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      Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP)

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          Abstract

          Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay’s detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

          Supplementary Information

          The online version contains supplementary material available at 10.1007/s00216-021-03436-y.

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          Most cited references57

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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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            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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              Rapid extraction of fungal DNA for PCR amplification.

              J L Cenis (1992)
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                Author and article information

                Contributors
                niessen@mytum.de
                Journal
                Anal Bioanal Chem
                Anal Bioanal Chem
                Analytical and Bioanalytical Chemistry
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                1618-2642
                1618-2650
                15 June 2021
                15 June 2021
                2021
                : 413
                : 19
                : 4801-4813
                Affiliations
                [1 ]GRID grid.5252.0, ISNI 0000 0004 1936 973X, Faculty of Veterinary Medicine, , Ludwig-Maximilians-University Munich, ; Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany
                [2 ]GRID grid.5252.0, ISNI 0000 0004 1936 973X, Institute for Infectious Diseases and Zoonoses, Faculty of Veterinary Medicine, , Ludwig-Maximilians-University Munich, ; Veterinaerstraße 13, 80539 Munich, Germany
                [3 ]GRID grid.6583.8, ISNI 0000 0000 9686 6466, Institute for Food Safety, Food Technology and Veterinary Public Health, Unit of Food Hygiene and Technology, , University of Veterinary Medicine, ; Veterinärplatz 1, A-1210 Vienna, Austria
                [4 ]GRID grid.6936.a, ISNI 0000000123222966, TUM School of Life Sciences, , Technical University of Munich, ; Gregor-Mendel-Str. 4, 85354 Freising, Germany
                Article
                3436
                10.1007/s00216-021-03436-y
                8318954
                34129076
                3ed213dc-5e2f-4ed5-a7cf-e24f17598218
                © The Author(s) 2021

                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/.

                Funding
                Funded by: Brigitte and Wolfram Gedek Foundation, Ismaning, Germany
                Categories
                Research Paper
                Custom metadata
                © Springer-Verlag GmbH Germany, part of Springer Nature 2021

                Analytical chemistry
                stachybotryotoxicosis,sick building syndrome,indoor air quality,water damage,diagnostic test kit,sat14 gene

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