Application of DNA Barcoding to Identify Medicinal Plants

Summary

The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

Abstract

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

Introduction

Medicinal plants have a wide range of pharmacological effects and are important substances for the treatment and prevention of diseases. The market demand for medicinal plants in herbal medicines and pharmaceutical products is huge and still growing. With the increasing market for medicinal plants, the problem of adulteration has hindered the development of medicinal plants. Currently, medicinal plant adulteration suffers from these reasons: 1) the similar morphology of medicinal plants makes it difficult to identify and use them correctly^1,2,3,4<....

Protocol

1. Sample preparation

1. The present study utilized two-year-old A. sinensis with low temperature and long sunshine at an altitude of 2800 m from Min County, Gansu Province, as experimental materials.Three plants were selected from which more than three roots were selected for experimentation. Rinse the roots first with sterilized water and then clean them with an alcohol sprayer or alcohol swabs to prevent contamination that could affect the accuracy of the experimental results. Cut th.......

Discussion

Molecular identification technology is easier to learn and master than traditional identification methods. It overcomes the limitations of traditional medicinal herb identification, as it is not affected by the plant's growth stage and does not rely on subjective judgment or the accumulation of specialized expertise³⁵. Other species are confused with A. sinensis due to its similar morphology and medicinal parts, but with the use of DNA barcoding, it can be definitively identified.......

Materials

Name	Company	Catalog Number	Comments
CTAB Rapid Plant Genomic DNA Extraction Kit	Shanghai Huiling Biotechnology Co.	NG411M	Suitable for rapid extraction of high quality genomic DNA from different tissues of a wide range of plants
DL5000 DNA Marker	Nanjing vazyme Bio-technology Co.	MD102-02	Ready-to-use product, take an appropriate amount of this product directly for electrophoresis when running the gel.
Electrophoresis	Beijing Liuyi Biotechnology Co.	DYY-6C	Adopt touch screen design, can display set voltage, set current at the same time, parallel output
Ethylenediaminetetraacetic acid	BeijingpsaitongBiotechnologyCo.,Ltd	E70015-100G	Nuclear Isolation Buffer formulation reagents.
Goldview Nucleic Acid Gel Stain(10,000×)	Yisheng Biotechnology (Shanghai) Co., Ltd	10201ES03	When using agarose gel electrophoresis to detect DNA, it binds to DNA and produces a strong fluorescent signal.
High-Speed Tabletop Centrifuge	Changsha High-tech Industrial Development Zone Xiangyi Centrifuge Instrument Co.	H1650	For fast and efficient separation of samples, this compact and lightweight centrifuge offers reliable safety
High-Throughput Tissue Grinder	Shanghai Jingxin Industrial Development Co.	Tiss-48	A high-frequency vibration instrument for grinding samples
http://www.gpgenome.com/	Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences	-	HerbGenomics database includes genome sequences, gene sets, organelle genomes, low coverage genome data and DNA barcode sequences.
https://www.ncbi.nlm.nih.gov/	U.S. National Library of Medicine	-	The National Center for Biotechnology Information advances science and health by providing access to biomedical and genomic information.
Kylin-Bell	Haimen Qilinbel Instrument Manufacturing Co.	VORTEX-5	Rapid mixing in the form of a high-speed vortex, mixing speed, uniformity, thoroughness
LifeTouch	Hangzhou BORI Technology Co.	TC-96/G/H(b)B	Adoption of advanced thermoelectric refrigeration technology and newly created TAS technology to enhance its overall performance
Multi-functional Gel Image Analysis System	Southwest Operation Center of Shanghai Tianneng Life Science Co.	Tanon-Mini Space 2000	Performs rapid gene amplification experiments with a gradient function for mapping amplification conditions and a gradient temperature range of up to 30°C
NaCl	Beijing Solarbio Science&Technology Co.,Ltd.	S8210-100	Nuclear Isolation Buffer formulation reagents.
Nanodrop One	Genes Ltd.	ND ONE	Quantify DNA, RNA and protein samples in seconds with just 1-2 µL of sample
Polyvinyl pyrrolidone	Shanghai yuanye Bio-Technology Co., Ltd	S30268-500g	Nuclear Isolation Buffer formulation reagents.
Snowflake Ice Maker	Shanghai Zhixin Experimental Instrument Technology Co.	ZX-60X	Adopting rotary extrusion ice making method, fast ice making speed and high efficiency of ice production
Stainless Steel Beads for Tissue Homogenizer	Beyotime Biotechnology.	F6623	Equipment for grinding and mixing of tissue and other samples by vibration
Tris Acetate-EDTA buffer	Beyotime Biotech Inc	ST716	TAE is a commonly used buffer for DNA electrophoresis, frequently employed in agarose gel electrophoresis.
Tris-HCl	Beijing Solarbio Science&Technology Co.,Ltd.	T8230	Nuclear Isolation Buffer formulation reagents.
Water bath Kettle	Shanghai Senxin Experimental Instrument Co.	DK-8D	Precise thermostat and temperature regulation, accurate and reliable temperature control
β-mercaptoethanol	Shanghai Eon Chemical Technology Co.	R054186-100ml	Nuclear Isolation Buffer formulation reagents.