title: Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay.
abstract: Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11M (190 amol), equivalent to 8.67 × 105copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.
authors.affiliation: Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Jauset-Rubio
authors.affiliation: Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, Spain.
authors.forename: Markéta
authors.initials: M
authors.lastname: Svobodová
authors.affiliation: Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Mairal
authors.affiliation: Institute of Cellular Medicine, Diagnostic and Therapeutic Technologies Group, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Calum
authors.initials: C
authors.lastname: McNeil
authors.affiliation: Institute of Cellular Medicine, Diagnostic and Therapeutic Technologies Group, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Keegan
authors.affiliation: National Research Centre, Cairo, Egypt.
authors.forename: Ayman
authors.initials: A
authors.lastname: Saeed
authors.affiliation: National Research Centre, Cairo, Egypt.
authors.forename: Mohammad Nooredeen
authors.initials: MN
authors.lastname: Abbas
authors.affiliation: Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia.
authors.forename: Mohammad S
authors.initials: MS
authors.lastname: El-Shahawi
authors.affiliation: Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia.
authors.forename: Abdulaziz S
authors.initials: AS
authors.lastname: Bashammakh
authors.affiliation: Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia.
authors.forename: Abdulrahman O
authors.initials: AO
authors.lastname: Alyoubi
authors.affiliation: Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, Spain.|Institució Catalana de Recerca I Estudis Avancats, Passeig Lluís Companys 23, 08010 Barcelona, Spain.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O Sullivan
mesh_terms: D004247:DNA / Q000032:analysis*; D006046:Gold / Q000737:chemistry; D057230:Limit of Detection; D053768:Metal Nanoparticles; D010209:Paper; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D006046:Gold; D004247:DNA