Evaluation of DNA barcoding coupled high resolution melting for discrimination of closely related species in phytopharmaceuticals

Maslin Osathanunkul; Chatmongkon Suwannapoom; Kitisak Osathanunkul; Panagiotis Madesis; Hugo de Boer

doi:10.1016/j.phymed.2015.11.018

Evaluation of DNA barcoding coupled high resolution melting for discrimination of closely related species in phytopharmaceuticals

Phytomedicine. 2016 Feb 15;23(2):156-65. doi: 10.1016/j.phymed.2015.11.018. Epub 2016 Jan 6.

Authors

Maslin Osathanunkul¹, Chatmongkon Suwannapoom², Kitisak Osathanunkul³, Panagiotis Madesis⁴, Hugo de Boer⁵

Affiliations

¹ Department of Biology, Faculty of Science, Chiang Mai University, 239 Huay Kaew Rd., Suthep, Muang, Chiang Mai 50200, Thailand. Electronic address: omaslin@gmail.com.
² State Key Laboratory of Genetic Resources and Evolution State, and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; School of Agriculture and Natural Resources, University of Phayao, Phayao 56000, Thailand.
³ Department of Computer Science, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand.
⁴ Institute of Applied Biosciences, Centre for Research & Technology Hellas (CERTH), Thessaloniki, Greece.
⁵ Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-75236 Uppsala, Sweden; The Natural History Museum, University of Oslo, P.O. Box 1172, NO-0318 Oslo, Norway.

PMID: 26926177
DOI: 10.1016/j.phymed.2015.11.018

Abstract

Background: Phytopharmaceuticals are increasingly popular as alternative medicines, but poorly regulated in many countries. The manufacturers of these products should be subject to strict controls regarding each product's quality and constituents. Routine testing and identification of raw materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use.

Hypothesis/purpose: We have applied DNA Barcoding - High Resolution Melting (Bar-HRM), an emerging method for identifying of medicinal plant species based on DNA dissociation kinetics and DNA barcoding, for the authentication of medicinal plant species.

Study design: Commonly commercialized Thai medicinal plants that are widely used for medicinal purposes were used in this study. Publicly available sequences of four plastid markers were used for universal primer design. Species discrimination efficiency of the designed primers was evaluated as single and multi-locus analyses by using the primers sets.

Methods: HRM analysis was performed in triplicate on each of the 26 taxa to establish the Tm for each primer set (matK, rbcLA, rbcLB, rbcLC, rpoC1, and trnL). The shapes of the melting curves were analyzed to distinguish the different plant species. Bar-HRM species identification success rates were assessed for each single-locus as well as for multi-locus combinations to establish the optimal combination of primer sets.

Results: In single locus analysis the rpoC1 primer set gave the highest discrimination (58%), and in multi locus analysis this could be increased from 87% to 99% depending on the total number of regions included. Different combinations proved to be more or less effective at discrimination, depending on the genus or family examined.

Conclusions: Bar-HRM has proven to be a cost-effective and reliable method for the identification of species in this study of Thai medicinal plants, and results show an identification success rate of 99% among species in the test set.

Keywords: Authentication; DNA barcoding; Herbal pharmacovigilance; High resolution melting; Medicinal plants.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Barcoding, Taxonomic*
DNA Primers / genetics
DNA, Chloroplast / genetics
DNA, Plant / genetics
Genetic Markers
Plants, Medicinal / chemistry
Plants, Medicinal / classification*
Quality Control
Thailand

Substances

DNA Primers
DNA, Chloroplast
DNA, Plant
Genetic Markers