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

Abstract

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.

Aptamer-Assisted Proximity Ligation Assay for Sensitive Detection of Infectious Bronchitis Coronavirus

“…Our study demonstrates that the homogenous and solid-phase PLAs provide greater sensitivity than the sandwich ELAA and perfect concordance with the qRT-PCR. The sensitivity and LOD of the developed PLA have the same range found in previous work, confirming the robustness of the assay ( 28 ). The developed PLA-based tests in this study can be a new tool for the direct detection of pathogens, with high sensitivity.…”

“…The DNA aptamer-assisted PLAs in homogenous and solid-phase formats were designed as described in our previous study, with some modifications ( 28 ). The two biotinylated aptamers, Apt_IBV02 and Apt_IBV05, were coupled to two streptavidin-conjugated DNA oligonucleotides to construct PLA probes.…”

“…PLA has proven to be a robust protein detection method and an exquisitely sensitive technique with a very low background. It was recently used for the detection of NDV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ( 28 , 42 ). Our study demonstrates that the homogenous and solid-phase PLAs provide greater sensitivity than the sandwich ELAA and perfect concordance with the qRT-PCR.…”

“…The PLA probes were constructed by connecting the two biotinylated aptamers (AptIBV05 and AptIBV02) against IBV ( Table 1 ) to the two streptavidin-conjugated DNA oligonucleotides (SCL1 and SCL2) ( Table 4 ) via a biotin-streptavidin interaction. The PLA probe preparation and aptamer immobilization on microplates were carried out as previously described ( 28 ). An aliquot of 10 5 EID 50 /mL −1 of the vaccine strain H120, diluted in PLA buffer, was used to prepare 2-fold serial dilutions.…”

Review detection of Newcastle disease virus

“…PLAs are high sensitivity, high efficiency, and reproducibility. They are reliable tools for detecting NDV and have the potential for a multiplex application for the detection of various avian viruses ( 88 ).…”