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      A Rapid and Efficient Loop-mediated Isothermal Amplification (LAMP) Assay for the Authentication of Food Supplements Based on Maitake ( Grifola Frondosa)

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          Abstract

          Grifola frondosa (“Maitake”) is an edible fungus with several nutraceutical properties, largely used in traditional medicine. The increased use of Maitake as a food supplements ingredient raised the need of accurate authentication methods since the morphological identification of G. frondosa is not feasible in formulated food supplements. We developed a diagnostic tool based on loop-mediated isothermal AMPlification (LAMP) for the detection of G. frondosa in food supplements. First, a modified CTAB protocol for DNA extraction from food supplements has been set up and it has been shown to be able to isolate amplifiable total genomic material from different types of commercial products. Subsequently, the LAMP assay confirmed high specificity and good analytical sensitivity, allowing to detect up to 0.62 pg of genomic DNA in less than 20 min. Ten related fungal species resulted negative, confirming the specificity of the assay. The presence of Maitake in commercial food supplements was confirmed, except for one, revealing a mislabeling (or a food fraud). This assay proved to be a rapid powerful tool for food authentication purposes and routine inspections at any level of the supply chain of Maitake-based products and it can be used as a model for other quality control assays of fungal food products.

          Supplementary Information

          The online version contains supplementary material available at 10.1007/s12161-022-02235-0.

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          Most cited references37

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          The Molecular Evolutionary Genetics Analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
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            A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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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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                Author and article information

                Contributors
                marco.arlorio@uniupo.it
                Journal
                Food Anal Methods
                Food Anal Methods
                Food Analytical Methods
                Springer US (New York )
                1936-9751
                1936-976X
                8 March 2022
                : 1-13
                Affiliations
                [1 ]GRID grid.16563.37, ISNI 0000000121663741, Dipartimento Di Scienze del Farmaco & Drug and Food Biotechnology (DFB) Center, , Università del Piemonte Orientale “A. Avogadro”, ; Largo Donegani 2, 28100 Novara, Italy
                [2 ]GRID grid.5326.2, ISNI 0000 0001 1940 4177, National Research Council - Institute for Sustainable Plant Protection (CNR-IPSP), ; Torino, Italy
                Author information
                http://orcid.org/0000-0002-0757-8089
                Article
                2235
                10.1007/s12161-022-02235-0
                8903311
                35282313
                d00127d6-2f56-4fb9-934f-fb4085fc75b5
                © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 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
                : 15 October 2021
                : 21 February 2022
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100009885, Regione Piemonte;
                Award ID: P.O.R. FESR 2014/2020 (Asse I – Azione I-1b.2.2)
                Award Recipient :
                Categories
                Article

                maitake,mushroom food supplements,food authentication,loop-mediated isothermal amplification,species identification

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