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Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real-time reverse transcription polymerase chain reaction assay

Correspondence: ¹Corresponding Author: Claudio L. Afonso, USDA ARS, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, GA 30605. Claudio.Afonso{at}ars.usda.gov

Prompt detection of virulent strains of Newcastle disease virus (vNDV) using real-time reverse transcription polymerase chain reaction (RT-PCR) is challenging because of the broad genetic variability across 2 clades comprising 18 recognized genotypes. A large proportion of class I low virulence ND viruses recently identified in samples recovered from wild birds and from poultry in live bird markets are not detected by the validated real-time RT-PCR assay that targets the matrix gene (M-gene assay). This study describes the identification and sequencing of a conserved region from the polymerase gene of class I NDV and the design and evaluation of a real-time RT-PCR assay (L-TET assay) that identifies a broad range of NDV, demonstrates a 10-fold increase in sensitivity over a previously reported L-gene assay, and works in conjunction with the existing M-gene assay using the same protocol. The L-TET assay detects 1 fg of homologous transcribed RNA from genotypes 5, 7, and 8 of class I, and from class II genotype II in either single- or multiplex format. Differential detection of mixed class I and II viruses down to 100 fg is possible because L-TET uses an alternate fluorophore from the M-gene assay. The multiplexed assay is capable of detecting a broad range of class I and II ND viruses with <1 threshold cycle decrease in sensitivity compared to the single probe. A total of 140 class I (n = 108, genotypes 1–2 and 4–9) and class II (n = 32, genotypes I–VII) were correctly identified by both the single- and multiplex formats.

Key Words: Diagnostic • multiplex • Newcastle disease virus • polymerase • real-time reverse transcription polymerase chain reaction