Method for identifying angelica sinensis by combining PCR (Polymerase Chain Reaction) with sequencing based on ITS2

Abstract

The invention discloses a method for identifying angelica sinensis based on ITS2 PCR combined with sequencing, and belongs to the field of traditional Chinese medicinal material detection. The method comprises the following steps: extracting DNA (Deoxyribonucleic Acid) of angelica sinensis, performing PCR (Polymerase Chain Reaction) amplification, adding A tail, performing nucleic acid electrophoresis, recovering gel, constructing a vector, converting, performing blue-white screening, extracting plasmids, performing PCR and enzyme digestion detection, finally sequencing, and comparing a gene bank to identify the authenticity of an angelica sinensis sample. The identification results of eight angelica sinensis samples show that four angelica sinensis samples, three angelica dahurica samples and one angelica dahurica sample, and the comparison similarities of ITS2 fragments of the angelica sinensis samples and a gene bank are 98.99%, 95.56% and 98.99% respectively. Therefore, the gene identification method established by the invention can accurately identify the authenticity of the angelica sinensis sample, and provides reference for angelica sinensis identification.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine material detection, and particularly to a method for identifying Angelica sinensis by ITS2-based PCR combined with sequencing. Background Art

[0002] Traditional Chinese medicine is a treasure of Chinese traditional culture. Since ancient times, authenticity identification has been an important topic in the research of traditional Chinese medicine materials. The identification methods of traditional Chinese medicine materials have been continuously improved and innovated, from the identification methods relying on sensory experience to the classical identification methods using instruments and reagents. With the development of modern science and technology, the usage methods and sources of traditional Chinese medicine materials have become more complex. There are more and more phenomena of mixing, substitution, passing off fakes as genuine, and passing off inferior goods as superior. Therefore, the identification methods of traditional Chinese medicine materials should be improved and innovated. At present, the main identification methods of traditional Chinese medicine materials include morphological identification, microscopic identification, physicochemical identification, etc. However, these traditional methods have certain defects. Recently, molecular systematics based on molecular biology and population genetics has developed, and more and more scientists have tried to apply it to the modern identification of traditional Chinese medicine materials. Gene methods can identify species through gene fragments and have been applied in the field of traditional Chinese medicine material identification. It is an effective supplement to traditional methods, avoiding the limitations of morphological identification and providing rich evidence directly at the gene level. With the increasingly wide use of traditional Chinese medicine materials, in order to seek profits, illegal merchants adulterate and forge traditional Chinese medicine materials, pass off inferior goods as superior, forge the origin, and use low-priced Chinese herbal medicines to counterfeit high-priced species or varieties. Consumers are paying more and more attention to the origin and authenticity of traditional Chinese medicine materials. The adulteration of traditional Chinese medicine materials has a wide range and various forms, and it is difficult to detect by conventional means. Advanced scientific and technological means such as spectral technology analysis, near-infrared spectroscopy analysis, characteristic chromatogram technology of traditional Chinese medicine, high-throughput DNA sequencing technology, and nucleic acid detection have been used to distinguish the authenticity of traditional Chinese medicine materials.

