colorado 80521,5 Animal Waste Pathogens Laboratory, USDA Agricultural Research Service, Beltsville, Maryland 20705,6 Technology and Diagnostics Development, Brooks Air Force Base, San Antonio, Texas 782357

Received 21 February 2002/ Returned for modification 2 June 2002/ Accepted 18 June 2002

A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene 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 limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 103 to 104 gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.

* Corresponding author. Mailing address: Southeast Poultry Research Laboratory, USDA-ARS, 934 College Station Rd., Athens, GA 30605. Phone: (706) 546-3434. Fax: (706) 546-3161. E-mail: dsuarez{at}seprl.usda.gov.

Journal of Clinical Microbiology, September 2002, p. 3256-3260, Vol. 40, No. 9
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.9.3256-3260.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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