SNP sites and identification methods for identifying biological components in Danggui Buxue Pills

Abstract

The present invention discloses SNP sites and identification methods for identifying biological components in Danggui Buxue Pills. The present invention discloses SNP sites for identifying biological components Danggui Buxue Pills, namely Danggui and / or Astragalus, and specific primers and applications thereof for identifying the above SNP sites. The identification method provided by the present invention is to identify biological components in Danggui Buxue Pills based on the above SNP sites or using the above specific primers. The present invention designs specific identification primers based on ITS gene fragments rich in variant sites of Danggui and its mixed and counterfeit products, and Astragalus and its mixed and counterfeit products, respectively, and uses specific multiplex PCR technology to simultaneously qualitatively identify two biological components in Danggui Buxue Pills: Danggui and Astragalus, in order to provide new ideas for the quality control of Danggui Buxue Pills and other Chinese patent medicines containing Danggui and / or Astragalus.

Description

Technical Field

[0001] The present invention relates to the technical field of identification, and in particular to a SNP site and an identification method for identifying biological components in Danggui Buxue Pills. Background Art

[0002] The main ingredients of Danggui Buxue Pills are angelica and astragalus, and it is a classic Chinese patent medicine. The 2020 edition of the "Chinese Pharmacopoeia" stipulates that astragalus is derived from the dried roots of Astragalus mongolica and Astragalus membranaceus of the Leguminosae family, and angelica is derived from the dried roots of Angelica sinensis of the Umbelliferae family. At present, the quality control methods for Danggui Buxue Pills are microscopic identification, thin-layer chromatography, and high-performance liquid chromatography. Because there are many counterfeit products of angelica and astragalus, and most of them are closely related species of the same genus, these methods have the problem of weak specificity in the identification of angelica and astragalus. The multiplex PCR technology based on specific SNP sites can simultaneously perform qualitative identification of multiple species in the same amplification reaction system. It has the advantages of rapidity, accuracy, strong specificity, and can save a lot of manpower, material resources and financial resources.

[0003] The quality of genomic DNA in Chinese patent medicines is the key to identifying biological components in Chinese patent medicines by PCR technology [non-patent literature 1-2]. Since the raw materials in Chinese patent medicines are processed through various processing techniques, such as decoction, calcination, frying, fermentation, etc. [non-patent literature 3-5], DNA is severely degraded, affecting the quality and quantity of DNA [non-patent literature 1,6-8]. At the same time, various pharmaceutical excipients added during the preparation of Chinese patent medicines, such as honey, starch and sugar coating, will also cause certain difficulties in DNA extraction [non-patent literature 9]. Therefore, the extraction method of genomic DNA needs to be determined according to the dosage form and specific preparation process of Chinese patent medicines. Commonly used methods for extracting DNA from Chinese medicines include CTAB method, SDS method, magnetic bead method, alkaline lysis method and other extraction methods. Chen, Jing Xiaotong, Cui Zhanhu and other researchers found that the CTAB method has the best overall effect in extracting DNA from plant-derived materials in solid preparations [non-patent literature 10,2,11]. In addition, DNA purity is another important factor affecting the effect of PCR amplification. Impurities such as polysaccharides, polyphenols and proteins mixed in genomic DNA can cause DNA contamination. CTAB, phenols, ethanol, isopropanol, etc. used in the extraction process can also interfere with the activity of Taq enzymes. These factors can lead to PCR amplification failure [non-patent literature 9, 12-14]. Among the many factors that affect the effective amplification of PCR, annealing temperature is the most important condition for PCR amplification. Increasing the annealing temperature can reduce the nonspecific binding between primers and templates, and lowering the annealing temperature can increase the sensitivity of the reaction. For multiplex PCR experiments, the requirements for annealing temperature are usually higher than those for single-plex PCR, because all target fragments need to be effectively amplified at the same time, and the designed primer pairs cannot bind to each other to produce nonspecific amplification [non-patent literature 15-17].

[0004] [Non-patent literature 1] Chen Rong, Wu Chengli, Deng Yun, et al. Application and research progress of DNA molecular identification technology in the authenticity identification of traditional Chinese medicines [J]. Traditional Chinese Medicine and Clinic, 2016, 7(2): 83-86.

[0005] [Non-patent literature 2] Jing Xiaotong, Huang Yong, Huang Yuting, et al. Research progress in DNA extraction methods for traditional Chinese medicines[J]. Chinese Journal of Pharmaceutical Science, 2021, 11(19): 47-50, 58.

[0006] [Non-patent literature 3] Chen Hongwei. The origin, development, transition and integration of Chinese medicine characteristic technologies[J]. Modern Distance Education of Traditional Chinese Medicine, 2018, 16(14):155-158.

[0007] [Non-patent literature 4] Yang Chunyu, Cao Hui, Wang Xiaotao, et al. Research on the current status of excipient standards for processing in my country and work suggestions [J]. Chinese Journal of Traditional Chinese Medicine, 2017: 1-6.

[0008] [Non-patent literature 5] Xue Xiechao, Shi Hongtao, Qiao Hongxing, et al. Research progress on fermented Chinese medicine processing [J]. Modern Animal Husbandry, 2017, 1(2): 26-29.

[0009] [[Non-patent document 6] Karni Moshe, Zidon Dolev, Polak Pazit, et al. Thermal degradation of DNA. [J]. DNA and cell biology, 2013, 32(6): 298-301.

[0010] [Non-patent document 7] Zhang Lianwen, Wu Qingyu. Single gene retrieval from thermally degraded DNA. [J]. Journal of biosciences, 2005, 30(5): 599-604.

[0011] [Non-patent document 8] Moreano Francisco, Busch Ulrich, Engel Karl-Heinz. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation. [J]. Journal of agricultural and food chemistry, 2005, 53(26): 9971-9979.

[0012] [Non-patent document 9] Sajali Nurhayatie, Wong Sie-Chuong, Hanapi Ummi-Kalthum, et al. The Challenges of DNA Extraction in Different Assorted Food Matrices: A Review. [J]. J Food Sci, 2018(10): 2409-2414.

[0013] [Non-patent literature 10] Cui Zhanhu, Jiang Chao, Yuan Yuan, et al. Molecular identification of raw material honeysuckle in 6 kinds of patent medicines [J]. Journal of Baotou Medical College, 2014: 1-3.

[0014] [Non-patent document 11] Chen Rong, Dong Juan, Cui Xin, et al. DNA based identification of medicinal materials in Chinese patent medicines. [Z], 2012: 958.

[0015] [Non-patent document 12] Hedman Johannes, Peter. Overcoming inhibition inreal-time diagnostic PCR.[J].Methods Mol Biol, 2013:17-48.

[0016] [Non-patent document 13] Hedman Johannes, Knutsson Rickard, Ansell Ricky, et al. Pre-PCR Processing in Bioterrorism Preparedness: Improved Diagnostic Capabilities for Laboratory Response Networks[J]. Biosecurity and bioterrorism: biodefensestrategy, practice, and science, 2013, 11(S1): S87-S101.

