Quantitative real-time PCR assays for the concurrent diagnosis of infectious laryngotracheitis virus, Newcastle disease virus and avian metapneumovirus in poultry

Abstract

Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log₁₀ dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%-98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.

Keywords: Newcastle disease virus; avian infectious laryngotracheitis virus; avian metapneumovirus; quantitative real-time PCR.

Fig. 1. Analytical sensitivity of qRT-PCR assays. Standard curves for (A) NDV-M, (B) NDV-L, (C) ILTV, (D) aMPV-A, (E) aMPV-B assays presenting the mean Ct plotted against the relative input copy numbers (log₁₀) of synthetic DNA standards that were tested in triplicate. Synthetic DNA standards were serially diluted by 10-fold at a 5 log₁₀ range, starting from 10⁵ copies down to 10 copies per reaction.

qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; NDV, Newcastle disease virus; ILTV, infectious laryngotracheitis virus; aMPV, avian metapneumovirus.