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      Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

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          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 −11 M (190 amol), equivalent to 8.67 × 10 5 copies 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.

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          Most cited references52

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          Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

          The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity. Copyright 2001 Academic Press.
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            Nucleic acid sequence-based amplification.

            J. Compton (1991)
            Nucleic acid sequence-based amplification (NASBA) is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature.
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              Advances in paper-based point-of-care diagnostics.

              Advanced diagnostic technologies, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), have been widely used in well-equipped laboratories. However, they are not affordable or accessible in resource-limited settings due to the lack of basic infrastructure and/or trained operators. Paper-based diagnostic technologies are affordable, user-friendly, rapid, robust, and scalable for manufacturing, thus holding great potential to deliver point-of-care (POC) diagnostics to resource-limited settings. In this review, we present the working principles and reaction mechanism of paper-based diagnostics, including dipstick assays, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (μPADs), as well as the selection of substrates and fabrication methods. Further, we report the advances in improving detection sensitivity, quantification readout, procedure simplification and multi-functionalization of paper-based diagnostics, and discuss the disadvantages of paper-based diagnostics. We envision that miniaturized and integrated paper-based diagnostic devices with the sample-in-answer-out capability will meet the diverse requirements for diagnosis and treatment monitoring at the POC. © 2013 Elsevier B.V. All rights reserved.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                25 November 2016
                2016
                : 6
                : 37732
                Affiliations
                [1 ]Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira I Virgili , 43007 Tarragona, Spain
                [2 ]Institute of Cellular Medicine, Diagnostic and Therapeutic Technologies Group, Newcastle University , Newcastle upon Tyne, NE2 4HH, UK
                [3 ]National Research Centre , Cairo, Egypt
                [4 ]Department of Chemistry, Faculty of Science, King Abdulaziz University , P. O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia
                [5 ]Institució Catalana de Recerca I Estudis Avancats , Passeig Lluís Companys 23, 08010 Barcelona, Spain
                Author notes
                Article
                srep37732
                10.1038/srep37732
                5123575
                27886248
                f48e2256-c9b5-475c-b399-9693a163c926
                Copyright © 2016, The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 14 September 2016
                : 31 October 2016
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