[0003] Angelica sinensis, sweet and pungent in taste, is the dried root of the umbelliferous plant Angelica sinensis (Oliv) Diels, which is a traditional species in China that can be used both as medicine and food. Its main producing areas are in the southeastern part of Gansu Province, with Min County having the largest output and the best quality, followed by Yunnan, Sichuan and other provinces. It has the functions of enriching blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relaxing bowel movements. It is commonly used for dizziness and palpitations, irregular menstruation, etc. The identification of Angelica sinensis has also developed greatly. In 2019, Fu Hong proposed to use identification methods such as character, microscopy, and thin layer to compare and analyze the identification of the medicinal materials and cut pieces of Angelica sinensis, Levisticum officinale, and Angelica acutiloba. In 2020, Che Surong et al. proposed to use the ultraviolet spectral group method to compare and identify the ultraviolet absorption spectrograms of Angelica sinensis, its adulterant Heracleum hemsleyanum, and Levisticum officinale in different polar solvents. In 2021, Shi Zhongfei et al. established a method based on site-specific PCR technology to quickly identify the adulterated Levisticum officinale in Angelica sinensis medicinal materials and cut pieces. Li Bo et al. analyzed the differences in the material basis of Angelica sinensis medicinal materials, cut pieces, and standard decoctions at low temperature drying based on the HPLC fingerprint feature correlation O2PLS-DA model. However, each of the above methods has its own advantages and disadvantages. Therefore, enriching the methods for identifying the authenticity of Angelica sinensis is of great significance for preventing the appearance of counterfeits from multiple angles. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for identifying Angelica sinensis by PCR combined with sequencing based on ITS2 to solve the problems existing in the above-mentioned prior art. The PCR method constructed by the primer pair designed based on ITS2 combined with gene sequencing can simply and highly sensitively identify the authenticity of Angelica sinensis samples, providing a reference for the gene identification of Angelica sinensis.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides the application of ITS2 in any of the following:

[0007] (1) Application in identifying the authenticity of Angelica sinensis;

[0008] (2) Application in preparing a kit for identifying the authenticity of Angelica sinensis;

[0009] Among them, the ITS2 is obtained by amplification using a primer pair with nucleotide sequences shown in SEQ ID NO.1-2.

[0010] Preferably, the ITS2 is used to identify Angelica sinensis and its adulterants Angelica dahurica and Peucedanum terebinthaceum.

[0011] Preferably, the identification method includes the following steps:

[0012] Using the DNA of the sample to be tested as a template, performing PCR amplification using the primer pair to obtain an amplification product;

[0013] Ligate the amplified product with a vector to construct a recombinant plasmid, and transform it into competent Escherichia coli. After culturing, pick positive bacteria; after lysing the positive bacteria, extract the plasmid, perform enzyme digestion, and sequence it;

[0014] Compare the sequencing results with ITS2 in the gene database to identify the authenticity of Angelica sinensis.

[0015] Preferably, the reaction system for PCR amplification includes: 12.5 μL of PrimeSTAR Max Premix (2×), 8.5 μL of ddH2O, 2 μL of total DNA, and 1 μL of each forward and reverse primer.

[0016] Preferably, the reaction program for PCR amplification is: denaturation at 98 °C for 5 s, annealing at 53 °C for 10 s, and extension at 72 °C for 5 s for one cycle, with 30 cycles.

[0017] Preferably, a PloyA is added to the 3' end of the nucleotide sequence of the amplified product; and / or the vector includes the pMD19-T vector.

[0018] Preferably, after enzyme digestion of the plasmid, samples larger than 3200 bp are selected for sequencing, or after PCR amplification of the plasmid, electrophoresis detection is performed, and samples showing 500 bp are selected for sequencing.

[0019] Preferably, ITS2 in the gene database includes the Angelica sinensis ITS2 gene with the gene accession number KC295071 on NCBI, the Angelica dahurica ITS2 gene with the gene accession number OK668234, and the Peucedanum terebinthaceum ITS2 gene with the gene accession number KC295082.

[0020] The present invention also provides a primer pair for identifying Angelica sinensis by PCR combined with sequencing based on ITS2, and the nucleotides of the primer pair are as shown in SEQ ID NO.1-2.

[0021] The present invention also provides a method for identifying Angelica sinensis by PCR combined with sequencing based on ITS2, including the steps of performing PCR amplification using the primer pair and sequencing the PCR amplification product.

