Detection of a Broad Range of Class I and II Newcastle Disease Viruses Using a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay

J Vet Diagn Invest. 2008 Jul;20(4):414-25. doi: 10.1177/104063870802000402.


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 <or=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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Newcastle disease virus / classification
  • Newcastle disease virus / isolation & purification*
  • RNA, Viral / isolation & purification
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / veterinary*
  • Sensitivity and Specificity


  • RNA, Viral