SSR fingerprints and identification methods of garden ginseng, forest ginseng and wild ginseng
Through SSR fingerprinting technology, DNA extraction, PCR amplification and electrophoresis analysis were used to solve the accuracy of identification of ginseng, under-forest ginseng and wild ginseng, and efficient and accurate variety identification was achieved.
Patent Information
- Application Number
- CN202011400012.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-12-02
- Publication Date
- 2025-08-22
- Estimated Expiration
- 2040-12-02
AI Technical Summary
The prior art is difficult to accurately distinguish between ginseng, undergrowth ginseng and wild ginseng. Especially the identification of ginseng slices and ginseng powder series of products has strong subjectivity and cannot meet the needs of efficient and accurate identification.
Using SSR fingerprinting technology, standard fingerprinting of ginseng, under-forest ginseng and wild ginseng was established through DNA extraction, PCR amplification and electrophoresis analysis. The primers were PCR amplified by 20 primers, and fingerprinting was constructed by non-denaturation polyacrylamide gel electrophoresis and capillary electrophoresis, and the identification was carried out in combination with similarity analysis.
It has achieved a high resolution, high sensitivity and good repeatability identification method, which can accurately distinguish between ginseng, undergrowth ginseng and wild ginseng, and is suitable for variety identification of ginseng samples.
Smart Images
Figure CN114959086B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of identification of origins of Chinese medicinal materials, and in particular to an SSR fingerprint spectrum and identification method of garden ginseng, forest ginseng and wild ginseng. Background Art
[0002] Ginseng, a plant of the genus Panax, belongs to the Araliaceae family. It effectively regulates the central nervous system, lowers blood sugar, enhances immunity, and combats the spread and metastasis of cancer cells. It is a commonly used and precious traditional Chinese medicine in my country. Depending on its growing environment, ginseng can be divided into garden ginseng, forest ginseng, and wild ginseng. Garden ginseng is cultivated artificially, typically 4-6 years old, with a high survival rate but average nutritional value. Forest ginseng, typically 10-20 years old, is artificially sown and naturally germinates in the mountains and forests. Its survival rate is lower, and its ginsenoside content is higher than that of garden ginseng, often mistaken for wild ginseng. Wild ginseng, which grows in deep mountain forests and ranges from decades to hundreds of years old, is the most pristine ginseng, with the highest ginsenoside content. It is the most common ginseng, with the highest ginsenoside content, but is extremely rare and endangered. Currently, ginseng varieties are primarily identified based on morphology, microscopic features, and physicochemical properties. However, these methods are highly subjective and cannot distinguish between ginseng slices and powdered products. Therefore, a method for authenticity verification is needed.
[0003] With the development of molecular biology, the application of DNA molecular marker technology has made it possible to directly analyze the genetic variation of medicinal plants, and has also made the identification of Chinese medicinal materials and Chinese medicinal products more accurate and efficient. At present, DNA molecular markers have gone through restriction fragment length polymorphism (RFLP) based on molecular hybridization, amplified fragment length polymorphism (AFLP) based on PCR technology, and SSR. In comparison, RFLP is time-consuming and labor-intensive, and AFLP is difficult to compare and analyze. SSR is widely used because of its simple operation, high repeatability and polymorphism. DNA fingerprints are identified based on the composition, order and length differences of DNA sequences. Just like human fingerprints, they are individual-specific. SSR fingerprints are a type of DNA fingerprint. Because of its advantages such as small amount of sample required, fast detection speed, high accuracy and reliability, and no influence from environmental factors, it has become the preferred method for distinguishing precious Chinese medicinal materials. Summary of the Invention
[0004] The purpose of the present invention is to provide an SSR fingerprint spectrum and identification method for garden ginseng, forest ginseng and wild ginseng. This method has high resolution, high sensitivity, good repeatability and accurate results, and can be used for variety identification of ginseng samples.
[0005] The purpose of the present invention is achieved by the following technical solutions:
[0006] A method for establishing SSR fingerprints of garden ginseng, forest ginseng and wild ginseng is characterized in that it comprises the following steps:
[0007] 1) DNA extraction: Genomic DNA was extracted from garden ginseng, forest ginseng, and wild ginseng using the modified CTAB method;
[0008] 2) PCR amplification: 20 pairs of primers were used to amplify the above DNA;
[0009] 3) PCR product identification: The amplified products were analyzed by native polyacrylamide gel electrophoresis and capillary electrophoresis, and the electrophoretic patterns were recorded;
[0010] 4) Establishment of SSR fingerprint: According to the amplification results of each primer in the electrophoresis pattern, representative polymorphic SSR primers were selected to construct the SSR fingerprint of garden ginseng, forest ginseng and wild ginseng.