[0017] [Non-patent document 14] Monteiro L, Bonnemaison D, Vekris A, et al. Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. [J]. J Clin Microbiol, 1997 (4): 995-8.

[0018] [Non-patent literature 15] Li Yingnuo, Wang Yanshuang, Yuan Guangxin, et al. Rapid detection of common adulterated animal-derived ingredients in beef by multiple site-specific PCR[J]. Food Industry Science and Technology, 2019, 40(24): 82-87.

[0019] [Non-patent literature 16] Dong Lei, Huang Xiaobo, Liu Jingxuan. Application and prospects of multiplex PCR technology in food detection[J]. Anhui Agricultural Sciences, 2020, 48(11): 15-18.

[0020] [Non-patent document 17] Lu yuanyuan SONG-YANBO-XUE-ZHIQUAN. Multiplex polymerase chain reaction detection of Curvularia lunata Curvularia lunata, Bipolarismaydis Bipolaris maydis, and Aureobasidium zeae Aureobasidium zeae in infected maize leaf tissues [J]. Journal of Basic Microbiology: An International Journal Morphology,Physiology,Genetics,and Ecology of Microorganisms,2019,59(9):862-866. Summary of the invention

[0021] In view of the above technical problems, the present invention provides a SNP site and an identification method for identifying biological components in Danggui Buxue Pills. The present invention designs specific identification primers based on ITS gene fragments rich in variable sites of Danggui and its mixed and counterfeit products, and Astragalus and its mixed and counterfeit products, and uses specific multiplex PCR technology to simultaneously qualitatively identify the two biological components (Danggui and Astragalus) in Danggui Buxue Pills, in order to provide a new method for quality control of Danggui Buxue Pills and other Chinese patent medicines containing Danggui and / or Astragalus.

[0022] To achieve the above object, the technical solution adopted by the present invention is:

[0023] In one aspect, the present invention provides a SNP site for identifying biological components in Danggui Buxue Wan, wherein the biological components are Danggui and / or Astragalus;

[0024] The SNP site of Angelica sinensis is selected from at least one of the 63rd site, 65th site, 67th site, 72nd site, 74th site, 78th site, 80th site, 398th site, 403th site and 409th site from the 5' end as shown in SEQ ID NO.1;

[0025] The SNP site of Astragalus is selected from at least one of the 41st, 43rd, 46th, 48th, 49th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 489th, 490th, 491st, 495th, 497th, 501st, 502nd, and 503rd sites from the 5' end shown in SEQ ID NO.2.

[0026] In yet another aspect, the present invention provides specific primers for identifying the above-mentioned SNP sites.

[0027] As a preferred embodiment, the specific primers include any one of the following specific primer pairs:

[0028] Angelica specific primer pair:

[0029] Upstream primer as shown in SEQ ID NO.3: 5'GGCTTTGGTCCCTTGTATG 3';

[0030] Downstream primer as shown in SEQ ID NO.4: 5'CACGAGGAGTGAGTGGTTG 3';

[0031] Astragalus-specific primer pairs:

[0032] Upstream primer as shown in SEQ ID NO.5: 5'GCACCACGACCTCCCTTTG 3';

[0033] The downstream primer is as shown in SEQ ID NO.6: 5'GCCATCATTCGCCCTAAA 3'.

[0034] In another aspect, the present invention provides an application of the above-mentioned SNP site or the above-mentioned specific primer, wherein the application is any one of the following (i) to (ii):

[0035] (i) identifying whether the sample to be tested is or whether the sample to be tested contains Angelica sinensis and / or Astragalus membranaceus;

[0036] (ii) preparing a kit for identifying whether a sample to be tested is or contains Angelica sinensis and / or Astragalus membranaceus.

[0037] In another aspect, the present invention provides a method for identifying biological components in Danggui Buxue Pills, wherein the method is to identify the biological components in Danggui Buxue Pills based on the above-mentioned SNP sites or using the above-mentioned specific primers.

[0038] Specifically, the method comprises the following steps:

[0039] (1) extracting genomic DNA from the sample to be tested;

[0040] (2) using the extracted genomic DNA of the sample to be tested as a template, designing specific primers based on the above SNP sites or using the above specific primers to perform PCR amplification reaction;

[0041] (3) Detect PCR amplification products.

[0042] As a preferred embodiment, the method for extracting genomic DNA of the sample to be tested is a modified CTAB method;

[0043] The improved CTAB method specifically comprises the following steps:

[0044] S1. Take 2-3 g of the sample to be tested and grind it;

[0045] S2. Add the fine powder obtained by grinding to 10-15 mL of 4×CTAB extraction buffer preheated at 65°C, wherein 2 / 1000 volume of 1M dithiothreitol (DTT) solution is added to the 4×CTAB extraction buffer before preheating, and incubate in a water bath at 65°C for 1.5 h;

[0046] S3. After cooling, add 1 to 1.5 times the volume of chloroform-isoamyl alcohol mixed solution, with a volume ratio of 24:1, and centrifuge;

[0047] S4. Take the supernatant, add 0.7 to 1 times volume of -20℃ precooled isopropanol, place at -20℃ to settle for 1 hour, centrifuge and discard the waste liquid to obtain crude DNA;

[0048] S5. The extracted crude DNA was washed with 70% ethanol solution and anhydrous ethanol respectively, and then purified using a universal DNA purification and recovery kit;

[0049] Preferably, step S3 is repeated twice.

[0050] As a preferred embodiment, in step (2), the annealing temperature of the PCR amplification reaction is 50°C to 67.6°C, preferably 65°C;

[0051] Preferably, in the PCR amplification reaction: the detection limit of genomic DNA of Angelica sinensis is ≥ 0.04 ng / μL; the detection limit of genomic DNA of Astragalus membranaceus is ≥ 0.004 ng / μL.

[0052] As a preferred embodiment, the detection of PCR amplification products is performed by electrophoresis detection.

[0053] The above technical solution has the following advantages or beneficial effects:

[0054] The present invention screens out the SNP sites of angelica and astragalus by comparing the distinguishing bases of ITS sequences of angelica and its mixed products and astragalus and its mixed products, designs an angelica-specific primer pair and an astragalus-specific primer pair that can at least identify one SNP site, and uses specific multiplex PCR technology to simultaneously qualitatively identify the angelica and astragalus biological components in angelica blood-enhancing pills.