[0022] The present invention discloses the following technical effects:

[0023] The authenticity of Angelica sinensis samples was identified by extracting genomic DNA from 8 groups of Angelica sinensis samples, performing PCR amplification, TA cloning, and DNA sequencing, and comparing the sequencing results with the gene bank. The results showed that 4 samples were Angelica sinensis, with a similarity of 98.99% to the ITS2 gene of Angelica sinensis in the database (GenBank number: KC295071); 3 samples were Angelica dahurica, with a similarity of 95.56% to the ITS2 gene of Angelica dahurica in the database (GenBank number: OK668234); 1 sample was Peucedanum terebinthaceum, with a similarity of 98.99% to the ITS2 gene of Peucedanum terebinthaceum in the database (GenBank number: KC295082). Therefore, the method for identifying the authenticity of Angelica sinensis based on the ITS2 sequence can accurately identify the authenticity of Angelica sinensis samples and provide a reference for the identification of Angelica sinensis. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 Amplification results of ITS2 primer sequences for Angelica sinensis samples L1 and X1; MKIII represents the standard DNA Marker, and L1 and X1 represent Angelica sinensis samples L1 and X1 respectively;

[0026] Figure 2 Amplification results of ITS2 primer sequences for Angelica sinensis samples M1, M2, and M3; MKIII represents the standard DNA Marker, and M1, M2, and M3 represent Angelica sinensis samples M1, M2, and M3 respectively;

[0027] Figure 3 Amplification results of ITS2 primer sequences for Angelica sinensis samples M4 and M5; MKIII represents the standard DNA Marker, and M4 and M5 represent Angelica sinensis samples M4 and M5 respectively;

[0028] Figure 4 Amplification results of ITS2 primer sequences for Angelica sinensis sample M6; MKIII represents the standard DNA Marker, and M6 is Angelica sinensis sample M6;

[0029] Figure 5 Restriction enzyme digestion results of plasmids of Angelica sinensis samples X1 and L1; MKIII represents the standard DNA Marker, X11-16 are six parallel samples after restriction enzyme digestion of plasmid X1, and L11-16 are 6 parallel samples after restriction enzyme digestion of plasmid L1;

[0030] Figure 6The results of plasmid digestion of Angelica sinensis sample M1; DL5000 represents the standard DNA Marker, M12-16 are five parallel samples after plasmid digestion of M1, and M22, M25 are two parallel samples after plasmid digestion of M2;

[0031] Figure 7 The results of plasmid digestion of Angelica sinensis samples M2 and M6; MKIII represents the standard DNA Marker, M21-26 are six parallel samples after plasmid digestion of M2, and M61-66 are six parallel samples after plasmid digestion of M6;

[0032] Figure 8 The results of plasmid digestion of Angelica sinensis sample M3; MKIII represents the standard DNA Marker, 31-35 are five parallel samples after plasmid digestion of M3;

[0033] Figure 9 The results of plasmid digestion of Angelica sinensis sample M4; MKIII represents the standard DNA Marker, M31-36 are six parallel samples after plasmid digestion of M3, and M41-46 are six parallel samples after plasmid digestion of M4;

[0034] Figure 10 The results of plasmid digestion of Angelica sinensis sample M5; MKIII represents the standard DNA Marker, M51-56 are six parallel samples after plasmid digestion of M5;

[0035] Figure 11 The partial ITS2 alignment results of Angelica sinensis samples X12 and X13;

[0036] Figure 12 The partial ITS2 alignment results of Angelica sinensis samples L15 and L16;

[0037] Figure 13 The partial ITS2 alignment results of Angelica sinensis samples M13 and M15;

[0038] Figure 14 The partial ITS2 alignment results of Angelica sinensis samples M22 and M23;

[0039] Figure 15 The partial ITS2 alignment results of Angelica sinensis samples M45 and M46;

[0040] Figure 16 The partial ITS2 alignment results of Angelica sinensis samples M53 and M54;

[0041] Figure 17 The partial ITS2 alignment results of Angelica sinensis samples M61 and M63;

[0042] Figure 18 The partial ITS2 sequence alignment results of eight groups of Angelica sinensis samples;

[0043] Figure 19 It is the sensitivity test result of the Angelica PCR method. Detailed implementation manners

[0044] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0048] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0049] Example 1

[0050] 1. Experimental materials

[0051] The sources of 7 groups of Angelica samples were purchased online (the sample from Shangri-La was named X1, the sample from Leshan City was named L1, and the 5 samples from Min County were named M1 to M5), and the Angelica from Min County provided by the laboratory in the 8th group was named M6.

[0052] The plant genomic DNA extraction kit (Plant Genomic DNA Kit, centrifugal column type), 10× Taq Buffer, Taq DNA Polymerase, and Marker III were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; competent E. coli DH5α and the Shengong column type DNA gel extraction kit were purchased from Shanghai Shengong Biological Engineering Co., Ltd.; the ITS2 universal primer was synthesized by General Biosystems (Anhui) Co., Ltd.; the reagents used for PCR amplification, A addition, and vector construction were all purchased from Baoruiyi Biotechnology Co., Ltd.; chloroform, absolute ethanol, isopropanol, etc. were of analytical grade and were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0053] The reagent preparation for plasmid extraction (SDS lysis method) is as follows:

[0054] Solution Ⅰ: Add 25 mL of 1 M Tris-HCl (pH 8.0), 45 mL of 20% Glucose (1.11 M), and 20 mL of 0.5 M EDTA (pH 8.0) into a 1 L beaker, and mix well. Add deionized water to make up to 1 L. Sterilize at 121 °C for 20 min under high temperature and high pressure, and store at 4 °C.

[0055] Solution Ⅱ: Add 50 mL of 10% SDS and 50 mL of 2 M NaOH into a 500 mL beaker, and mix well. Add deionized water to make up to 500 mL, and store at room temperature.

[0056] Solution Ⅲ: Weigh 147 g of potassium acetate into a 500 mL beaker, then add 57.5 mL of glacial acetic acid, and mix well. Add deionized water to make up to 500 mL. Sterilize at 121 °C for 20 min under high temperature and high pressure, and store at 4 °C.

[0057] 2. Experimental methods

[0058] 2.1 Angelica sinensis DNA extraction

[0059] Pretreatment: Gently wipe the surface of the Angelica sinensis medicinal materials with 75% ethanol to remove dust, place them in a ventilated place or use a hair dryer to blow until the absolute ethanol has completely evaporated. Take some of the dried medicinal materials and grind them into powder in a high-speed grinder.

[0060] Extract Angelica sinensis DNA: Weigh 30 mg of the ground Angelica sinensis powder, use the plant DNA extraction kit to extract DNA (operate according to the instructions), measure the concentration and purity of the extracted DNA with a ultra-micro nucleic acid and protein analyzer, and store the prepared sample at -20 °C for later use.

[0061] 2.2 PCR amplification and A-tailing

[0062] PCR Amplification: Angelica sinensis is a traditional Chinese medicine. Due to a series of processing treatments such as sun drying and poor storage conditions or long storage time, the DNA is degraded to varying degrees, thus affecting the PCR amplification efficiency. Therefore, the ITS2 fragment of Angelica sinensis is selected for amplification in this invention, and the primers designed with ITS2 as the target gene are as follows:

[0063] ITS2-F (SEQ ID NO.1): 5’-ACACCCAGGCAGACGTGCCC-3’;

[0064] ITS2-R (SEQ ID NO.2): 5’-ACCATCGAGTCTTTGAACGC-3’.

[0065] A 25 μL PCR reaction system is adopted: 12.5 μL of PrimeSTAR Max Premix (2×), 8.5 μL of ddH2O, 2 μL of total DNA, and 1 μL each of forward and reverse primers (10 μM). It is placed in a PCR amplifier, and the reaction program is set: denaturation at 98 °C for 5 s, annealing at 53 °C for 10 s, and extension at 72 °C for 5 s for one cycle, with 30 cycles in total.

[0066] Adding A Tail: To facilitate the subsequent TA cloning experiment, an A is added to the DNA end. The sample is taken out from the PCR amplifier, and the following are added to it: 0.5 μL of dATP, 0.5 μL of Taq enzyme, and 2.5 μL of 10× Buffer. The new system with a total volume of 28.5 μL is placed in the PCR amplifier again and reacted at 72 °C for 30 min.

