Reverse Transcription Recombinase-Aided Amplification Assay for Newcastle Disease Virus in Poultry

Newcastle disease (ND) is a highly contagious and economically significant viral infection that affects poultry globally, with recurrent outbreaks occurring even among vaccinated flocks in Egypt. Caused by the Newcastle disease virus (NDV), the disease results in substantial losses due to high mortality rates, decreased productivity, and the imposition of trade restrictions. This study aimed to develop a rapid, sensitive, and field-deployable diagnostic assay based on real-time reverse transcription recombinase-aided amplification (RT-RAA) for the detection of all NDV genotypes in clinical avian specimens. Primers and an exo-probe were designed based on the most conserved region of the NDV matrix gene. After testing ten primer combinations, the pair NDV RAA-F1 and RAA-R5 demonstrated the highest sensitivity, detecting as low as 6.89 EID₅₀/mL (95% CI). The RT-RAA assay showed excellent clinical sensitivity and specificity, with no cross-reactivity to other common respiratory pathogens such as avian influenza virus, infectious bronchitis virus, Mycoplasma gallisepticum or infectious laryngotracheitis virus. All 25 field samples that were tested positive by real-time RT-PCR, including those with high CT values (~35), were detected by RT-RAA in 2-11 min, indicating superior sensitivity and speed. The assay requires only basic equipment and can be performed under isothermal conditions, making it highly suitable for on-site detection in resource-limited or rural settings. The successful implementation of RT-RAA can improve NDV outbreak response, support timely vaccination strategies, and enhance disease control efforts. Overall, the assay presents a promising alternative to conventional diagnostic methods, contributing to the sustainability and productivity of the poultry sector in endemic regions.