[0055] The SNP site of angelica provided by the present invention is located at the distinguishing bases of the ITS sequence of angelica and its counterfeits, namely, Angelica sinensis, Peucedanum purpurogenum and Angelica dahurica. The designed specific primer pair can identify at least one of the above SNP sites, wherein the upstream primer covers 7 specific SNP sites of the angelica ITS, and the downstream primer covers 3 specific SNP sites of the angelica ITS. The primer pair is complementary to the bases of the SNP sites on the angelica ITS, and the specific amplification of the angelica ITS is achieved through allele-specific PCR to obtain an amplified fragment with a length of 349 bp. No amplified band is generated for the counterfeits of angelica, so that it is possible to detect whether the angelica base source is contained in the angelica blood-enriching pills, realize the authenticity identification of the angelica in the angelica blood-enriching pills, and the identification result has high accuracy and stability.

[0056] The SNP site of the astragalus provided by the present invention is located at the distinguishing bases between the ITS sequence of the astragalus and its counterfeits, such as flat stem astragalus, caragana, hibiscus and alfalfa. The designed specific primer pair can identify at least one of the above SNP sites, wherein the upstream primer covers 14 specific SNP sites of the angelica ITS, and the downstream primer covers 8 specific SNP sites of the angelica ITS. The primer pair is complementary to the bases of the SNP sites on the astragalus ITS, and the specific amplification of the astragalus ITS is achieved through allele-specific PCR to obtain an amplified fragment with a length of 381bp. No amplified band is generated for the counterfeit astragalus, so that it is possible to detect whether the angelica blood-enhancing pills contain the astragalus base source, realize the authenticity identification of the astragalus in the angelica blood-enhancing pills, and the identification result has high accuracy and stability.

[0057] The method for identifying biological components in Danggui Buxue Pills provided by the present invention uses a modified CTAB method to extract DNA in Danggui Buxue Pills. The method for extracting DNA in Danggui Buxue Pills in the prior art has the problem of difficulty in PCR amplification, and cannot be effectively amplified and detected. After a large number of experimental tests, the present invention finds that the DNA extraction method provided by the present invention increases the amount of the sample to be tested to 2-3g, and uses a universal DNA purification and recovery kit to purify the extracted crude DNA, so that stable quality DNA that can be used for amplification can be extracted, making it possible to apply DNA molecular identification technology to identify Danggui Buxue Pills; the identification method provided by the present invention specifically amplifies the ITS genes of Danggui and Astragalus in Danggui Buxue Pills, and obtains the DNA only when the raw material pieces of Danggui Buxue Pills are genuine Danggui and Astragalus. The PCR products are a 349bp gene fragment (corresponding to Angelica sinensis) and a 381bp gene fragment (corresponding to Astragalus membranaceus); if the raw material pieces are counterfeit Angelica sinensis, the 349bp gene fragment cannot be amplified; if the raw material pieces are counterfeit Astragalus membranaceus, the 381bp gene fragment cannot be amplified; if the raw material pieces are both counterfeit Angelica sinensis and counterfeit Astragalus membranaceus, the amplified gene fragment cannot be obtained; therefore, the identification method provided by the present invention has the advantages of high accuracy and good stability, provides a basis for tracing the raw medicinal materials of Danggui Buxue Pills, realizes effective control of the authenticity of the raw medicinal materials, is conducive to establishing a unified quality management standard for Danggui Buxue Pills, standardizes the supervision of the production, circulation and medication process of Danggui Buxue Pills, provides guarantee for the drug safety of Danggui Buxue Pills, and provides a basis for establishing and improving the quality standard system of the Chinese patent medicine industry chain;

[0058] The identification method provided by the present invention optimizes the experimental screening of the reaction program of PCR amplification and the reaction system of PCR amplification. In the PCR amplification of alleles for detecting SNP molecular markers, the PCR reaction system, annealing temperature, etc. directly affect the accuracy and specificity of specific multiplex PCR identification. In the present invention, the annealing temperature, the number of cycles, etc. are investigated to obtain the reaction program of PCR amplification and the reaction system of PCR amplification that are mutually compatible with the specific primer pairs of Angelica sinensis and Astragalus membranaceus, which provides a basis for the accuracy and stability of Danggui Buxue Wan identification.

[0059] The identification method provided by the present invention can be used to simultaneously identify all biological components in Danggui Buxue Pills, and has the characteristics of high efficiency, time-saving, labor-saving, and economical. This study provides a new methodological reference for the qualitative identification of Chinese patent medicines with multiple biological components. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the DNA gel electrophoresis diagram of some plant samples in Example 1.

[0061] Figure 2It is the DNA gel electrophoresis diagram of some homemade and commercially available Danggui Buxue Pills in Example 1.

[0062] Figure 3 This is the gel electrophoresis diagram of PCR amplification using specific primers for Angelica sinensis and Astragalus membranaceus in Example 2.

[0063] Figure 4 It is the gel electrophoresis diagram of the PCR amplification products at different annealing temperatures using the homemade Danggui Buxue Pills DNA as a template in Example 3.

[0064] Figure 5 This is a graph showing the detection limit test results of the PCR amplification reaction of the homemade Danggui Buxue Pills in Example 3.

[0065] Figure 6 This is the gel electrophoresis diagram of the PCR amplification product of the commercially available Danggui Buxue Pills in Example 4. DETAILED DESCRIPTION

[0066] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0067] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0068] Embodiment 1:

[0069] 1. Experimental Materials and Reagents

[0070] 1.1 Experimental Materials

[0071] The research team went to Gansu, Hebei, Guizhou, Shanxi, Shanghai, Beijing and other places to collect angelica and its common counterfeits, including Angelica sinensis, Peucedanum purpurogenum, and Angelica dahurica, and Astragalus membranaceus (Astragalus mongoliaensis) and its counterfeits, Medicago truncatula, Althaea officinalis, and Caragana. All samples were identified by Associate Professor Tian Enwei and Professor Chao Zhi from the Medicinal Plant and Chinese Medicine Identification Laboratory of the School of Chinese Medicine of Southern Medical University, and the samples were preserved in the specimen museum of the School of Chinese Medicine of Southern Medical University (Table 1). The dried roots of authentic angelica and Astragalus membranaceus were taken according to the plasticizing method specified in the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China·Chinese Medicine Formula Preparations" [Pharmacopoeia Committee of the People's Republic of China. Pharmaceutical Standards of the Ministry of Health of the People's Republic of China·Chinese Medicine Formula Preparations. [S]. Beijing: People's Medical Publishing House, 1995.] to make the authentic Danggui Buxue Pills (ZP). Eight batches (BX1-BX8) of Danggui Buxue Wan samples were purchased from manufacturer A (Lin Zixin Pharmaceutical Co., Ltd.), and six batches (BX9-BX14) of Danggui Buxue Wan samples were purchased from manufacturer B (Shangqiu Jinma Pharmaceutical Co., Ltd.) (Table 2).

