Development of a real-time reverse-transcription PCR for detection of newcastle disease virus RNA in clinical samples.
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 10(3), 10(2), and 10(4) genome copies, respectively, with in vitro-transcribed RNA. Both M gene assays could detect approximately 10(1) 50% egg infective doses (EID(50)), and the F gene assay could detect approximately 10(3) EID(50). 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.
Wise
Mark G
MG
Southeast Poultry Research Laboratory, Agricultural Research Service, US Department of Agriculture, Athens, Georgia 30605,USA.
Suarez
David L
DL
Seal
Bruce S
BS
Pedersen
Janice C
JC
Senne
Dennis A
DA
King
Daniel J
DJ
Kapczynski
Darrell R
DR
Spackman
Erica
E
D000818:Animals; D002642:Chick Embryo; D002645:Chickens; D017931:DNA Primers; D009522:Newcastle disease virus / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral