Method for qualitatively / quantitatively detecting astragalus membranaceus component in Chinese patent medicine as well as primer and probe thereof

Abstract

The invention provides a method, a primer and a probe for qualitatively / quantitatively detecting an astragalus component in a Chinese patent medicine. The method for qualitatively or quantitatively detecting the astragalus membranaceus component in the Chinese patent medicine is constructed on the basis of the specific primer pair, the probe and ddPCR. The quantitative detection method comprises the following steps: extracting DNA (Deoxyribose Nucleic Acid) of a Chinese patent medicine which has a known feed ratio and contains astragalus membranaceus components in different proportions, and carrying out ddPCR amplification reaction based on a specific primer pair and a probe to determine copy number ranges corresponding to the astragalus membranaceus components in different proportions; dNA of the Chinese patent medicine to be detected is extracted, ddPCR amplification reaction is carried out based on the specific primer pair and the probe, and the target DNA copy number of astragalus membranaceus is calculated; and determining the content of the astragalus component in the Chinese patent medicine to be detected through comparison. The method provided by the invention can qualitatively and quantitatively detect the astragalus membranaceus component in the Chinese patent medicine in a high-specificity and high-sensitivity manner, and a new technical means is provided for quality control of Chinese patent medicine bulk drugs.

Description

Technical Field

[0001] This invention belongs to the field of identification technology, and in particular relates to a method for qualitative / quantitative detection of Astragalus membranaceus components in traditional Chinese medicine, as well as its primers and probes. Background Technology

[0002] Traditional Chinese medicine (TCM) preparations are drugs processed into different dosage forms, including pills, tablets, capsules, granules, and injections, using Chinese medicinal herbs and various excipients as raw materials and following prescribed methods, under the guidance of TCM theory (Non-Patent Literature 1). Due to the complexity of TCM ingredients and the uncertainty of active components, their quality is easily affected by various factors such as raw materials (APIs), origin, processing technology, storage methods, and intended use (Non-Patent Literature 2-3). Among the many production stages affecting the quality of TCM preparations, API quality control is the most important link in ensuring their safety and efficacy (Non-Patent Literature 4). Therefore, how to strictly control the authenticity of API input (adulteration, substitution, insufficient input, etc.) in TCM preparation production is a key issue that urgently needs to be addressed in current TCM quality control.

[0003] Currently, the main quality control methods for traditional Chinese medicine (TCM) preparations include microscopic identification, physicochemical identification, and molecular identification. Microscopic identification relies primarily on the characteristics of cell morphology, tissue structure, and intracellular contents of plant and animal raw materials for qualitative identification, as well as quantitative identification based on microscopic characteristic constants (Non-Patent Literature 5). However, the raw materials in TCM preparations often undergo complex processing and preparation, which frequently alters their specific cell or tissue structures, leading to a decrease in the accuracy of qualitative and quantitative identification. Furthermore, microscopic identification is highly dependent on the experience of the operators. Physicochemical identification methods, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC), are now widely used in the quality control of TCM preparations (Non-Patent Literature 6-7). These methods mainly involve qualitative and / or quantitative analysis of one or more major chemical components of the raw materials. However, this method sometimes struggles to distinguish between raw materials derived from closely related species and species containing the same major chemical components (such as Angelica sinensis and Angelica dahurica, ginseng and American ginseng, honeysuckle and honeysuckle buds), resulting in poor specificity (Non-Patent Literature 8-9). In recent years, with the rapid development of molecular biology techniques, DNA molecular identification technology has also been applied to the quality control of traditional Chinese medicine (TCM) preparations, making up for some shortcomings of microscopic and physicochemical identification methods (Non-Patent Literature 10). There are two main methods of DNA molecular identification: one is to identify a specific component in TCM preparations using target fragment sequencing or specific PCR (Non-Patent Literature 11). The other is to identify components in TCM preparations using cloning sequencing, metabarcoding, or metagenomics technologies (Non-Patent Literature 12). However, these methods are primarily for qualitative identification of raw materials in TCM preparations; issues such as the amount of raw materials used and the presence of adulteration remain unresolved.