[0067] 2.3 Nucleic Acid Electrophoresis and Gel Cutting

[0068] The amplified product is detected by agarose gel electrophoresis. The voltage is set at 130 V, the current is set at 130 mA, and the time is set at 20 min.

[0069] After nucleic acid electrophoresis, the agarose gel is placed in a gel imaging system. The ultraviolet lamp is turned on, and the complete gel block is cut with a knife for the next step of DNA gel recovery.

[0070] 2.4 Gel Recovery

[0071] A column-type DNA gel recovery kit (Sangon Biotech, Shanghai) is used for DNA gel recovery.

[0072] Measuring the DNA Concentration of Gel Recovery: The DNA gel recovery solution is measured for its DNA concentration using an ultraviolet spectrophotometer, and the remaining solution is stored in a 4 °C refrigerator for future use.

[0073] 2.5 Vector Construction

[0074] Construct the vector ligation system (6 μL): 0.5 μL of pMD19T-vector, 2.5 μL of gel-extracted DNA, and 3 μL of Solution I. Store it overnight at 16 °C in a dry incubator.

[0075] 2.6 Transformation

[0076] Add 50 μL of competent E. coli DH5α to the 6 μL plasmid system on the ultra-clean workbench.

[0077] Incubate the mixture in an ice bath for 20 min first, then heat shock it in a 42 °C metal bath for 45 s, and finally incubate it in an ice bath for 3 min again.

[0078] Add 800 μL of SOC medium to the mixture on the ultra-clean workbench, and incubate it in a shaker at 200 rmp and 37 °C for 1 h. Then centrifuge it at 4000 rmp for 1 min at room temperature.

[0079] On the ultra-clean workbench, when the supernatant is discarded to about 100 μL, mix the remaining supernatant and the precipitate evenly with a pipette, and spread the solution on an LB plate with antibiotic resistance using a sterile spreading rod that has been sterilized by flaming. Culture it in an environment at 37 °C for 10 - 12 h.

[0080] Observe the colony status, and perform the next step of picking colonies when there are colonies that can be picked.

[0081] 2.7 Colony Picking

[0082] On the ultra-clean workbench, pick colonies with a sterilized toothpick and transfer them to a 15 mL centrifuge tube containing 4 mL of culture medium (the ratio of Amp100 - LB is 1:1000, that is, the mass - volume ratio of 100 mg / mL ampicillin and LB medium is 1:1000) (do 6 parallels), and culture them in a shaker at 37 °C and 200 rmp for 12 h.

[0083] 2.8 Plasmid Extraction (SDS Alkaline Lysis Method)

[0084] On the ultra-clean workbench, take 1 mL of the culture and transfer it to a 1.5 mL EP tube, and centrifuge it at 12000 rmp for 30 s at room temperature. Discard the supernatant, invert the EP tube on the toilet paper to remove all the supernatant, add 200 μL of Solution I, suspend the cells vigorously by shaking, then add 400 μL of Solution II and invert the centrifuge tube 5 - 6 times to lyse the bacterial solution. At this time, the DNA denatures and the solution becomes clear, and there is a DNA drawing phenomenon when opening the tube mouth.

[0085] Add 300 μL of Solution III to the EP tube, invert it 5 - 6 times up and down. Add 10 μL of chloroform to the EP tube and mix well. At room temperature, place it in a centrifuge and set the rotation speed to 13000 rmp for centrifugation for 8 minutes.

[0086] Carefully transfer the supernatant to a new 1.5 mL EP tube, add 500 μL of isopropanol, shake it vigorously to mix well. Place the EP tube in a -20 °C freezer for 10 minutes, then take it out, place it in a centrifuge, set the rotation speed to 15000 rmp, and centrifuge at 4 °C for 10 minutes.

[0087] Pour the supernatant into the waste liquid tank, add 1 mL of 75% ethanol to wash the precipitate, place it in a centrifuge, set the rotation speed to 12000 rmp for centrifugation for 2 minutes, discard the alcohol, and suck it dry with a 10 μL pipette tip.