[0072] Table 1 Information on Chinese medicine materials

[0073]

[0074] Table 2 Danggui Buxue Pills sample information

[0075]

[0076]

[0077] 1.2 Instruments

[0078] PCR instrument (ExCell Bio G3, Shanghai Yikesai Biological Products Co., Ltd.); high-speed refrigerated centrifuge (HC-3018R, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); electrophoresis instrument (DYY-6C, Beijing Liuyi Instrument Factory); gel imaging system (Js-380, Shanghai Peiqing Technology Co., Ltd.); analytical balance (ALC-210.4, ACCULAB), ultra-micro-ultraviolet spectrophotometer (DS-11, DeNovix, USA); digital display constant temperature water tank (HH-W240, Jintan District Xicheng Xinrui Instrument Factory); ultrapure water instrument (Biosafer-20TA, Saifei (China) Co., Ltd.); micropipette (Eppendorf, Germany Eppendorf AG).

[0079] 1.3 Reagents and related preparation methods

[0080] Reagent: TaKaRa Taq TMPCR enzyme (TaKaRa, 9151AM); D2000 DNA Marker (Tiangen; MD114)); 50bp Ladder DNA Marker (Biomed, MD111); 10×PCR Buffer (Mg2+free) (TaKaRa, 9151AM); MgCl 2 (TaKaRa, 9151AM); dNTP Mixture (TaKaRa, 4030); 2× Taq PCR Mix (Tiangen; KT201); 6× DNA loading buffer (TaKaRa, AJ71987A); SuperRed / GelRed (Biosharp, 68110160); 2× Taq PCR Mix (TaKaRa, KT201); agarose (VetectM ​​REAGENT grade Sigma-Aldrich, 111860); anhydrous ethanol (Guangdong Guanghua Technology Co., Ltd.); methanol (Guangdong Guanghua Technology Co., Ltd.); chloroform (Guangzhou Chemical Reagent Factory); isopentanol (Shanghai MacLean Biochemical Technology Co., Ltd.); dithiothreitol (DTT, Beijing Mengyimei Biotechnology Co., Ltd.), ethylenediaminetetraacetic acid disodium salt (EDTA-Na 2 , Beijing Zhongkangbo Biotechnology Co., Ltd.), Tris base (Lambolide (Fuzhou) Biotechnology Co., Ltd.), NaOH (Guangzhou Chemical Reagent Factory), acetic acid (Guangzhou Chemical Reagent Factory), hydrochloric acid (Guangzhou Chemical Reagent Factory), sodium chloride (Guangzhou Chemical Reagent Factory), Tris-HCl (Shanghai McLean Biochemical Technology Co., Ltd.), hexadecyltrimethylammonium bromide (CTAB, Guangzhou Solaibao Biotechnology Co., Ltd.), sodium acetate trihydrate (NaAc·3H 2 O, Guangzhou Chemical Reagent Factory), etc., universal DNA purification and recovery kit (Tian Gen, DP214-03, centrifugal column type).

[0081] The preparation process of DNA extraction reagent is shown in Table 3.

[0082] Table 3 Preparation method of DNA extraction reagent

[0083]

[0084] Example 1: DNA extraction and quality testing

[0085] (1) DNA extraction from plant samples

[0086] The modified CTAB method [Guillemaut Pierre, Maréchal-drouard Laurence. Isolation of plant DNA: A fast, inexpensive, and reliable method [J]. Plant Molecular Biology Reporter (1): 60-65.] was used to extract the original plant DNA of Angelica sinensis, Astragalus membranaceus and their mixed products. The specific steps are as follows:

[0087] ① Weigh about 20 mg of the collected roots or leaves of Angelica sinensis, Astragalus membranaceus and their mixtures, clean the surface with tap water, disinfect with 75% alcohol and dry in the air;

[0088] ② After cleaning, cut the sample into pieces with scissors and grind it into fine powder in a mortar. Add 2 mL of preheated 4×CTAB (add 1 M DTT at a concentration of 0.2% of the mass of CTAB before preheating and mix well) for grinding. Then transfer it to a 7 mL centrifuge tube and place it in a 65°C water bath for 1.5 h, shaking it intermittently during the period.

[0089] ③ After the water bath, cool the sample to room temperature or place it on ice to cool to room temperature, add an equal volume of chloroform isoamyl alcohol, turn it upside down for about 5 minutes, place it in a centrifuge, and centrifuge it at 12000rpm for 8 minutes; then use a pipette to transfer the supernatant to a 2mL centrifuge tube;

[0090] ④ Add an equal volume of chloroform and isoamyl alcohol to the supernatant, mix and shake for 5-8 minutes, place in a centrifuge, centrifuge at 12000rpm for 8 minutes, and use a pipette to transfer the supernatant to a new 2mL centrifuge tube;

[0091] ⑤ Add 0.7 times the volume of -20℃ precooled isopropanol to the centrifuge tube, and place it in a -20℃ refrigerator to settle for 1 hour; then, place the centrifuge tube in a centrifuge, centrifuge at 12000rpm for 8 minutes, discard the waste liquid, and obtain genomic DNA;

[0092] ⑥ Add 1 mL of 70% alcohol to the centrifuge tube, invert it to clean the DNA, then centrifuge it at 12,000 rpm for 3 minutes, discard the waste liquid, and repeat the operation once;

[0093] ⑦ Add 1 mL of anhydrous ethanol to the centrifuge tube, wash the DNA by inverting it, and then centrifuge it at 12,000 rpm for 3 minutes. Discard the waste liquid and repeat the operation once;

[0094] ⑧The obtained DNA was placed at room temperature to dry, and then 100 μL ddH 2O in a centrifuge tube to dissolve the DNA, and the obtained DNA solution was stored in a -20℃ refrigerator for later use.

[0095] (2) DNA extraction from Danggui Buxue Pills

[0096] The DNA in Danggui Buxue Pills was extracted by using the modified CTAB method combined with column purification [Lin Yihua, Yang Junjie, Zhang Tianxiang, Cao Minghua, Lin Zongkeng. Comparison of two methods for DNA extraction from Moringa oleifera [J]. Fujian Tropical Crops Science and Technology, 2017, 42(01): 33-35+38.] The extraction steps are as follows:

[0097] (1) Grind Danggui Buxue Pills into fine powder and weigh 2 g;

[0098] (2) Add the fine powder obtained in step (1) to 10 mL of 4×CTAB extraction buffer preheated at 65° C., add 2 / 1000 of the volume of CTAB to 1 M DTT before preheating and mix well, place in a 50 mL centrifuge tube, and place in a 65° C. water bath for 1.5 h, shaking intermittently during the process;

[0099] (3) After the water bath, the sample was cooled to room temperature or on ice, and an equal volume of chloroform-isoamyl alcohol mixed solution (volume ratio of 24:1) was added. The sample was inverted for about 5 minutes and centrifuged at 12000 rpm for 8 minutes. The supernatant was then transferred to a 50 mL centrifuge tube using a pipette.

