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      An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus

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          Abstract

          Abstract

          Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming.

          Key points

          •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity.

          •Fifty field samples have been visualized using DNA-nanoprobe validation.

          •The developed system is a reliable, fast, and cost-effective option for POCT.

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          Supplementary Information

          The online version contains supplementary material available at 10.1007/s00253-024-13243-x.

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          Most cited references36

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          The Vienna RNA Websuite

          The Vienna RNA Websuite is a comprehensive collection of tools for folding, design and analysis of RNA sequences. It provides a web interface to the most commonly used programs of the Vienna RNA package. Among them, we find folding of single and aligned sequences, prediction of RNA–RNA interactions, and design of sequences with a given structure. Additionally, we provide analysis of folding landscapes using the barriers program and structural RNA alignments using LocARNA. The web server together with software packages for download is freely accessible at http://rna.tbi.univie.ac.at/.
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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
                lbenitez@ucm.es
                rimadrid@ucm.es
                Journal
                Appl Microbiol Biotechnol
                Appl Microbiol Biotechnol
                Applied Microbiology and Biotechnology
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0175-7598
                1432-0614
                10 July 2024
                10 July 2024
                2024
                : 108
                : 1
                : 414
                Affiliations
                [1 ]BioAssays SL. Parque Científico de Madrid, Madrid, Spain
                [2 ]GRID grid.4795.f, ISNI 0000 0001 2157 7667, Research Group of “Animal Viruses” of Complutense University of Madrid, ; Madrid, Spain
                [3 ]Deparment of Animal Health, Veterinary Faculty, Complutense University of Madrid (UCM), ( https://ror.org/02p0gd045) Madrid, Spain
                [4 ]Centro de Sanidad Avícola de Cataluña y Aragón (CESAC), Reus, Spain
                [5 ]Department of Genetics, Physiology, and Microbiology, School of Biology, Complutense University of Madrid (UCM), ( https://ror.org/02p0gd045) Madrid, Spain
                Author information
                http://orcid.org/0000-0002-1350-9864
                Article
                13243
                10.1007/s00253-024-13243-x
                11236856
                38985204
                72e5f90b-7bf3-4c0f-b831-d682a44c9d8a
                © The Author(s) 2024

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 14 February 2024
                : 6 June 2024
                : 20 June 2024
                Categories
                Applied Genetics and Molecular Biotechnology
                Custom metadata
                © Springer-Verlag GmbH Germany, part of Springer Nature 2024

                Biotechnology
                molecular detection,reverse transcription loop mediated isothermal amplification (rt-lamp),nanoprobes,avian metapneumovirus (ampv),point of care (poc) test

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