[0004] Danggui Buxue Wan (Angelica Blood-Nourishing Pill) is a traditional Chinese medicine developed based on Danggui Buxue Tang (Angelica Blood-Nourishing Decoction), a representative formula for invigorating qi and nourishing blood, created by the famous Jin Dynasty physician Li Dongyuan. It has the effect of nourishing qi and blood and is widely used in gynecological clinical practice (Non-patent literature 13). The formula of Danggui Buxue Wan is simple, consisting of Astragalus membranaceus and Angelica sinensis in a 5:2 mass ratio. Astragalus membranaceus is a commonly used and precious traditional Chinese medicinal herb in my country. In recent years, the resources of wild Astragalus membranaceus have decreased dramatically, and its original plant, Astragalus membranaceus, is becoming increasingly scarce. Astragalus membranaceus (Fisch.) and Mongolian Astragalus Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao has been listed as an endangered species (Non-Patent Literature 14). In the market, Astragalus membranaceus is often adulterated or substituted, such as with Astragalus membranaceus var. flattened. Phyllolobium chinense Fisch., Alfalfa Medicago sativa L., Hollyhock Alcea rosea L. and Golden Pheasant Caragana sinica The root of *Astragalus membranaceus* (Buc'hoz) Rehde is often substituted or adulterated (Non-Patent Literature 15-17). These substitutes or adulterants are very similar in appearance to genuine *Astragalus membranaceus* root, but their clinical applications and efficacy differ greatly; some adulterants are even toxic. Furthermore, according to the latest filing information from the National Medical Products Administration, there are currently 255 registered traditional Chinese medicine products containing *Astragalus membranaceus* in my country. If *Astragalus membranaceus* in *Dang Gui Bu Xue Wan* or other traditional Chinese medicine products is intentionally or unintentionally adulterated or substituted, it may seriously affect clinical efficacy and even cause medication safety issues. The quality control methods for *Dang Gui Bu Xue Wan* recorded in the *Drug Standards of the Ministry of Health of the People's Republic of China: Traditional Chinese Medicine Preparations* are microscopic examination and thin-layer chromatography (Non-Patent Literature 13), but both of these identification methods lack specificity and corresponding quantitative standards. Therefore, there is an urgent need to establish a method for qualitative and quantitative detection of *Astragalus membranaceus* to ensure the safety and effectiveness of *Dang Gui Bu Xue Wan* in clinical application.

[0005] Non-patent literature 1: Zhuang W, Fan Z, Chu YQ, et al. Chinese Patent Medicines in the Treatment of Coronavirus Disease 2019 (COVID-19) in China, FrontPharmacol, 2020,11:1066.

[0006] Non-patent literature 2: Liu Jing, Zhu Jialiang, Feng Lei, et al. Analysis of the quality status of traditional Chinese medicine preparations in the national drug sampling inspection in 2020 [J]. China Modern Chinese Medicine, 2021, 23(5): 755-759.

[0007] Non-patent literature 3: Liu Jing, Wang Chong, Feng Lei, et al. Analysis of quality and safety issues of traditional Chinese medicine preparations based on national drug sampling inspection work [J]. Modern Chinese Medicine, 2019, 21(3): 279-283.

[0008] Non-patent literature 4: Yan Qianru, Wu Weikui, Song Wei. Analysis of standardization issues in the addition of raw materials for traditional Chinese medicine preparations and strategies for quality risk prevention and control [J]. Journal of South-Central University for Nationalities (Natural Science Edition), 2024, 43(02):189-193.

[0009] Non-patent literature 5: Zhao Minjia, Wu Di, Wang Xu, et al. Microscopic identification study of Huanglian Yanggan Pills from different manufacturers [J]. Journal of Harbin University of Commerce (Natural Science Edition), 2020, 36(5): 528-531, 541.

[0010] Non-patent literature 6: Sun, LJ, Guo, DL., and Li, CB TLC identification of rhizoma cyperi and HPLC determination of ferulic acid in xin tong ning pill. Chin. J. Microecol., 2009, 21, 620-621.

[0011] Non-patent literature 7: Gao, P., and Niu, YJ Simultaneous determination of puerarin, rosmarinic acid, praeruptorin A, praeruptorin B and praeruptorin Ein shensu tablets by HPLCQAMS. China Pharm. 23, 2020, 341-345.

