title: Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1.
abstract: Aptamers are artificial oligonucleotides (DNA or RNA) that can bind to a broad range of targets. In diagnostic and detection assays, aptamers represent an alternative to antibodies as recognition agents. The objective of this study was to select and characterize DNA aptamers that can specifically bind to avian influenza virus (AIV) H5N1 based on Systematic Evolution of Ligands by EXponential enrichment (SELEX) and surface plasmon resonance (SPR). The selection was started with an ssDNA (single-stranded DNA) library of 10¹⁴ molecules randomized at central 74 t. For the first four selection cycles, purified hemagglutinin (HA) from AIV H5N1 was used as the target protein, and starting from the fifth cycle, entire H5N1 virus was applied in order to improve the specificity. After 13 rounds of selection, DNA aptamers that bind to the H5N1 were isolated and three aptamer sequences were characterized further by sequencing and affinity binding. Dot blot analysis was employed for monitoring the SELEX process and conducting the preliminary tests on the affinity and specificity of aptamers. With the increasing number of selection cycles, a steady increase in the color density was observed, indicating that the aptamers with good binding affinity to the target were enriched. The best aptamer candidate had a dissociation constant (KD) of 4.65 M as determined by SPR, showing a strong binding between the HA and the selected aptamer. The specificity was determined by testing non-target AIV H5N2, H5N3, H5N9, H9N2 and H7N2. Negligible cross-reactivity confirmed the high specificity of selected aptamers. The developed aptamer was then applied for detection of AIV H5N1 in spiked poultry swab samples. The obtained aptamers could open up possibilities for the development of aptamer-based medical diagnostics and detection assays for AIV H5N1. (The H5N1 used in this study was inactivated virus.).
authors.affiliation: Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Ronghui
authors.initials: R
authors.lastname: Wang
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhao
authors.forename: Tieshan
authors.initials: T
authors.lastname: Jiang
authors.forename: Young M
authors.initials: YM
authors.lastname: Kwon
authors.forename: Huaguang
authors.initials: H
authors.lastname: Lu
authors.forename: Peirong
authors.initials: P
authors.lastname: Jiao
authors.forename: Ming
authors.initials: M
authors.lastname: Liao
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000302:isolation & purification*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis*; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology; D007700:Kinetics; D011200:Poultry; D011485:Protein Binding; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance
chemical_list: D052157:Aptamers, Nucleotide; D019267:Hemagglutinin Glycoproteins, Influenza Virus