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Title: Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real time RT-PCR assay

Author
item Kim, L
item Suarez, David
item Afonso, Claudio

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2008
Publication Date: 7/7/2008
Citation: Kim, L.M., Suarez, D.L., Afonso, C.L. 2008. Detection of a broad range of class I and II Newcastle disease viruses using a multiplex real-time reverse transcription polymerase chain reaction assay. Journal of Veterinary Diagnostic Investigation. 20:414-425.

Interpretive Summary: Newcastle disease virus (NDV) is of significant concern to poultry producers worldwide. Prompt detection of virulent strains of Newcastle disease virus (vNDV) which cause disease in poultry and differentiation of these viruses from those of low virulence (loNDV) are challenging due to the broad genetic variability of the viruses. Rapid detection of NDV in the U.S. is currently accomplished by two validated real time reverse transcription (RT)-PCR assays: the M-gene assay which detects both loNDV and vNDV, and the F-gene assay which detects virulent isolates only. A large number of loNDV viruses have been recently identified in samples recovered from wild birds and from poultry in live bird markets (LBMs); however, the M-gene assay failed to detect the majority of these viruses. The ability to detect endemic loNDV strains would enhance our ability to identify the presence of loNDV viral circulating in poultry, predict which viruses represent a potential outbreak risk, and prevent future outbreaks. Here we describe the development and evaluation of a real time RT-PCR assay that allows identification of a broad range of viruses and has been designed to work in conjunction with the existing M-gene assay as a multiplex assay that is capable of detecting both virulent and loNDV.

Technical Abstract: Prompt detection of virulent strains of Newcastle disease virus (vNDV) using real time RT-PCR (rRT-PCR) is challenging due to the broad genetic variability across two clades comprising 18 recognized genotypes. A large proportion of class I low virulence Newcastle disease viruses (loNDV) recently identified in samples recovered from wild birds and from poultry in live bird markets are not detected by the validated rRT-PCR assay which 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 rRT-PCR assay (L-TET assay) that identifies a broad range of NDV, demonstrates a ten-fold increase in sensitivity over a previously reported L-gene assay, identifies previously missed isolates, 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 NDV with <1 Ct 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 to VII) were correctly identified by both the single- and multiplex formats.