[0100] (4) Add an equal volume of chloroform-isoamyl alcohol mixed solution (volume ratio of 24:1) to the supernatant, shake well for 5 min, centrifuge at 12000 rpm for 8 min, and transfer the supernatant to a new 50 mL centrifuge tube with a pipette;

[0101] (5) Add 0.7 times the volume of -20°C precooled isopropanol to the centrifuge tube and place it in a -20°C refrigerator to settle for 1 hour; then, place the centrifuge tube in a centrifuge and centrifuge at 12000 rpm for 8 minutes, discard the waste liquid, and obtain genomic DNA;

[0102] (6) Add 1 mL of 70% alcohol to the centrifuge tube, invert the tube to clean the DNA, and then centrifuge at 12,000 rpm for 3 min. Discard the waste liquid and repeat the operation once.

[0103] (7) Add 1 mL of anhydrous ethanol to the centrifuge tube, invert the tube to clean the DNA, and then centrifuge at 12,000 rpm for 3 min. Discard the waste liquid and repeat the operation once;

[0104] (8) After purifying DNA using a universal DNA purification and recovery kit (Tian Gen, DP 214), add 200uL ddH2 Dissolve in 0.5% O and store at -20°C.

[0105] (3) UV spectrophotometer to detect DNA concentration and purity

[0106] The A260 and A280 values ​​of the extracted DNA solution were measured by UV spectrophotometer. When the A260 / A280 ratio was about 1.8, it indicated that the DNA quality was good. If A260 / A280>1.8, RNA contamination may exist. If A260 / A280<1.8, it may contain impurities such as proteins and polysaccharides.

[0107] (4) Detection of genomic DNA integrity by agarose gel electrophoresis

[0108] The extracted genomic DNA was subjected to agarose gel electrophoresis to determine the integrity of the genomic DNA. The specific steps are as follows: take 5 μL of the reaction solution after PCR amplification in step 2, stain it with 6× loading buffer, use 2% agarose gel stained with SuperRed / GelRed for electrophoresis, turn on the power and perform electrophoresis at a constant voltage of 120V for 1.5 hours. After the electrophoresis is completed, place the gel on a gel imager or a UV transilluminator for imaging.

[0109] Result analysis:

[0110] In this example, the concentration of genomic DNA of Angelica sinensis, Astragalus membranaceus and their counterfeits extracted by the modified CTAB method was 20-300 ng / μL, and A260 / 280 was between 1.6-2.0, indicating that the quality of the extracted samples was good. The total DNA of the genomic DNA of Angelica sinensis, Astragalus membranaceus and their counterfeits was analyzed by 1.2% agarose gel electrophoresis ( Figure 1 ) The total genomic DNA bands can be observed, the main bands of the samples are clear, and the brightness is distinct, but some samples have obvious degradation and tailing phenomena.

[0111] In this example, the concentration of genomic DNA in the homemade Danggui Buxue Pills ZP and the commercially available Danggui Buxue Pills samples was 10 ng / μL to 100 ng / μL, and the A260 / 280 was between 1.5 and 1.7, indicating that some samples were contaminated with protein. The total DNA was detected in 1.2% agarose gel electrophoresis ( Figure 2 ) There is no obvious main band, and the overall bands are evenly dispersed and light in color, indicating that the genomic DNA has been fragmented.

[0112] In this embodiment, after DNA is extracted using the improved CTAB method, the extracted genomic DNA is purified using a DNA purification recovery kit. In the prior art, when extracting DNA from a Chinese medicine preparation, a Chinese medicine preparation sample generally requires more than 100 mg. In order to ensure that a sufficient amount of genomic DNA is extracted, this embodiment, through multiple experiments, finally determines that the amount of Danggui Buxue Wan sample is 2 g. This sample amount can obtain a higher purity of genomic DNA to meet the needs of subsequent PCR amplification. The purified DNA sample in this embodiment has relatively few impurities and higher purity, which is conducive to subsequent species-specific primer PCR amplification.

[0113] Example 2: Design and screening of specific primers for angelica and astragalus components in Danggui Buxue Pills

[0114] (1) Specific site screening

[0115] In order to select appropriate DNA barcodes to design species-specific primers, this example downloaded the sequences of four universal barcodes (ITS, matK, rbcL and trnH psbA) of Angelica sinensis and its counterfeits, Angelica dahurica, Peucedanum purpurogenum, Angelica dahurica, Astragalus membranaceus and its counterfeits, Medicago truncatula, Astragalus membranaceus, Caragana chinensis, and Hibiscus truncatula through the GenBank database of the NCBI (https: / / www.ncbi.nlm.nih.gov) website, and analyzed the sequence variation between Angelica sinensis, Astragalus membranaceus and the counterfeits respectively. Among them, the ITS sequence has more variable and information-rich sites than other barcodes. Therefore, the ITS sequence was selected to design species-specific primers, and the ITS gene sequence is shown in Table 4. The downloaded sequence files were imported into the mega7.0.14 software [Kumar Sudhir, Stecher Glen, Tamura Koichiro. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. [J]. Mol Biol Evol, 2016, 33 (7): 1870-1874.] and the sequence alignment was performed by using the Alignment by clustal W function. The non-overlapping sequences before and after were deleted, and the sequence information of Angelica sinensis and its pseudo-products, and Astragalus membranaceus and its pseudo-products after alignment was analyzed. The regions rich in SNP sites in the Angelica sinensis and Astragalus membranaceus sequences were found as the site range for the design of specific primers.

[0116] Table 4 Species sequence information downloaded from GenBank

[0117]

[0118] The ITS sequences of Angelica sinensis, Astragalus membranaceus and their counterfeits were analyzed by Mega software, and the site information shown in Table 5 was obtained.

[0119] Table 5 ITS sequence characteristics of Angelica sinensis and Astragalus membranaceus

[0120]

[0121]

[0122] The SNP sites screened are as follows:

[0123] Angelica:

[0124] The 63rd base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker I (polymorphism: G / C).

[0125] The 65th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker II (polymorphism: T / C).

[0126] The 67th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker III (polymorphism: T / C).

[0127] The 72nd base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker IV (polymorphism: C / T).

[0128] The 74th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker V (polymorphism: T / C),

[0129] The 78th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker VI (polymorphism: A / T / C),

[0130] The 80th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker VII (polymorphism: T / C),

[0131] The 398th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker VIII (polymorphism: C / G).

[0132] The 403rd base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker IX (polymorphism: T / A).

[0133] The 409th base of the nucleotide sequence shown in SEQ ID NO.1 is Angelica SNP molecular marker X (polymorphism: T / A);

[0134] The 63rd base of Angelica ITS is G, the 65th base is T, the 67th base is T, the 72nd base is C, the 74th base is T, the 78th base is A, the 80th base is G, the 398th base is C, the 403rd base is T, and the 409th base is T.

[0135] Astragalus:

[0136] The 31st base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker I (polymorphism: G / T / A).

[0137] The 33rd base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker II (polymorphism: A / G / C).

[0138] The 36th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker III (polymorphism: A / T).

[0139] The 38th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker IV (polymorphism: G / A).

[0140] The 39th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker V (polymorphism: A / G).

