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      Loop-mediated isothermal amplification (LAMP): An advanced molecular point-of-care technique for the detection of Leishmania infection

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          Abstract

          Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.

          Author summary

          Developing sensitive point-of-care diagnostic tests is vital for enhancing Leishmania-infection control programs and treatment. This review provides information on the development of the loop-mediated isothermal amplification (LAMP) diagnostic test and highlights recent advances in the field of molecular diagnosis of leishmaniasis and the needs for future research. Furthermore, we elaborate of the future potential of LAMP as a rapid point-of-care (in-clinic and in the field) test for diagnosis and entomological monitoring of Leishmania infection, including evaluation of control programs in Leishmania-endemic areas.

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          Most cited references62

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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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            Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.

            Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3'-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.
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              Nucleic acid isothermal amplification technologies: a review.

              Nucleic acid amplification technologies are used in the field of molecular biology and recombinant DNA technologies. These techniques are used as leading methods in detecting and analyzing a small quantity of nucleic acids. The polymerase chain reaction (PCR) is the most widely used method for DNA amplification for detection and identification of infectious diseases, genetic disorders and other research purposes. However, it requires a thermocycling machine to separate two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitro isothermal DNA amplification methods, mimicking these in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. In this article, we review these isothermal nucleic acid amplification technologies and their applications in molecular biological studies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                7 November 2019
                November 2019
                : 13
                : 11
                : e0007698
                Affiliations
                [1 ] Snyder Institute for Chronic Diseases, Departments of Microbiology, Immunology, and Infectious Diseases, Cumming School of Medicine and Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
                [2 ] Division of Medical Zoology, Department of Infection and Immunity, Jichi Medical University, Tochigi, Japan
                Pasteur Institute of Iran, ISLAMIC REPUBLIC OF IRAN
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-2548-9581
                Article
                PNTD-D-19-00431
                10.1371/journal.pntd.0007698
                6837287
                31697673
                97a18c07-f284-4b1c-986f-5bdf75cf2d36
                © 2019 Nzelu et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Page count
                Figures: 4, Tables: 2, Pages: 21
                Funding
                This work was supported by funding from the Infections, Inflammation and Chronic Diseases Research Strategy, Vice-President of Research Office, University of Calgary ( https://ucalgary.ca/iicd/) and the Canadian Institutes of Health Research ( http://www.cihr-irsc.gc.ca/e/193.html), grant MPO 142302, to NCP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Review
                Medicine and Health Sciences
                Tropical Diseases
                Neglected Tropical Diseases
                Leishmaniasis
                Medicine and Health Sciences
                Parasitic Diseases
                Protozoan Infections
                Leishmaniasis
                Medicine and Health Sciences
                Infectious Diseases
                Zoonoses
                Leishmaniasis
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protozoans
                Parasitic Protozoans
                Leishmania
                Medicine and Health Sciences
                Parasitic Diseases
                Medicine and Health Sciences
                Infectious Diseases
                Disease Vectors
                Insect Vectors
                Sand Flies
                Biology and Life Sciences
                Species Interactions
                Disease Vectors
                Insect Vectors
                Sand Flies
                Medicine and Health Sciences
                Diagnostic Medicine
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Kinetoplasts

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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