Specific detection of avian influenza H5N2 whole virus particles on lateral flow strips using a pair of sandwich-type aptamers.

We report a selection of a cognate pair of aptamers for whole avian influenza virus particles of H5N2 by using graphene-oxide based systemic evolution of ligands by exponential enrichment (GO-SELEX), and the application of a pair of sandwich-type binding aptamers on the lateral flow strips. The aptamers were characterized by GO-FRET assay, and Kd values of the selected aptamers were estimated to be from 6.913 × 10 ⁵ to 1.27 × 10 ⁶ EID ₅₀ /ml (EID ₅₀ /ml: 50% egg infective dose). Based on the evidence from confocal laser scanning microscope (CLSM), surface plasmon resonance (SPR), and circular dichroism (CD) spectrum analysis, the aptamers, J ₃ APT and JH ₄ APT, were found to be working as a cognate pair that binds to the target virus at the different sites simultaneously. This cognate pair of aptamers then was successfully applied on the lateral flow strips, clearly showing sandwich-type binding images with the presence of the certain numbers of H5N2 virus particles. On the newly developed lateral flow strips, the target virus was detectable down to 6 × 10 ⁵ EID ₅₀ /ml in the buffer and 1.2 × 10 ⁶ EID ₅₀ /ml in the duck's feces, respectively, by the naked eye. By using the ImageJ software, the LOD was found to be 1.27 × 10 ⁵ EID ₅₀ /ml in the buffer and 2.09 × 10 ⁵ EID ₅₀ /ml in the duck's feces, respectively. Interestingly, on the lateral flow strips, enhanced specificity towards the target virus (H5N2) appeared over other subtypes of H5Nx. To the best of our knowledge, this is the first report about the application of the cognate pair of aptamers for the detection of influenza virus on the lateral flow strips. This study shows the promising perspective of a cognate pair of aptamers for the on-site detection system which could be useful for rapid detection of avian influenza viruses for preventing the pandemic influenza viruses from spreading.
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