[0141] The 41st base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker VI (polymorphism: C / T).

[0142] The 42nd base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker VII (polymorphism: T / C).

[0143] The 43rd base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker VIII (polymorphism: C / A / T),

[0144] The 44th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker IX (polymorphism: C / A).

[0145] The 45th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker X (polymorphism: C / T),

[0146] The 46th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker Ⅺ (polymorphism: T / C),

[0147] The 47th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XII (polymorphism: T / C / G).

[0148] The 48th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XIII (polymorphism: T / G / C),

[0149] The 49th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XIV (polymorphism: G / T).

[0150] The 395th base of the nucleotide sequence shown in SEQ ID NO.2 is the Astragalus SNP molecular marker XV (polymorphism: T / C / G).

[0151] The 396th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XVI (polymorphism: T / C / G).

[0152] The 397th base of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XVIII (polymorphism: A / T).

[0153] The base at position 401 of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XVIII (polymorphism: C / T).

[0154] The base at position 403 of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XIX (polymorphism: A / G).

[0155] The base at position 405 of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XX (polymorphism: T / A),

[0156] The base at position 406 of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XXI (polymorphism: G / A).

[0157] The base at position 407 of the nucleotide sequence shown in SEQ ID NO.2 is Astragalus SNP molecular marker XXII (polymorphism: A / T);

[0158] The 31st base of Astragalus ITS is G, the 33rd base is A, the 36th base is A, the 38th base is G, the 39th base is A, the 41st base is C, the 42nd base is T, the 43rd base is C, the 44th base is C, the 45th base is C, the 46th base is T, the 47th base is T, the 48th base is T, the 49th base is G, the 395th base is T, the 396th base is T, the 397th base is A, the 401st base is C, the 403rd base is A, the 405th base is T, the 406th base is G, and the 407th base is A.

[0159] (2) Specific primer design

[0160] In this example, primer design software Primer Premier 5 was used to design specific primers for Angelica sinensis (GenBank sequence number: GU289658) and Astragalus mongolica (GenBank sequence number: AB787166). The design parameters were as follows: primer length was between 18 and 25 bp; GC content was between 40% and 60%; base duplication was avoided, especially the presence of four consecutive G bases; three G or C base duplications were avoided at the 3′ end; the upstream and downstream design regions were SNP-rich regions; complementary sequences were avoided in the primers themselves; and the product length was 300 to 500 bp. Finally, the designed specific primers were handed over to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0161] The specific process is as follows: after deleting the non-overlapping sequences before and after the ITS of Angelica sinensis (GenBank sequence number GU289658, SEQ ID NO.1), the upstream and downstream primer design regions were set to 1-240bp and 370-600bp, and finally 5 pairs of specific primers with higher scores were designed. After deleting the non-overlapping sequences before and after the ITS of Astragalus membranaceus (GenBank sequence number AB787166, SEQ ID NO.2), the upstream and downstream primer design regions were set to 1-300bp and 370-563bp, and finally 5 pairs of specific primers with higher scores were designed.

[0162] (3) Specific primer screening

[0163] In this example, the DNA samples extracted from Angelica sinensis and Astragalus membranaceus and their counterfeits (Table 1) were respectively added with the designed primers for PCR amplification (PCR reaction system and conditions are shown in Tables 6 and 7), and the amplified products were analyzed by electrophoresis. According to the imaging results, it was determined whether the primers could only amplify the target fragments of Angelica sinensis or Astragalus membranaceus, and the specific primers of Angelica sinensis and Astragalus membranaceus were screened out.

[0164] The specific operation of this embodiment is: using the designed species-specific primers to amplify the angelica, angelica sinensis, peucedanum purpurogenum, angelica dahurica, astragalus, astragalus flat-stem, alfalfa, hibiscus, and caragana, respectively, and using ddH 2 O was used as a blank control, and the amplification results were detected by agarose gel electrophoresis. It was found that among the above 5 pairs of specific primer pairs for Angelica sinensis and the above 5 pairs of specific primer pairs for Astragalus membranaceus, there was one pair each: the primer pair DG-F / R of Angelica sinensis and the primer pair HQ-F / R of Astragalus membranaceus could stably and clearly amplify the bands of the target species, while the other false products and blank controls had no target bands.

[0165] in:

[0166] The primer pair DG-F / R of Angelica sinensis recognizes SNP molecular markers I to X of Angelica sinensis, and the upstream primer DG-F is complementary to the base of Angelica sinensis ITS at the 7th SNP site (Angelica sinensis SNP molecular marker I corresponds to the 1st base at the 5' end of the DG-F nucleotide sequence, Angelica sinensis SNP molecular marker II corresponds to the 3rd base at the 5' end of the DG-F nucleotide sequence, Angelica sinensis SNP molecular marker III corresponds to the 5th base at the 5' end of the DG-F nucleotide sequence, Angelica sinensis SNP molecular marker IV corresponds to the 10th base at the 5' end of the DG-F nucleotide sequence, Angelica sinensis SNP molecular marker V corresponds to the 12th base at the 5' end of the DG-F nucleotide sequence, Angelica sinensis SNP molecular marker VI corresponds to the 16th base at the 5' end of the DG-F nucleotide sequence, Angelica sinensis SNP molecular marker Molecular marker VII corresponds to the 18th base at the 5' end of the DG-F nucleotide sequence), and the downstream primer DG-R is complementary to the bases of Angelica ITS at the three SNP sites (Angelica SNP molecular marker VIII corresponds to the 7th base at the 5' end of the DG-R nucleotide sequence, Angelica SNP molecular marker IX corresponds to the 12th base at the 5' end of the DG-R nucleotide sequence, and Angelica SNP molecular marker X corresponds to the 18th base at the 5' end of the DG-R nucleotide sequence). When DG-F and DG-R are used for gene DNA amplification of Danggui Buxue Pills, they can specifically bind to Danggui ITS for PCR amplification to obtain a gene fragment with a length of 349bp, while it is impossible to identify and amplify Danggui counterfeit species. Therefore, the use of the above-mentioned first primer pair DG-F and DG-R can achieve allele-specific PCR amplification of Danggui ITS and inhibit PCR amplification of Danggui counterfeit species, so as to detect whether Danggui Buxue Pills contain Danggui gene source and realize the authenticity identification of the raw medicinal materials of Danggui Buxue Pills.

