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      Super-assembly of integrated gold magnetic assay with loop-mediated isothermal amplification for point-of-care testing

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          Abstract

          With the increasing global threat of various diseases and infections, it is essential to develop a fast, low-cost, and easy-to-use point-of-care testing (POCT) system for inspections at all levels of medical institutions and self-examination at home. In this work, gold magnetic nanoparticles (GMNPs) are used as the key material, and a rapid visual detection method is designed through integrating loop-mediated isothermal amplification (LAMP) and lateral flow assay (LFA) biosensor for detecting a variety of analytes which includes whole blood, buccal swabs, and DNA. It is worth to note that the proposed method does not need DNA extraction. Furthermore, uracil DNA glycosylase (UDG) is employed to eliminate carrier contamination for preventing false positive results. The whole detection process can be finished within 25 min. The accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) C677T. The detection limit of the newly developed extraction-free detection system for MTHFR C677T is 0.16 ng/μL. A preliminary clinical study of the proposed method is carried out by analyzing 600 clinical samples (including 200 whole blood samples, 100 buccal swabs, and 300 genomic DNA samples). The results indicate that the proposed method is 100% consistent with the sequencing results which provides a new choice for POCT and shows a broad application prospect in all levels of medical clinics and at home.

          Electronic Supplementary Material

          Supplementary material (details for MTHFR C677T primer sequences, the cell count results of samples at different dilution ratios, genotyping results and frequency samples, a Hardy—Weinberg equilibrium test, the sensitivity of the system, detection results of multiple samples, and optimization of the system) is available in the online version of this article at 10.1007/s12274-022-4692-9.

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          Most cited references56

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          Loop-mediated isothermal amplification of DNA.

          T. Notomi (2000)
          We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
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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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              Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.

              Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.
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                Author and article information

                Contributors
                yalicui@nwu.edu.cn
                bkong@fudan.edu.cn
                huiwl@nwu.edu.cn
                Journal
                Nano Res
                Nano Res
                Nano Research
                Tsinghua University Press (Beijing )
                1998-0124
                1998-0000
                4 August 2022
                : 1-10
                Affiliations
                [1 ]GRID grid.412262.1, ISNI 0000 0004 1761 5538, The College of life science, , Northwest University, ; Xi’an, 710069 China
                [2 ]Shaanxi Provincial Engineering Research Center for Nano-Biomedical Detection, Xi’an, 710077 China
                [3 ]GRID grid.8547.e, ISNI 0000 0001 0125 2443, Department of Chemistry, Shanghai Key Lab of Molecular Catalysis and Innovative Materials, Collaborative Innovation Center of Chemistry for Energy Materials, , Fudan University, ; Shanghai, 200438 China
                [4 ]GRID grid.412262.1, ISNI 0000 0004 1761 5538, Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, , Northwest University, ; Xi’an, 710069 China
                [5 ]GRID grid.412262.1, ISNI 0000 0004 1761 5538, Provincial Key Laboratory of Biotechnology of Shaanxi Province, , Northwest University, ; Xi’an, 710069 China
                [6 ]GRID grid.1005.4, ISNI 0000 0004 4902 0432, School of Chemical Engineering, Graduate School of Biomedical Engineering, and Australian Centre for NanoMedicine, , University of New South Wales, ; Sydney, NSW 2052 Australia
                Article
                4692
                10.1007/s12274-022-4692-9
                9362447
                f2086574-f472-46b5-8299-317a537204c2
                © Tsinghua University Press 2022

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 25 May 2022
                : 21 June 2022
                : 21 June 2022
                Categories
                Research Article

                gold magnetic nanoparticles,loop-mediated isothermal amplification,lateral flow assay system,free extraction,single-nucleotide polymorphisms genotyping

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