[0088] Dry the precipitate at 65 °C for 2 minutes, add 20 μL of 1×TE (pH 8.0, containing 20 μg / mL of Rnase) to dissolve it, and incubate at 37 °C for 30 minutes.

[0089] Electrophoresis: Take 2 μL of plasmid, add 2 μL of 6×Loding buffer, mix well on a 1% agarose gel, and add it to the gel wells. Set the program: voltage 120 V; current 130 mA; time 20 minutes for electrophoresis. If there are bands, it can be used for constructing the plasmid digestion system. After the electrophoresis detection is completed, store the qualified plasmid at -20 °C.

[0090] 2.9 Plasmid Digestion and PCR

[0091] Construct a general digestion system (20 μL): 16 μL of ddH2O, 2 μL of 10×Buffer, 1 μL of QuikCut EcoR I, 1 μL of plasmid DNA, place it in an incubator at 37 °C for 1 hour. Electrophoresis again, add Marker III to the first well, load the samples in sequence behind, and put it into a gel imaging system to observe the ligation situation. Select samples larger than 3200 bp for detection. If there are no available samples, try plasmid PCR.

[0092] Construct a plasmid PCR system (20 μL): 0.25 μL of Taq, 2 μL of 10×buffer, 0.5 μL each of ITS2 forward and reverse primers, 2 μL of dNTP, 0.2 μL of plasmid, and 14.55 μL of ddH2O; put it into a PCR amplifier, the reaction program is the same as the total DNA PCR amplification, perform electrophoresis, observe the results. If the sample meets 500 bp, the bacterial liquid can be retained for detection.

[0093] 2.10 Preservation and Submission of Bacterial Liquid

[0094] Bacterial liquid preservation: Take 0.6 mL of bacterial liquid and 0.6 mL of 40% glycerol in a laminar flow hood, add them to a 1.5 mL EP tube, label the number, wrap it with sealing film, and place it in a -80 °C refrigerator for later use.

[0095] Submission for inspection: Take 1 mL of bacterial liquid and send it to General Biosystems (Anhui) Co., Ltd. for DNA sequencing.

[0096] 3. Results and Analysis

[0097] 3.1 Angelica Genome DNA Concentration Results

[0098] Extract the genomic DNA of eight groups of Angelica samples using a plant DNA extraction kit, and then measure its concentration and purity. The results are shown in Table 1.

[0099] Table 1 Measurement Results of DNA Concentration and Purity of Eight Groups of Angelica Samples

[0100]

[0101]

[0102] 3.2 ITS2 Sequence Amplification Results

[0103] After PCR amplification and adding A tail to eight groups of Angelica samples, nucleic acid electrophoresis detection was carried out. The amplified bands were all around 500 bp ( Figures 1 - 4 ), and it can be preliminarily determined that the amplified nucleic acid fragment is the Angelica ITS2 sequence.

[0104] 3.3 Plasmid Digestion and Plasmid PCR Results

[0105] The electrophoresis results after plasmid digestion and plasmid PCR are shown in Figures 5 - 10 (X11 - 16 represents 6 parallels of sample X1, and the numbering rules of other samples are the same). Select the bands that can produce around 3200 bp and have high brightness after plasmid digestion in eight groups of Angelica samples, and the bands that can produce around 500 bp in the original plasmid PCR, indicating that the ITS sequence cloning is successful. The successfully cloned samples are Figure 5 X12, X13, L15, L16 in Figure 6 M13 and M15 in Figure 7 M22, M23, M61, M63 in Figure 8 M35 in Figure 9 M45, M46 in Figure 10 M53, M54 in

[0106] 3.4 Comparison between the Results of Submission for Sequencing

[0107] Using the DNAMAN software, the partial ITS2 fragments of the above 15 Angelica sinensis samples were compared among the same samples. The results showed that there were no differences among the same samples ( Figures 11 - 17 ). Then, among all 15 groups of samples, eight samples such as X1 and L1 were randomly selected and compared. As Figure 18 shown, from the base alignment in the first row, it can be seen that the eight groups of samples were roughly divided into three categories. The first category was X1, M2, M5, and M6; the second category was M1, M3, and M4; the third category was L1.

