Species discrimination and hybrid detection in terrestrial orchids using Bar-HRM: A case of the Calanthe group

Buddhachat, Kittisak; Sripairoj, Nattaporn; Punjansing, Tasanai; Kongbangkerd, Anupan; Inthima, Phithak; Tanming, Wattana; Kosavititkul, Prasuk

doi:10.1016/j.plgene.2021.100349

Cited by 3 publications

(1 citation statement)

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“…DNA mini-barcoding is the development of shorter molecular markers (usually 100-300 bp) based on the nucleotide profile of a specific species or genus group, effectively solving the problem of identifying herbs with varying degrees of DNA degradation (Yeo et al, 2020;Parveen et al, 2022). High-resolution melting (HRM) is a classical melting curve analysis method based on PCR fragments, which, compared to other molecular identification methods, allows the sample to be identified, even if a single-base variation occurs in the DNA sequence, facilitating the quantitative determination of the rate of adulteration (Wu et al, 2008;Ganopoulos et al, 2011;Duan et al, 2018;Buddhachat et al, 2021). HRM represents a simple, sensitive, low cost, and specific method that can realize closed-tube operation (Reed et al, 2007;Kalivas et al, 2014;Kim et al, 2023).…”

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confidence: 99%

Successful development of molecular diagnostic technology combining mini-barcoding and high-resolution melting for traditional Chinese medicine agarwood species based on single-nucleotide polymorphism in the chloroplast genome

Feng,

Liu,

Xie

et al. 2024

Front. Plant Sci.

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Agarwood is a valuable traditional medicine and fragrance. The production process is a typical injury-induced defense response. Currently, there are approximately 22 known species in the genus Aquilaria Lam., all of which can produce agarwood, whereas there are only two legal species of traditional Chinese medicinal agarwood, Aquilaria sinensis (Lour.) Spreng. and Aquilaria agallocha (Lour.) Roxb. The Taiwan herbal Pharmacopoeia of China stipulates that the medicinal agarwood species are A. sinensis and its relatives in the same genus. Moreover, there are five species of agarwood available for clinical medicinal use in Japan, including A. agallocha and A. sinensis, which are often confused with each other or used in a mixed way in the trade process. Therefore, accurate identification of traditional Chinese medicinal agarwood species is important to ensure the authenticity of traditional medicines and to guide the safety of clinical medication. In this study, 59 specific single-nucleotide polymorphism loci were screened and obtained from the chloroplast genomes of 12 species of the genus Aquilaria Lam. We established an identification method for traditional Chinese medicinal agarwood using mini-barcoding combined with high-resolution melting (HRM) and designed and validated 10 pairs of primers from the psbM-trnD, psbA, rps16, petN, ndhE-psaC, rps4, atpE, ycf1, rps15-trnN, and matK regions. The amplification products were all less than 200 bp, with a high success rate of amplification. The method was applied to successfully identify traditional Chinese medicinal agarwood species from commercial agarwood samples. Overall, the sensitivity of this method was sufficient to detect 1% of adulterants in medicinal agarwood products, proving that mini-barcoding HRM is a powerful and flexible tool. This method can be used as a fast and effective high-throughput method for authenticity testing of traditional Chinese medicinal agarwood and its raw materials containing agarwood-containing proprietary Chinese medicines and is recommended for industrial applications.

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