[0167] The primer pair HQ-F / R of Astragalus membranaceus recognizes the Astragalus membranaceus SNP molecular markers I to XII, wherein the upstream primer HQ-F and the Astragalus membranaceus ITS bases at the 14 SNP sites are paired with each other (Astragalus membranaceus SNP molecular marker I recognizes the first base at the 5' end of the HQ-F nucleotide sequence, and Astragalus membranaceus SNP molecular marker II recognizes the third base at the 5' end of the HQ-F nucleotide sequence). Astragalus SNP molecular marker III recognizes the 6th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker IV recognizes the 8th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker V recognizes the 9th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker VI recognizes the 11th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker VII (recognizes the 12th base at the 5' end of the HQ-F nucleotide sequence, and Astragalus SNP molecular marker VIII recognizes the 13th base at the 5' end of the HQ-F nucleotide sequence. Astragalus SNP molecular marker IX recognizes the 14th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker X recognizes the 15th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker XI recognizes the 16th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker XII recognizes the 17th base at the 5' end of the HQ-F nucleotide sequence, Astragalus SNP molecular marker XIII recognizes the 18th base at the 5' end of the HQ-F nucleotide sequence, and Astragalus SNP molecular marker XIV recognizes the 19th base at the 5' end of the HQ-F nucleotide sequence. The downstream primer HQ-R and the Astragalus ITS were complementary to each other at the 8 SNP sites (Astragalus SNP molecular marker XV corresponds to the 2nd base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XVI corresponds to the 3rd base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XVIII corresponds to the 4th base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XVIII corresponds to the 8th base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XIX corresponds to the 10th base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XX corresponds to the 11th base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XXI corresponds to the 12th base at the 5' end of the HQ-R nucleotide sequence, Astragalus SNP molecular marker XIX corresponds to the 13th base at the 5' end of the HQ-R nucleotide sequence, Molecular marker XXII corresponds to the 13th base at the 5' end of the HQ-R nucleotide sequence). When HQ-F and HQ-R are used for the gene DNA amplification of Danggui Buxue Pills, they can specifically bind to Astragalus ITS for PCR amplification to obtain a gene fragment with a length of 381 bp, while the pseudo Astragalus species cannot be identified and amplified. Therefore, the second primer pair HQ-F and HQ-F can realize the allele-specific PCR amplification of Astragalus ITS and inhibit the PCR amplification of the pseudo Astragalus species, so as to detect whether Danggui Buxue Pills contain Astragalus source and realize the authenticity identification of the raw medicinal materials of Danggui Buxue Pills.

[0168] Figure 3 A is the amplification result of the DG-F / R primer pair of Angelica sinensis in this example, Figure 3 B is the amplification result of the HQ-F / R primer pair of Astragalus membranaceus in this example (the other four primer pairs are not shown because of poor results), which shows that both pairs of primers have good species specificity. Therefore, DG-F / R and HQ-F / R primers are selected as specific amplification primers for the two biological components of Danggui Buxue Pills in subsequent experiments in Example 3. The sequence information of DG-F / R and HQ-F / R primers is shown in Table 8, and the primer sequence design site information is shown in Tables 11-14.

[0169] In this embodiment, the raw materials of Danggui Buxue Wan were only crushed, and the genomic DNA was not completely degraded into short fragments, so the length of the target product was set to 300 bp to 500 bp. In the experiment, the amplification success rates of the two pairs of primers DG-F / R and HQ-F / R (the size of the Danggui-specific amplification product was 349 bp and the size of the Astragalus-specific amplification product was 381 bp) were very high, and the species specificity of the primers was good.

[0170] Table 6 PCR reaction system

[0171]

[0172] Table 7 PCR reaction conditions

[0173]

[0174] Table 8 Specific identification primer sequences of Angelica sinensis and Astragalus membranaceus

[0175]

[0176] Example 3 Optimization of multiplex PCR identification conditions for two biological components of homemade Danggui Buxue Pills

[0177] (1) Annealing temperature

[0178] Using homemade Danggui Buxue Pill DNA (ZP) as a template, multiplex PCR annealing temperature optimization experiments were conducted, and the selected Danggui and Astragalus specific primers were amplified, and the annealing temperature gradient was set to 50-72℃ (50℃, 51℃, 52.4℃, 54.6℃, 57.2℃, 59.7℃, 62℃, 65.1℃, 67.6℃, 69.6℃, 71.1℃, 72℃), respectively, to investigate the effect of different annealing temperatures on amplification, and screen out the optimal temperature or temperature range for multiplex PCR amplification of homemade Danggui Buxue Pill biological components. The results are shown in Figure 4. It can be seen from the figure that: within the annealing temperature range of 50℃ to 67.6℃, the two pairs of species-specific primers can detect the corresponding target fragment bands, but the amplification signal at 67.6℃ is weak. Under the annealing condition of 65℃, the brightness of the amplification products of the two pairs of primers is similar, so the annealing temperature of multiplex PCR was selected as 65℃ in subsequent studies.

[0179] (2) Detection sensitivity

[0180] The DNA of homemade Danggui Buxue Pills sample (ZP) was used as template. The initial concentration was 40.5 ng / μL. Sterile ddH 2 O 10-fold dilution (10 0 ~10 5 times), the annealing temperature was optimized at 65°C, and the detection limit of multiplex PCR was obtained according to the amplification results. Figure 5 ), the minimum detection limit of the specific band of Angelica sinensis in the multiplex PCR system was 4×10 -2 The minimum detection limit of Astragalus membranaceus was 4×10 -3 ng / μL. The amplification system and procedure of multiplex PCR for angelica and astragalus components in Danggui Buxue Pills were finally determined through optimization.

[0181] In this example, the 20 μL multiplex PCR reaction system and conditions were finally determined through the above experiments and are shown in Tables 9 and 10.

[0182] Table 9 Multiplex PCR reaction system

[0183]

[0184]

[0185] Table 10 Multiplex PCR reaction conditions

[0186]

[0187] Example 4 Qualitative identification of commercially available Danggui Buxue Pills by multiplex PCR

[0188] In order to verify the accuracy and stability of the established multiplex PCR method, this example uses the multiplex PCR method established above to perform PCR amplification on 14 batches of commercially available Danggui Buxue Pills DNA. After the PCR amplification reaction is completed, 5 μL of the PCR reaction product is taken, 1 μL of 6× Loading buffer is added and mixed, and then the mixture is subjected to 2% agarose gel electrophoresis stained with Super Red / GelRed, and the gel is observed and photographed under a gel imaging system. The results show ( Figure 6 ), in 14 batches of commercially available samples labeled as Danggui Buxue Wan, the target amplified fragments (349 bp and 381 bp) of Danggui and Astragalus species were simultaneously obtained, indicating that the established multiplex PCR method is accurate, stable and specific.

