Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

AbstractMitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

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Differentiation of Mitragyna Speciosa, a Narcotic Plant, From Allied Mitragyna Species Using DNA Barcoding-high-resolution Melting (Bar-HRM) Analysis

10.21203/rs.3.rs-109985/v1 ◽

2020 ◽

Author(s):

Chayapol Tungphatthong ◽

Santhosh Kumar J Urumarudappa ◽

Supita Awachai ◽

Thongchai Sooksawate ◽

Suchada Sukrong

Keyword(s):

High Resolution ◽

Dna Barcoding ◽

High Resolution Melting ◽

Dna Barcode ◽

Herbal Medicines ◽

Efficient Tool ◽

Hrm Analysis ◽

Mitragyna Speciosa ◽

Leaf Powder

Abstract Mitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

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A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Genes ◽

10.3390/genes12020199 ◽

2021 ◽

Vol 12 (2) ◽

pp. 199

Author(s):

Xiaochun Zhang ◽

Huan Yu ◽

Qi Yang ◽

Ziwei Wang ◽

Ruocheng Xia ◽

...

Keyword(s):

High Resolution ◽

Dna Barcoding ◽

High Resolution Melting ◽

Its Region ◽

Its Sequencing ◽

Melting Temperatures ◽

Hrm Analysis ◽

National Drug ◽

Species Specific ◽

Its1 And Its2

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

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Microsatellite and DNA-barcode regions typing combined with High Resolution Melting (HRM) analysis for food forensic uses: A case study on lentils (Lens culinaris)

Food Research International ◽

10.1016/j.foodres.2011.12.013 ◽

2012 ◽

Vol 46 (1) ◽

pp. 141-147 ◽

Cited By ~ 58

Author(s):

Irene Bosmali ◽

Ioannis Ganopoulos ◽

Panagiotis Madesis ◽

Athanasios Tsaftaris

Keyword(s):

High Resolution ◽

Lens Culinaris ◽

High Resolution Melting ◽

Dna Barcode ◽

Hrm Analysis

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Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis

Genes ◽

10.3390/genes9080408 ◽

2018 ◽

Vol 9 (8) ◽

pp. 408 ◽

Cited By ~ 1

Author(s):

Nur Fadzil ◽

Alina Wagiran ◽

Faezah Mohd Salleh ◽

Shamsiah Abdullah ◽

Nur Mohd Izham

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Dna Barcode ◽

Pcr Amplification ◽

Herbal Products ◽

Reliable Technique ◽

Safety Issues ◽

Eurycoma Longifolia ◽

Hrm Analysis ◽

Melting Analysis

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.

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DNA barcode ITS2 coupled with high resolution melting (HRM) analysis for taxonomic identification of Sideritis species growing in Greece

Molecular Biology Reports ◽

10.1007/s11033-014-3381-5 ◽

2014 ◽

Vol 41 (8) ◽

pp. 5147-5155 ◽

Cited By ~ 34

Author(s):

Apostolos Kalivas ◽

Ioannis Ganopoulos ◽

Aliki Xanthopoulou ◽

Paschalina Chatzopoulou ◽

Athanasios Tsaftaris ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Dna Barcode ◽

Taxonomic Identification ◽

Hrm Analysis

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In vitro chondroprotective potential of Senna alata and Senna tora in porcine cartilage explants and their species differentiation by DNA barcoding-high resolution melting (Bar-HRM) analysis

PLoS ONE ◽

10.1371/journal.pone.0215664 ◽

2019 ◽

Vol 14 (4) ◽

pp. e0215664 ◽

Cited By ~ 1

Author(s):

Siriwan Ongchai ◽

Chatchadawalai Chokchaitaweesuk ◽

Patiwat Kongdang ◽

Siriwadee Chomdej ◽

Kittisak Buddhachat

Keyword(s):

High Resolution ◽

Dna Barcoding ◽

High Resolution Melting ◽

Cartilage Explants ◽

Species Differentiation ◽

Hrm Analysis ◽

Senna Alata

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Genotyping of GATA4 Gene Variant (G296S) in Malaysian Congenital Heart Disease Subjects by Real-Time PCR High Resolution Melting Analysis

Journal of Medical Biochemistry ◽

10.2478/jomb-2013-0006 ◽

2013 ◽

Vol 32 (2) ◽

pp. 152-157

Author(s):

Nora Fawzi ◽

Ramachandran Vasudevan ◽

Patimah Ismail ◽

Mazeni Alwi ◽

Ahmad Fazli Abdul Aziz ◽

...

Keyword(s):

Congenital Heart Disease ◽

Heart Disease ◽

High Resolution ◽

Congenital Heart ◽

High Resolution Melting ◽

New Technology ◽

Cost Effective ◽

Study Cohort ◽

Hrm Analysis ◽

Melting Analysis

Summary Background: Congenital heart disease (CHD) is the most common birth defect; however, the underlying etiology is unrecognized in the majority of cases. GATA-binding protein 4 (GATA4), a cardiac transcription factor gene, has a crucial role in the cardiogenesis process; hence, a number of heterozygote sequence variations were identified as a cause of CHD. G296S heterozygote variant is the most frequently reported GATA4 gene sequence alteration. This study aims to investigate the role of G296S variant of the GATA4 gene in Malaysian CHD subjects. Methods: We have investigated 86 Malaysian CHD subjects with cardiac septation defects for the presence of the GATA4 gene heterozygote variant (G296S) by the new technology of high resolution melting (HRM) analysis. Results: Genotyping of G296S (c.886G>A) by HRM analysis shows that all the sample genotypes were of the wild GG type genotype and the heterozygote mutant GA genotype was totally absent from this study cohort. Conclusions: The results of our study showed that the G296S variant of the GATA4 gene was not associated with the development of CHD in Malaysian subjects. The use of HRM analysis proved a cost-effective, high-throughput, specific and sensitive genotyping technique which eliminates the need for unnecessary sequencing.

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