[0108] 3.5 Comparison of sequencing results with the database

[0109] From the above results, it can be seen that the eight Angelica sinensis samples can be divided into three categories. For each category of samples, a randomly selected available sample was compared with the gene database. The results showed that the similarity between samples X1, M2, M5, and M6 and the ITS2 gene of Angelica sinensis in the database (GenBank number: KC295071) was 98.99%, that is, these 4 samples were Angelica sinensis; the similarity between samples M1, M3, and M4 and the ITS2 gene of Angelica dahurica in the database (GenBank number: OK668234) was 95.56%, that is, these 3 samples were Angelica dahurica; the similarity between sample L1 and the ITS2 gene of Peucedanum terebinthaceum in the database (GenBank number: KC295082) was 98.99%, that is, this 1 sample was Peucedanum terebinthaceum.

[0110] 3.6 Method sensitivity test

[0111] The identified Angelica sinensis DNA was serially diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, and ddH2O was used as a negative control to test the sensitivity of the PCR method. The test results are shown in Figure 12 . As can be seen from Figure 19 , amplification reactions occurred for Angelica sinensis DNA at 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, while no amplification phenomenon occurred for Angelica sinensis DNA at 0.1 pg / μL and the negative control. Thus, the detection sensitivity of the established Angelica sinensis PCR method for Angelica sinensis DNA was 1 pg / μL.

[0112] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of ITS2 in any of the following: (1) Application in identifying the authenticity of Angelica sinensis; (2) Application in the preparation of a kit for identifying the authenticity of Angelica sinensis; in, The ITS2 is amplified using a primer pair with a nucleotide sequence as shown in SEQ ID NO.1-2.

2. The use according to claim 1, characterized in that The ITS2 is used to identify Angelica sinensis and its counterfeits such as Angelica dahurica and Saposhnikovia divaricata.

3. The use according to claim 2, characterized in that The identification method comprises the following steps: Using the DNA of the sample to be tested as a template, performing PCR amplification using the primer pair to obtain an amplified product; The amplified product is connected to a vector to construct a recombinant plasmid, and then transformed into competent Escherichia coli. After cultivation, positive bacteria are picked; after the positive bacteria are lysed, the plasmid is extracted, enzyme digested, and sequenced; The sequencing results were compared with ITS2 in the gene database to identify the authenticity of Angelica sinensis.

4. The use according to claim 3, characterized in that The PCR amplification reaction system includes: PrimeSTARMax Premix (2×) 12.5 μL, ddH2O 8.5 μL, total DNA 2 μL, and forward and reverse primers 1 μL each.

5. The use according to claim 3, characterized in that The reaction procedure of the PCR amplification is: denaturation at 98° C. for 5 s, annealing at 53° C. for 10 s, and extension at 72° C. for 5 s as one cycle, and the cycle is repeated 30 times.

6. The use according to claim 3, characterized in that PloyA is added to the 3' end of the nucleotide sequence of the amplified product; and / or the vector includes a pMD19-T vector.

7. The use according to claim 3, characterized in that After the plasmid is digested by enzymes, samples larger than 3200 bp are selected for sequencing, or after the plasmid is amplified by PCR, electrophoresis is performed and samples with 500 bp are selected for sequencing.

8. The use according to claim 3, characterized in that The ITS2 in the gene database includes the Angelica sinensis ITS2 gene with the gene accession number KC295071 on NCBI, the Angelica dahurica ITS2 gene with the gene accession number OK668234, and the Saposhnikovia divaricata ITS2 gene with the gene accession number KC295082.

9. A primer pair for identifying Angelica sinensis by PCR combined with sequencing based on ITS2, characterized in that: The nucleotides of the primer pair are shown in SEQ ID NO.1-2.

10. A method for identifying Angelica sinensis based on ITS2 PCR combined with sequencing, characterized in that: The method comprises the steps of performing PCR amplification using the primer pair described in claim 9, and sequencing the PCR amplification product.

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