Advertisement

High-Resolution Melting Analysis as a Tool for Plant Species Authentication

Protocol
First Online:
  • 610 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2264)

Abstract

High-resolution melting (HRM) analysis is a cost-effective, specific, and rapid tool that allows distinguishing genetically related plants and other organisms based on the detection of small nucleotide variations, which are recognized from melting properties of the double-stranded DNA. It has been widely applied in several areas of research and diagnostics, including botanical authentication of several food commodities and herbal products. Generally, it consists of the main steps: (1) in silico sequence analysis and primer design; (2) DNA extraction from plant material; (3) amplification by real-time PCR with an enhanced fluorescent dye targeting a specific DNA barcode or other regions of taxonomic interest (100–200 bp); (4) melting curve analysis; and (5) statistical data analysis using a specific HRM software. This chapter presents an overview of HRM analysis and application, followed by the detailed description of all the required reagents, instruments, and protocols for the successful and easy implementation of a HRM method to differentiate closely related plant species.

Key words

HRM Species identification Authenticity Botanical origin Food Herbal products 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgment

This work was supported by FCT (Fundação para a Ciência e Tecnologia) under the Partnership Agreements UIDB/50006/2020 and UIDB/00690/2020. L. Grazina is grateful to FCT grant (SFRH/BD/132462/2017) financed by POPH-QREN (subsidized by FSE and MCTES).

References

  1. 1.
    Villa C, Costa J, Oliveira MBPP, Mafra I (2017) Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus). Food Chem 229:680–687.  https://doi.org/10.1016/j.foodchem.2017.02.136CrossRefPubMedGoogle Scholar
  2. 2.
    Böhme K, Calo-Mata P, Barros-Velazquez J, Ortea I (2019) Review of recent DNA-based methods for main food-authentication topics. J Food Agric Chem 67:3854–3864.  https://doi.org/10.1021/acs.jafc.8b07016CrossRefGoogle Scholar
  3. 3.
    Costa J, Campos B, Amaral JS, Nunes ME, Oliveira MBPP, Mafra I (2016) HRM analysis targeting ITS1 and matK loci as potential DNA mini-barcodes for the authentication of Hypericum perforatum and Hypericum androsaemum in herbal infusions. Food Control 61:105–114.  https://doi.org/10.1016/j.foodcont.2015.09.035CrossRefGoogle Scholar
  4. 4.
    Grazina L, Amaral JS, Mafra I (2020) Botanical origin authentication of dietary supplements by DNA-based approaches. Compr Rev Food Sci Food Saf 19:1080–1109.  https://doi.org/10.1111/1541-4337.12551CrossRefGoogle Scholar
  5. 5.
    Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860.  https://doi.org/10.1373/49.6.853CrossRefPubMedGoogle Scholar
  6. 6.
    Druml B, Cichna-Markl M (2014) High resolution melting (HRM) analysis of DNA – its role and potential in food analysis. Food Chem 158:245–254.  https://doi.org/10.1016/j.foodchem.2014.02.111CrossRefPubMedGoogle Scholar
  7. 7.
    Sun W, Li J-J, Xiong C, Zhao B, Chen S-L (2016) The potential power of Bar-HRM technology in herbal medicine identification. Front Plant Sci 7:367.  https://doi.org/10.3389/fpls.2016.00367CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Pereira L, Gomes S, Barrias S, Fernandes JR, Martins-Lopes P (2018) Applying high-resolution melting (HRM) technology to olive oil and wine authenticity. Food Res Int 103:170–181.  https://doi.org/10.1016/j.foodres.2017.10.026CrossRefPubMedGoogle Scholar
  9. 9.
    Montgomery JL, Sanford LN, Wittwer CT (2010) High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev Mol Diagn 10:219–240.  https://doi.org/10.1586/erm.09.84CrossRefPubMedGoogle Scholar
  10. 10.
    Hollingsworth PM, Forrest LL, Spouge JL, Hajibabaei M, Ratnasingham S, van der Bank M et al (2009) A DNA barcode for land plants. Proc Natl Acad Sci U S A 106:12794–12797.  https://doi.org/10.1073/pnas.0905845106CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS One 6:e19254.  https://doi.org/10.1371/journal.pone.0019254CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Techen N, Parveen I, Pan Z, Khan IA (2014) DNA barcoding of medicinal plant material for identification. Curr Opin Biotechnol 25:103–110.  https://doi.org/10.1016/j.copbio.2013.09.010CrossRefPubMedGoogle Scholar
  13. 13.
    Soares S, Grazina L, Costa J, Amaral JS, Oliveira MBPP, Mafra I (2018) Botanical authentication of lavender (Lavandula spp.) honey by a novel DNA-barcoding approach coupled to high resolution melting analysis. Food Control 86:367–373.  https://doi.org/10.1016/j.foodcont.2017.11.046CrossRefGoogle Scholar
  14. 14.
    Villa C, Costa J, Meira L, Oliveira MBPP, Mafra I (2016) Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.). Food Control 65:21–31.  https://doi.org/10.1016/j.foodcont.2016.01.008CrossRefGoogle Scholar
  15. 15.
    Osathanunkul M, Osathanunkul R, Madesis P (2018) Species identification approach for both raw materials and end products of herbal supplements from Tinospora species. BMC Complem Altern Med 18:111.  https://doi.org/10.1186/s12906-018-2174-0CrossRefGoogle Scholar
  16. 16.
    Costa J, Mafra I, Oliveira MBPP (2012) High resolution melting analysis as a new approach to detect almond DNA encoding for Pru du 5 allergen in foods. Food Chem 133:1062–1069.  https://doi.org/10.1016/j.foodchem.2012.01.077CrossRefGoogle Scholar
  17. 17.
    Martín-Fernández B, Costa J, de-los-Santos-Álvarez N, López-Ruiz B, Oliveira MBPP, Mafra I (2016) High resolution melting analysis as a new approach to discriminate gluten-containing cereals. Food Chem 211:383–391.  https://doi.org/10.1016/j.foodchem.2016.05.067CrossRefPubMedGoogle Scholar
  18. 18.
    Kress WJ (2017) Plant DNA barcodes: applications today and in the future. J Syst Evol 55:291–307.  https://doi.org/10.1111/jse.12254CrossRefGoogle Scholar
  19. 19.
    Lipp M, Brodmann P, Pietsch K, Pauwels J, Anklam E (1999) IUPAC collaborative trail study of a method to detect genetically modified soy beans and maize in dried powder. J AOAC Int 82:923–928CrossRefPubMedGoogle Scholar
  20. 20.
    Mafra I, Silva SA, Moreira EJMO, da Silva CSF, Oliveira MBPP (2008) Comparative study of DNA extraction methods for soybean derived food products. Food Control 19:1183–1190.  https://doi.org/10.1016/j.foodcont.2008.01.004CrossRefGoogle Scholar
  21. 21.
    Christensen H, Olsen JE (2018) Primer design. In: Christensen H (ed) Introduction to bioinformatics in microbiology. Springer International Publishing, Cham, Switzerland, pp 81–102CrossRefGoogle Scholar
  22. 22.
  23. 23.
    Primer design for PCR. https://www.addgene.org/protocols/primer-design/. Accessed Jan 2020

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.REQUIMTE-LAQV, Faculdade de FarmáciaUniversidade do PortoPortoPortugal
  2. 2.Centro de Investigação de Montanha (CIMO)Instituto Politécnico de BragançaBragançaPortugal

Personalised recommendations

Cite protocol

Buy options