[0011] A method for SSR fingerprint identification of garden ginseng, forest ginseng and wild ginseng is characterized in that it comprises the following steps:
[0012] 1) First, establish the DNA standard fingerprint of garden ginseng, forest ginseng and wild ginseng as follows:
[0013] (a) DNA extraction: DNA was extracted from garden ginseng, forest ginseng, and wild ginseng as a control using the modified CTAB method;
[0014] (b) PCR amplification: Using the DNA extracted in step (1) as a template, PCR amplification reaction was performed using 20 pairs of primers;
[0015] (c) PCR product identification: The amplified products were analyzed by native polyacrylamide gel electrophoresis and capillary electrophoresis, and the electrophoretic patterns were recorded;
[0016] (d) Establishment of the SSR standard fingerprint of ginseng: Based on the amplification results of each primer in the electrophoresis pattern, the representative P35 primer was selected to construct the DNA standard fingerprint of garden ginseng, forest ginseng and wild ginseng;
[0017] 2) Determine the SSR fingerprint of the sample: Determine the SSR fingerprint of the garden ginseng, forest ginseng and wild ginseng samples using the same method as above;
[0018] 3) Result identification: Compare the similarity between the SSR fingerprint of the sample and the SSR standard fingerprint of garden ginseng, forest ginseng and wild ginseng, and identify the ginseng variety based on the similarity. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Non-denaturing polyacrylamide gel electrophoresis SSR standard fingerprints of garden ginseng, forest ginseng and wild ginseng
[0020] Figure 2Capillary electrophoresis SSR standard fingerprint of ginseng (garden ginseng, forest ginseng and wild ginseng) DETAILED DESCRIPTION
[0021] The present invention will be further described below with reference to the examples. The following examples are only used to illustrate the present invention but are not intended to limit the present invention.
[0022] Example 1
[0023] 1. DNA Extraction
[0024] Thirty garden ginseng samples, 15 forest ginseng samples and 16 wild ginseng samples were collected from different production areas. 0.1 g of sample was taken from each sample, and the genomic DNA of the samples was extracted using the modified CTAB method.
[0025] Table 1. Ginseng sample names and origins
[0026]
[0027] 2. PCR Amplification
[0028] The PCR reaction system was as follows: 1 μL 10 μmol / L SSR upper and lower primers, 4 μL 10 ng / μL template DNA, 10 μL rTaq DNA polymerase (TaKaRa TaqTM Version 2.0), and 4 μL deionized water. The reaction was performed on a gene amplification instrument.
[0029] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at a suitable temperature for 30 s; extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min, and storage at 4°C.
[0030] 3. Establishment of SSR fingerprints of garden ginseng, forest ginseng and wild ginseng
[0031] The PCR amplification products in step 2 were analyzed by native polyacrylamide gel electrophoresis and capillary electrophoresis. The specific steps are as follows:
[0032] 3.1 Non-denaturing polyacrylamide gel electrophoresis
[0033] PCR products were identified on an 8% non-denaturing polyacrylamide gel (10.5 ml ddH2O, 3.3 ml 30% acrylamide, 4 ml 5× TBE, 140 μL 10% ammonium persulfate, and 13 μL TEMED). 5 μL of PCR product was mixed with 1 μL 6× Loading Buffer and added to the sample wells. Electrophoresis was performed in an electrophoresis tank containing 1× TBE buffer. First, run at 120 V for 15 minutes to allow the sample to be compressed and produce more uniform bands. Then, run at 90 V until the dye has completely run out, which takes approximately 70-100 minutes. AgNO3 was used for staining, and the slides were placed under a film viewing light to photograph and record the bands.