[0012] Non-patent literature 8: Huang Luqi, Yuan Yuan, Yuan Qingjun, et al. Discussion on several issues in the development of molecular identification of traditional Chinese medicine. China Journal of Traditional Chinese Medicine, 2014, 39(19): 3663 3667.

[0013] Non-patent literature 9: Yang Jin. Quality problems of traditional Chinese medicine urgently need to be solved [J]. Democracy and Science, 2016, (1): 32-33, 36.

[0014] Non-patent literature 10: Zhou Shilin, Zhang Yanmei, Zhou Ting, et al. Research progress on the application of molecular identification technology in the identification of medicinal ingredients in traditional Chinese medicine formulations [J / OL]. Modern Chinese Materia Medica, 1-7 [2025-04-04].

[0015] Non-patent literature 11: Chen R, Dong J, Cui X, et al. DNA based identification of medicinal materials in Chinese patent medicines. Sci. Rep., 2012, 2:958.

[0016] Non-patent literature 12: Cheng XW, Su XQ, Chen XH, et al. Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughputsequencing: the story for Liuwei Dihuang Wan. Sci. Rep., 2014, 4: 5147.

[0017] Non-patent literature 13: Pharmacopoeia Commission of the Ministry of Health of the People's Republic of China. Drug Standards of the Ministry of Health of the People's Republic of China: Traditional Chinese Medicine Compound Preparations. [S]. 1989.

[0018] Non-patent literature 14: Liu Jianquan, Yang Jianhong, Zhu Gao. Life history analysis of endangered plant Astragalus membranaceus population [J]. Grassland Science, 2011, 028(2):180-184.

[0019] Non-patent literature 15: Wang Yuqi, Li Ruiming, Ma Yanbin, et al. Identification and application of Astragalus membranaceus and its adulterants Althaea sylvestris and Glycyrrhiza uralensis [J]. Modern Distance Education of Chinese Medicine, 2013, 11(16): 128-129.

[0020] Non-patent literature 16: Shang Huaihai, Wu Qiong, Sun Yuping. DNA molecular identification of Astragalus membranaceus and analysis of its adulterants [J]. China Coal Industry Medical Journal, 2016, 10(1): 72-75.

[0021] Non-patent literature 17: Wang Wei, Yuan Ye, Nan Peng. Molecular identification of Astragalus membranaceus Radix and its counterfeit processed products [J]. Journal of Fudan University (Natural Science Edition), 2020, 59(01): 64-72. Summary of the Invention

[0022] To address the aforementioned technical problems, this invention provides a method for qualitative detection of Astragalus membranaceus (Huang Qi) components in traditional Chinese medicine (TCM) preparations, a method for quantitative detection of Astragalus membranaceus (Huang Qi) components in TCM preparations, and corresponding primers and probes. This invention utilizes ddPCR for qualitative or quantitative detection of Astragalus membranaceus (Huang Qi) components in TCM preparations, providing a simple method for quality control of TCM preparations.

[0023] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides a specific primer pair for detecting Astragalus membranaceus in traditional Chinese medicine preparations, comprising: For example, the upstream primer shown in SEQ ID NO.1: GCACCACGACCTCCCTTTG; The downstream primer shown in SEQ ID NO.2 is: GCCATCATTCGCCCTAAA.

[0024] In another aspect, the present invention provides a probe for detecting Astragalus membranaceus in traditional Chinese medicine, the sequence of which is shown in SEQ ID NO:3: SEQ ID NO:3 AACTAAAATTCGATCAATGTGCCCCGTC.

[0025] In another aspect, the present invention provides a method for qualitative detection of Astragalus component in traditional Chinese medicine, the method being based on the above-mentioned specific primer pair and probe to detect Astragalus component in traditional Chinese medicine.

[0026] In a preferred embodiment, the 5' end of the specific probe is modified with the fluorescent group 6-FAM, and the 3' end is modified with the fluorescent quenching group BHQ1.

[0027] In a preferred embodiment, the method includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted genomic DNA of the sample to be tested as a template, ddPCR amplification reaction was performed based on the above-mentioned specific primer pairs and probes; (3) Detect ddPCR amplification products.

[0028] In the technical solution of the present invention, whether the sample to be tested contains Astragalus membranaceus is determined by whether positive droplets are detected in the ddPCR amplification product.

