Development of a real-time reverse-transcription PCR for detection of newcastle disease virus RNA in clinical samples

Abstract

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.

FIG. 1.

Phylogenetic analysis of nucleotide sequences from Newcastle disease virus fusion protein genes. Isolates include those examined by RRT-PCR as listed in Table 2. The phylogenetic tree was generated with the neighbor-joining method (28) following alignment of sequences. Numbers represent bootstrap confidence limits following 2,000 replications (12) with the avian paramyxovirus type 2 (APMV-2) sequence as an outgroup.

FIG. 2.

SmartCycler fluorographs illustrating 10-fold serial dilutions used to determine limits of detection of the RRT-PCR assays for viral nucleic acid. For the APMV-1 matrix gene with in vitro-transcribed game chicken/US(CA)/02 RNA (A), the limit of detection was approximately 10³ copies of the target gene, and the assay could detect approximately 10 EID₅₀ (B). For the fusion gene assay, the limit of detection as measured with in vitro-transcribed game chicken/CA/02 RNA (C) was approximately 10⁴ target copies, and the assay could detect approximately 10³ EID₅₀ (D). The N.A. pre-1960 genotype specific assay could detect approximately 10² target copies of chicken/US/B1/48 RNA (E) and approximately 10 EID₅₀ (F).