Accurate detection of Newcastle disease virus using proximity-dependent DNA aptamer ligation assays

Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid-phase formats are widely used for high-performance protein detection in medical research. However, the affinity reagents used, which are mainly poly- and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid-phase proximity-dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real-time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid-phase PLAs, which use NDV-selective DNA aptamers, are more sensitive than the sandwich enzymatic-linked aptamer assay (ELAA), and have a comparable sensitivity to real-time reverse transcription PCR (rRT-PCR) as the gold standard detection method. In addition, the solid-phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper- and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT-PCR. The specificity of PLA is shown to be concordant with rRT-PCR.