[0029] In a preferred embodiment, step (1) involves the following steps for extracting genomic DNA from the sample to be tested: ① Grind the sample to be tested; then add it to 4×CTAB extraction buffer preheated at 65℃; before preheating the 4×CTAB extraction buffer, add 0.2% volume of 1 M dithiothreitol (DTT) solution; after mixing, incubate in a 65℃ water bath for 1.5~2 h; ② After cooling, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1) mixed solution, mix well, and centrifuge; ③ Take the supernatant, add 0.7~1 volume of isopropanol pre-cooled at -20℃, precipitate at -20℃ for 1~2 h, centrifuge and discard the waste liquid to obtain crude DNA; repeat this step twice. ④ 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.

[0030] Preferably, in step ①, every 2 g of the sample to be tested is ground and added to 10~15 mL of 4×CTAB extraction buffer.

[0031] In a preferred embodiment, in step (2), the annealing temperature of the ddPCR amplification reaction is 55~65℃, preferably 59℃.

[0032] In another aspect, the present invention provides a method for quantitatively detecting Astragalus components in traditional Chinese medicine preparations, comprising the following steps: DNA was extracted from traditional Chinese medicine preparations containing different proportions of Astragalus membranaceus (TCM) components with known feed ratios. ddPCR amplification was performed using the specific primer pairs and probes described above to determine the copy number range corresponding to different proportions of Astragalus membranaceus. DNA was extracted from the TCM preparation to be tested and ddPCR amplification was performed using the specific primer pairs and probes described above to calculate the target DNA copy number of Astragalus membranaceus. The content of Astragalus membranaceus in the TCM preparation to be tested was confirmed by comparison.

[0033] In the technical solution of the present invention, the content of Astragalus component in the tested traditional Chinese medicine can be determined by comparing the target DNA copy number concentration of Astragalus in the tested traditional Chinese medicine with the copy number range of traditional Chinese medicine with a known Astragalus feed ratio.

[0034] In a preferred embodiment, the method for extracting DNA from traditional Chinese medicine preparations or the method for extracting DNA from traditional Chinese medicine preparations to be tested includes the following steps: ① Grind the sample to be tested; then add it to 4×CTAB extraction buffer preheated at 65℃; before preheating the 4×CTAB extraction buffer, add 0.2% volume of 1 M dithiothreitol (DTT) solution; after mixing, incubate in a 65℃ water bath for 1.5~2 h; ② After cooling, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1) mixed solution, mix well, and centrifuge; ③ Take the supernatant, add 0.7~1 volume of isopropanol pre-cooled at -20℃, precipitate at -20℃ for 1~2 h, centrifuge and discard the waste liquid to obtain crude DNA; repeat this step twice. ④ 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.

[0035] Preferably, in step ①, every 2 g of the sample to be tested is ground and added to 10~15 mL of 4×CTAB extraction buffer.

[0036] In a preferred embodiment, the annealing temperature of the ddPCR amplification reaction is 55~65℃, preferably 59℃.

[0037] The above technical solution has the following advantages or beneficial effects: This invention provides primers and probes for detecting Astragalus membranaceus (Huang Qi) components in traditional Chinese medicine (TCM) preparations. Furthermore, it establishes a method for qualitative and quantitative detection of Astragalus membranaceus components in TCM preparations based on ddPCR technology. The method provided by this invention can specifically identify Astragalus membranaceus under optimal annealing temperatures of 55℃ to 59℃, and detect the content of Astragalus membranaceus components in TCM preparations based on the copy number of the Astragalus membranaceus target gene. The ddPCR quantitative detection method and HPLC quantitative detection results provided by this invention show good consistency, providing a new technology for the qualitative and quantitative quality control of raw materials for TCM preparations and serving as a powerful supplement to current methods.

