Recombinase polymerase amplification–nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection

Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) are two poultry pathogens affecting the respiratory tract of chickens, and cause major economic losses in the industry. Rapid detection of these viruses is crucial to inform implementation of appropriate control measures. The objective of our study is developing a simple, rapid and field applicable recombinase polymerase amplification (RPA)–nucleic acid lateral flow (NALF) immunoassay for detection of NDV and IBV. Isothermal amplification of the matrix protein (M) gene of NDV and the nucleoprotein (N) gene of IBV was implemented via recombinase polymerase amplification at 38 °C for 40 min and 20 min, respectively using modified labeled primers. NALF device used in this study utilizes antibodies for detection of labeled RPA amplicons. The results revealed that RPA-NALF immunoassays can detect both viruses after 40 min at 38 °C and only NDV after 20 min. The limit of detection (LOD) was 10 genomic copies/RPA reaction. The assays results on clinical samples collected from diseased chicken farms demonstrated a good performance in comparison with quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). The assays established in this study can facilitate rapid, on-site molecular diagnosis of suspected cases of ND and IB viral infections as the results can be detected by the naked eye without the need for measuring fluorescence. Furthermore, the NALF device could be adapted to detect other infectious agents.