[0189] Table 11 Angelica sinensis primer DG-F site information

[0190]

[0191]

[0192] Table 12 DG-R site information of Angelica sinensis primers

[0193]

[0194] Table 13 Astragalus primer HQ-F site information

[0195]

[0196]

[0197] Table 14 Astragalus primer HQ-R site information

[0198] SEQUENCE LISTING <110> Southern Medical University <120> SNP sites and identification methods for identifying biological components in Danggui Buxue Pills <130> 2022 <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 599 <212> DNA <213> Angelica sinensis <400> 1 tcgaatcctg cgatagcaga acgacccgct aacatgtaaa catattgggc aagtgttcgg 60 tcgaatcctg cgatagcaga acgacccgct aacatgtaaa catattgggc aagtgttcgg 60 gggctttggt cccttgtatg cgaacctggt aggtggcccc tctcgggtgg ccactggcct 120 gggctttggt cccttgtatg cgaacctggt aggtggcccc tctcgggtgg ccactggcct 120 gcgaaatcat tcgggcgcgg aatgcgccaa ggaacttaaa attgaattgt acgtcggcat 180 gcgaaatcat tcgggcgcgg aatgcgccaa ggaacttaaa attgaattgt acgtcggcat 180 cccgttagcg ggcatcgacg tcattccaaa acacaacgac tctcgacaac ggatatctcg 240 cccgttagcg ggcatcgacg tcattccaaa acacaacgac tctcgacaac ggatatctcg 240 gctctcgcat cgatgaagaa cgtagcgaaa tgcgatactt ggtgtgaatt gcagaatccc 300 gctctcgcat cgatgaagaa cgtagcgaaa tgcgatactt ggtgtgaatt gcagaatccc 300 gtgaaccatc gagtctttga acgcaagttg cgcccgaagc cattaggctg agggcacgtc 360 gtgaaccatc gagtctttga acgcaagttg cgcccgaagc cattaggctg agggcacgtc 360 tgcctgggtg tcacgcatca tctttgccca caaccactca ctcctcgtgg agctgtactg 420 tgcctgggtg tcacgcatca tctttgccca caaccactca ctcctcgtgg agctgtactg 420 gtatgggggc ggaaattggc ctcccgtgcc ttgttgtgcg gttggcgcaa aagtgagtct 480 gtatgggggc ggaaattggc ctcccgtgcc ttgttgtgcg gttggcgcaa aagtgagtct 480 ccggcgacgg acgtcgtgac attggtggtt gtaaaatacc ctcatgtctt gtcgcgcgaa 540 ccggcgacgg acgtcgtgac attggtggtt gtaaaatacc ctcatgtctt gtcgcgcgaa 540 tccgcgtcat cttagtgagc tcaaggaccc ttaggcggca cacactttgt gcacttcga 599 tccgcgtcat cttagtgagc tcaaggaccc ttaggcggca cacactttgt gcacttcga 599 <210> 2 <210> 2 <211> 563 <211> 563 <212> DNA <212> DNA <213> 蒙古黄芪(Astragalus mongholicus) <213> Astragalus mongholicus <400> 2 <400> 2 ttgaatactt agggatggct ggggtgtttt gcaccacgac ctccctttgg gtggggggtg 60 gtgcgcaatg cgttccccct cctgcccgaa cacaaacccc ggcgctcaat gcgccaagga 120 actaaaattc gatcaatgtg ccccgtcggc ccggagacgg tgcttcggcg gtggtgcctt 180 gtcacatgat acagaatgac tctcggcaac ggatatctag gctcttgcat cgatgaagaa 240 cgtagcgaaa tgcgatactt ggtgtgaatt gcagaatccc gtgaaccatc gagtctttga 300 acgcaagttg cgcccgaagc cattaggttg agggcacgtc tgcctgggcg tcacatatcg 360 ttgcccgatg cctattgcag tgtgatagga atttttaggg cgaatgatgg cttcccgtga 420 gcgttgttgc ctcgcggctg gttgaaaatt gagtccttgg tggggtgtgc catgatagat 480 ggtggtcgag ttagcacgag acccatcatg tgtacgctcc ccataatatg gcttcgatga 540 cccacatgcg tcttttgact ctc 563 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <400> 3 ggctttggtc ccttgtatg 19 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <400> 4 cacgaggagt gagtggttg 19 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <400> 5 gcaccacgac ctccctttg 19 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <400> 6 gccatcattc gccctaaa 18

Claims

1. A method for identifying biological components in Danggui Buxue Pills, It is characterized in that The method is to identify biological components in Danggui Buxue Wan based on SNP sites, wherein the biological components include Danggui and Astragalus; The method comprises the following steps: (1) Extracting genomic DNA from the sample to be tested; (2) Using the extracted genomic DNA of the sample to be tested as a template, specific primers are designed based on the SNP site for PCR amplification reaction; (3) Detection of PCR amplification products; The SNP sites include: The SNP site of Angelica sinensis is selected from at least one of the 63rd site, 65th site, 67th site, 72nd site, 74th site, 78th site, 80th site, 398th site, 403th site and 409th site from the 5' end as shown in SEQ ID NO.1; The SNP site of Astragalus membranaceus is selected from at least one of the 41st, 43rd, 46th, 48th, 49th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 489th, 490th, 491st, 495th, 497th, 501st, 502nd and 503rd sites from the 5' end as shown in SEQ ID NO.2; The specific primers include: Angelica specific primer pair: Upstream primer as shown in SEQ ID NO.3: 5'GGCTTTGGTCCCTTGTATG3'; Downstream primer as shown in SEQ ID NO.4: 5'CACGAGGAGTGAGTGGTTG3'; Astragalus-specific primer pairs: Upstream primer as shown in SEQ ID NO.5: 5'GCACCACGACCTCCCTTTG3'; Downstream primer as shown in SEQ ID NO.6: 5'GCCATCATTCGCCCTAAA3'; The method for extracting genomic DNA of the sample to be tested is a modified CTAB method, which specifically includes the following steps: S1. Grind 2-3 g of the sample to be tested; S2. Add the fine powder obtained by grinding to 10-15 mL of 4×CTAB extraction buffer preheated at 65°C, wherein 0.2 volume of 1M dithiothreitol solution is added to the 4×CTAB extraction buffer before preheating, and incubate in a water bath at 65°C for 1.5 h; S3. After cooling, add 1 to 1.5 times the volume of chloroform-isoamyl alcohol mixed solution, with a volume ratio of 24:1, and centrifuge; S4. Take the supernatant, add 0.7~1 times volume of -20℃ precooled isopropanol, place at -20℃ to settle for 1 hour, centrifuge and discard the waste liquid to obtain crude DNA; S5. The extracted crude DNA was washed with 70% ethanol solution and anhydrous ethanol respectively, and then purified using a universal DNA purification and recovery kit.

2. The method according to claim 1, It is characterized in that Step S3 is repeated twice.

3. The method according to claim 1, It is characterized in that In step (2), the annealing temperature of the PCR amplification reaction is 50°C to 67.6°C.

4. The method according to claim 3, It is characterized in that In step (2), the annealing temperature of the PCR amplification reaction is 65°C.

5. The method according to claim 1, It is characterized in that In step (2), in the PCR amplification reaction: the detection limit of genomic DNA of Angelica sinensis is ≥ 0.04 ng / μL; the detection limit of genomic DNA of Astragalus membranaceus is ≥ 0.004 ng / μL.

6. The method according to claim 1, It is characterized in that The detection of PCR amplification products is performed by electrophoresis detection.

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