Generation of ssDNA aptamers as diagnostic tool for Newcastle avian virus

Abstract

Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing. After three rounds of selections, a highly enriched ssDNA pool was sequenced, and the results were analyzed using FASTAptamer Toolkit. Sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. Top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (ELAA) for rapid and sensitive detection of NDV in farm samples. The selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. Following optimization of the sandwich ELAA method, the results demonstrated that both selected aptamers Apt_NDV01 and Apt_NDV03 with dissociation constant values of 31 nM and 78.1 nM, respectively, showed the highest specificity and affinity for NDV detection. The ELAA results were verified by quantitative real-time PCR, demonstrating strong concordance, and showing outstanding accuracy for detection of NDV in field sample. In summary, combination of SELEX with high-throughput DNA sequencing allowed rapid screening and selection of aptamers. The selected aptamers allowed recognition of NDV with high affinities. This is the first report that uses a validated sandwich ELAA for rapid and specific detection of NDV in poultry samples.

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

“…The binding affinity, specificity, and compatibility of selected aptamers were described in our previous work [15]. To determine the limit of detection (LOD) of the sandwich Enzyme Linked Aptamer Assay (ELAA) test, LaSota vaccine strain titrating 10 6 EID 50 ·mL −1 was used, demonstrating that using such aptamers, the sandwich ELAA was able to detect as low as 1.2 (EID 50 ·mL −1 ), as compared to the gold standard for NDV detection, the reverse real‐time PCR (rRT‐PCR) that provides a LOD of 0.6 (EID 50 ·mL −1 ), when using the aptamers as affinity reagents.…”

“…1. The PLA probes were constructed by connecting the two biotinylated aptamers against the NDV [15] to two streptavidin‐conjugated DNA oligonucleotides via biotin‐streptavidin interaction. The DNA oligonucleotides have previously been designed and empirically evaluated for use in PLA [16].…”

“…The affinity, the specificity, and the compatibility of these two selected aptamers used in the sandwich assays have been evaluated in our previous study [15]. The binding analysis revealing that both aptamers recognize NDV in field clinical samples with high specificity and nanomolar affinities [15]. The developed PLA‐based tests for the detection of NVD were further evaluated on the farm samples previously analyzed by rRT‐PCR and sandwich ELAA.…”

“…Both Apt_NDV01 and Apt_NDV03 were selected using LaSota vaccine as a target along with SELEX process combined with high‐throughput sequencing. The affinity, the specificity, and the compatibility of these two selected aptamers used in the sandwich assays have been evaluated in our previous study [15]. The binding analysis revealing that both aptamers recognize NDV in field clinical samples with high specificity and nanomolar affinities [15].…”

“…Aptamer production and validation was previously described by Marnissi et al . [15]. Briefly, selection was performed using three rounds of systematic evolution of ligands by exponential enrichment (SELEX).…”