Design of a multiplex real time RT-PCR assay to detect Newcastle disease viruses from classes I and II

Authors: Kim, L; Suarez, David; Afonso, Claudio

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2007
Publication Date: 6/15/2007
Citation: Kim, L.M., Suarez, D.L., Afonso, C.L. 2007. Design of a multiplex real time RT-PCR assay to detect Newcastle disease viruses from classes I and II [abstract]. 10th International Conference for Biodetection Technologies 2007, June 14-15, 2007, Atlanta, Georgia. Poster #8.

Interpretive Summary:

Technical Abstract: Newcastle disease (ND) is a major concern for poultry producers around the world and the rapid diagnosis of an outbreak is crucial to any control program. Prompt detection of the causative agent of ND, virulent strains of Newcastle disease virus (vNDV), and differentiation of these viruses from those of low virulence (loNDV) are challenging due to their broad genetic variability and because these viruses are serologically indistinguishable. Both vNDV and loNDV belong to avian paramyxovirus serotype-1 and have recently been classified into two sister clades, class I and II, which contain several genotypes in each. Virulent viruses are primarily found in class II, while loNDV predominate class I. Rapid identification of NDV in the U.S. is currently accomplished by two validated real time RT-PCR assays. The M-gene assay, which targets the matrix gene, is used as an initial screening technique for detecting any NDV and the F-gene assay directed at the fusion gene is employed to distinguish virulent isolates from those of low virulence. A large number of class I NDV viruses have been recently identified in samples recovered from waterfowl and shorebirds, and also from poultry in live bird markets (LBMs); however, it was discovered that the M-gene assay failed to detect the majority of these viruses. Comparative nucleotide analysis of the 24 bp M-gene assay probe-site identified significant genomic variability between class I and II isolates that are likely responsible for the decreased sensitivity of the M-gene assay to the class I viruses. Because loNDV found in waterfowl and shorebirds represent a large and highly mobile reservoir that has previously been shown to transmit to domestic poultry, there is the possibility that these viruses could increase in virulence upon introduction to poultry. The increase in virulence from loNDV to vNDV was seen in outbreaks in the Republic of Ireland during 1990 and Australia during 1998-2000. In the Australian outbreak, the progenitor virus was a local strain prone to greater genetic variation that evolved from a loNDV into vNDV while circulating in domestic poultry. To predict and prevent future outbreaks caused by NDV strains circulating in wild birds and poultry from LBMs, accurate monitoring assays are needed. The monitoring assay should detect all endemic strains from class I and II, because mixed class I and II infections have been recovered from wild birds and poultry. Here we describe the identification of conserved regions suitable for assay development using bioinformatics tools, and the evaluation of a novel real time RT-PCR assay targeting the polymerase gene (L-gene) to identify class I viruses. Preliminary results demonstrate significant improvement in detection of the class I viruses with the L-gene primer/probe set over the M-gene assay, with the limit of detection consistently at 102 copies of the target gene or 2 EID50. The assay is designed to be compatible with the current protocol for the M-gene assay, allowing for detection of a broad range of class I and II NDV recovered from wild birds and poultry in LBMs with a single multiplex assay. In addition, the L-gene set uses TET fluorescence instead of FAM (used by the M-gene assay) which allows for clear distinction between class I and II viruses, and the ability to detect mixed infections within a single sample.