Submitted to: Journal of Clinical Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/6/2003
Publication Date: 1/1/2004
Citation: Wise, M., Suarez, D.L., Seal, B.S., Janice, P.C., Senne, D.A., King, D.J., Kapczynski, D.R., Spackman, E. Development Of A Real-Time Reverse-Transcription, Polymerase Chain Reaction For Detection Of Newcastle Disease Virus RNA In Clinical Samples. Journal of Clinical Microbiology. Vol. 42, No.1, p329-338. 2004 Interpretive Summary: In October 2002, the state of California reported an outbreak of exotic Newcastle disease, a highly contagious avian disease that can cause severe economic harm to the poultry industry. Present methods for the detection of the causative agent, Newcastle disease virus (NDV), are based on isolation of the virus in embryonated chicken eggs. Although virus isolation is a sensitive and reliable method, it can be expensive and more importantly, time consuming. It normally takes several days for a routine diagnosis. As such, a more rapid detection test would be extremely useful for controlling outbreaks of this potentially devastating disease. We have developed assays for the detection of Newcastle disease virus RNA using a real-time reverse-transcription polymerase chain reaction (RRT-PCR) procedure. One RRT-PCR assay has broad specificity and could detect every member of a diverse collection of worldwide isolates. A second assay was developed to target potentially virulent NDV strains, and a third test can detect the vaccine strains that are currently being used in the United States. The efficacy of the procedures was demonstrated on experimentally infected chickens. These tests should be useful for the rapid screening of at-risk poultry flocks.
Technical Abstract: A real-time reverse-transcription polymerase chain reaction (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 utilizes 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 utilizing in vitro transcribed RNA, respectively. Both M gene assays could detect approx 101 50% egg infective doses (EID50) and the F gene assay could detect approximately 103 EID50. The RRT-PCR test was utilized 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.