title: Label-free detection of gliadin food allergen mediated by real-time apta-PCR.
abstract: Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2 nM (100 ng mL(-1)). The specificity of the selected aptamer was demonstrated and no cross-reactivity was observed with streptavidin, bovine serum albumin or anti-gliadin IgG.
authors.affiliation: Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, Tarragona, 43007, Spain.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pinto
authors.forename: Pedro Nadal
authors.initials: PN
authors.lastname: Polo
authors.forename: Olivier
authors.initials: O
authors.lastname: Henry
authors.forename: M Carmen Bermudo
authors.initials: MC
authors.lastname: Redondo
authors.forename: Marketa
authors.initials: M
authors.lastname: Svobodova
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O'Sullivan
mesh_terms: D000485:Allergens / Q000032:analysis*; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D002417:Cattle; D003001:Cloning, Molecular; D002523:Edible Grain / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005903:Gliadin / Q000032:analysis*; D006801:Humans; D007074:Immunoglobulin G; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique; D012710:Serum Albumin, Bovine; D019809:Streptavidin; D020349:Surface Plasmon Resonance
chemical_list: D000485:Allergens; D052157:Aptamers, Nucleotide; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine; D005903:Gliadin; D019809:Streptavidin