[0034] 3.2 Capillary electrophoresis
[0035] Add 2 μL of sodium acetate solution and 40 μL of anhydrous ethanol to 20 μL of PCR product and place at -80°C for 1 hour. Centrifuge at 12,000 r / min at 4°C for 10 minutes to precipitate the DNA. Add 750 μL of 90% ethanol to the EP tube, centrifuge at 12,000 r / min at 4°C for 2 minutes, discard the supernatant, and air dry overnight. Add an appropriate amount of ddH2O to dissolve the DNA, measure the concentration and dilute it to 0.5-2 ng / μL. Load the capillary with 15 μL of sample. Start recording the sample band data when the 20 bp internal reference is detected. When the 1000 bp internal reference is detected, the system stops recording the data and the electrophoresis ends.
[0036] Example 2
[0037] The SSR fingerprint identification method of the present invention is used to identify a certain ginseng sample
[0038] Assume that there is a batch of garden ginseng samples and it is uncertain whether they are genuine garden ginseng. Therefore, they are identified according to the following method.
[0039] After the SSR standard fingerprint of Panax notoginseng is established, when a sample is to be identified, the genomic DNA of the sample is first extracted using the modified CTAB method; the extracted DNA is used as a template and PCR amplified using the P35 primer; the amplified products are analyzed by non-denaturing polyacrylamide gel electrophoresis and capillary electrophoresis, and the electrophoresis pattern is recorded to obtain the DNA fingerprint of the batch of samples to be tested; the similarity between the DNA fingerprint of the sample to be tested and the standard DNA fingerprint of Panax notoginseng is compared, and the sample to be tested is identified based on the similarity.
[0040] Identification results: If the similarity between the non-denaturing polyacrylamide gel electrophoresis and capillary electrophoresis patterns of the test sample and the standard is greater than 95% (including 95%), this batch of samples is genuine garden ginseng; if the similarity between the two is less than 95%, then this batch of samples is not genuine garden ginseng.
[0041] Example 3
[0042] The DNA fingerprint identification method of the forest ginseng of the present invention is used to identify a forest ginseng sample
[0043] Assume that there is a batch of forest ginseng samples and it cannot be confirmed whether they are genuine forest ginseng. Therefore, they are identified according to the following method.
[0044] After the SSR standard fingerprint of Panax notoginseng is established, when a sample is to be identified, the genomic DNA of the sample is first extracted using the modified CTAB method; the extracted DNA is used as a template and PCR amplified using the P35 primer; the amplified products are analyzed by non-denaturing polyacrylamide gel electrophoresis and capillary electrophoresis and the electrophoresis pattern is recorded to obtain the SSR fingerprint of the batch of samples to be tested; the similarity between the SSR fingerprint of the sample to be tested and the standard fingerprint of Panax notoginseng DNA is compared, and the sample to be tested is identified based on the similarity.
[0045] Identification results: If the similarity between the non-denaturing polyacrylamide gel electrophoresis and capillary electrophoresis patterns of the test sample and the standard is greater than 95% (including 95%), this batch of samples is genuine forest ginseng; if the similarity between the two is less than 95%, then this batch of samples is not genuine forest ginseng.
[0046] Example 4
[0047] The wild ginseng DNA fingerprint identification method of the present invention is used to identify a wild ginseng sample
[0048] Assume that there is a batch of wild ginseng samples and it is uncertain whether they are genuine wild ginseng. Therefore, they are identified according to the following method.
[0049] After the wild ginseng SSR standard fingerprint is established, when a sample is to be identified, the genomic DNA of the sample is first extracted using the modified CTAB method; the extracted DNA is used as a template and PCR amplified using the P35 primer; the amplified products are analyzed by non-denaturing polyacrylamide gel electrophoresis and capillary electrophoresis and the electrophoresis pattern is recorded to obtain the DNA fingerprint of the batch of samples to be tested; the similarity between the SSR fingerprint of the sample to be tested and the wild ginseng SSR standard fingerprint is compared, and the sample to be tested is identified based on the similarity.
[0050] Identification results: If the similarity between the non-denaturing polyacrylamide gel electrophoresis and capillary electrophoresis patterns of the test sample and the standard is greater than 95% (including 95%), this batch of samples is genuine wild ginseng; if the similarity between the two is less than 95%, then this batch of samples is not genuine wild ginseng.
Claims
1. A method for identifying garden ginseng, forest ginseng and wild ginseng, characterized by: Primer P35 was used to amplify garden ginseng, forest ginseng and wild ginseng. The upstream and downstream primers of primer P35 are: Upstream primer: 5′-AAAGCAGGGTGTGCTCACTT-3′; Downstream primer: 5′-AGGGAGACCGGAGCATTATT-3′.