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Journal of Clinical Microbiology, January 2004, p. 329-338, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.329-338.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples

Mark G. Wise,1 David L. Suarez,1 Bruce S. Seal,1 Janice C. Pedersen,2 Dennis A. Senne,2 Daniel J. King,1 Darrell R. Kapczynski,1 and Erica Spackman1*

Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia,1 National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa2

Received 3 June 2003/ Returned for modification 14 September 2003/ Accepted 5 October 2003

A real-time reverse-transcription PCR (RRT-PCR) was developed to detect avian paramyxovirus 1 (APMV-1) RNA, also referred to as Newcastle disease virus (NDV), in clinical samples from birds. The assay uses a single-tube protocol with fluorogenic hydrolysis probes. Oligonucleotide primers and probes were designed to detect sequences from a conserved region of the matrix protein (M) gene that recognized a diverse set (n = 44) of APMV-1 isolates. A second primer-probe set was targeted to sequences in the fusion protein (F) gene that code for the cleavage site and detect potentially virulent NDV isolates. A third set, also directed against the M gene, was specific for the North American (N.A.) pre-1960 genotype that includes the common vaccine strains used in commercial poultry in the United States. The APMV-1 M gene, N.A. pre-1960 M gene, and F gene probe sets were capable of detecting approximately 103, 102, and 104 genome copies, respectively, with in vitro-transcribed RNA. Both M gene assays could detect approximately 101 50% egg infective doses (EID50), and the F gene assay could detect approximately 103 EID50. The RRT-PCR test was used to examine clinical samples from chickens experimentally infected with the NDV strain responsible for a recent epizootic in the southwestern United States. Overall, a positive correlation was obtained between the RRT-PCR results and virus isolation for NDV from clinical samples.

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

Journal of Clinical Microbiology, January 2004, p. 329-338, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.329-338.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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