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Title: DEVELOPMENT OF MULTIPLEX REAL-TIME RT-PCR AS A DIAGNOSTIC TOOL FOR AVIAN INFLUENZA

Author
item Spackman, Erica
item SENNE, DENNIS - NVSL-APHIS-USDA
item BULAGA, LESLIE - APHIS-USDA-NJ
item TROCK, SUSAN - CORNELL UNIV-ITHACA,NY
item Suarez, David

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2002
Publication Date: 9/1/2003
Citation: Spackman, E., Senne, D.A., Bulaga, L.L., Trock, S., Suarez, D.L. 2003. Development Of Multiplex Real-Time RT-PCR As A Diagnostic Tool For Avian Influenza. Avian Diseases. 47:1087-1090, 2003.

Interpretive Summary: Not required.

Technical Abstract: A multiplex real-time RT-PCR (RRT-PCR) assay for the simultaneous detection of the H5 and H7 hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro transcribed RNA templates, to have a reproducible detection limit for H7 of 104 HA gene copies and 104-105 HA gene copies of H5. A direct comparison of H5-H7 multiplex RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR and virus isolation positive tracheal and cloacal swab samples obtained from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79 (95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either assay.