[0038] The primers and probes provided by this invention have high specificity and can qualitatively identify Astragalus membranaceus and its adulterants; ddPCR shows good linearity. Y = 0.884 X -1.195, R 2 = 0.9937), the detection limit for DNA concentration can reach 2.0 × 10⁻⁶. -3 ng / μL; target gene copy number detected by ddPCR ( Y = 5.5649 X , R 2 = 0.9941) and the astragaloside A content detected by HPLC ( Y =0.0045 X +0.015, R 2 = 0.9824) all showed a significant linear relationship with the proportion of Astragalus membranaceus used; Pearson correlation analysis showed that the copy number of Astragalus membranaceus target genes in Danggui Buxue Wan was significantly positively correlated with the content of its quality marker, astragaloside A. r =0.986, P<0.001); Within-group correlation coefficient analysis showed that the ddPCR and HPLC quantitative detection results had good consistency (ICC = 0.816). Therefore, the ddPCR method provided by this invention can perform qualitative and quantitative detection of Astragalus membranaceus in traditional Chinese medicine with high specificity and sensitivity, providing a new technical means for the quality control of raw materials of traditional Chinese medicine. Attached Figure Description

[0039] Figure 1 This is a diagram showing the results of the specificity verification experiment of the primers and probes in Example 2 of this invention.

[0040] Figure 2 This is a graph showing the experimental results of ddPCR annealing temperature optimization in Example 2 of this invention.

[0041] Figure 3 This is a graph showing the experimental results of the linear response of the ddPCR instrument in Embodiment 2 of the present invention.

[0042] Figure 4 This is a graph showing the results of the sensitivity test of ddPCR in Example 2 of this invention.

[0043] Figure 5 This is a graph showing the quantitative results of Astragalus membranaceus molecules in Danggui Buxue Wan (Angelica Blood-Nourishing Pill) in Example 2 of this invention.

[0044] Figure 6 This is a linear relationship graph between the content of astragaloside IV in Danggui Buxue Pills and the amount of astragalus added in Example 2 of the present invention.

[0045] Figure 7 This is a PP graph showing the normal distribution of astragaloside IV content and target DNA copy number in Example 2 of this invention. Detailed Implementation

[0046] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0047] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0048] Example 1 1.1 Experimental Materials 1.1.1 Collection of Plant Materials This embodiment collected 17 batches of plant samples from different regions across the country, including the original plants of genuine Astragalus membranaceus and the original plants of common adulterants. Sample collection information is shown in Table 1. All plant voucher specimens were identified by Associate Professor Tian Enwei of the School of Traditional Chinese Medicine, Southern Medical University, and are preserved in the Herbarium of Southern Medical University. Fresh plant leaves were dried and preserved with silica gel for genomic DNA extraction and primer and probe specificity verification; plant roots were naturally air-dried and used in the preparation of Dang Gui Bu Xue Wan (Angelica Blood-Nourishing Pill) with different Astragalus membranaceus dosage ratios.

[0049] Table 1. Collection information of the original plants of Astragalus membranaceus and its adulterants

[0050] 1.1.2 Preparation of Angelica Blood-Nourishing Pills with Different Astragalus Root Proportions Following the preparation process outlined in the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China - Traditional Chinese Medicine Compound Preparations" (Non-Patent Literature 13), six batches of Dang Gui Bu Xue Wan (Angelica Blood-Nourishing Pills) with different Astragalus membranaceus (Huang Qi) proportions were prepared. The amount of Angelica sinensis (Dang Gui) in each batch remained constant at 160g, while the amounts of Astragalus membranaceus (Huang Qi) were 400g, 320g, 240g, 160g, 80g, and 0g, respectively. Simultaneously, dried roots of Caragana korshinskii were selected as adulterant and added in amounts of 0g, 80g, 160g, 240g, 320g, and 400g, respectively. The medicinal samples were pulverized into fine powder, sieved, and mixed thoroughly. For every 100g of powder, 100-130g of refined honey was added to form large honey pills, or 30-40g of refined honey and an appropriate amount of water were added to form pills, which were then dried.

[0051] Table 2. Ratio of Astragalus and Adulteration in Six Batches of Dang Gui Bu Xue Wan (Angelica Blood-Nourishing Pills)

[0052] Example 2 2.2.1 DNA Extraction In this embodiment, total genomic DNA was extracted from silica gel-dried leaf samples using the 4×CTAB method. Genomic DNA was then extracted from six batches of Danggui Buxue Wan prepared in Example 1 using the CTAB method combined with a silica-based purification column DNA purification kit (Tiangen, Beijing, China). To address the issue of raw material DNA degradation during the preparation of Danggui Buxue Wan, the amount of Danggui Buxue Wan used for DNA extraction was increased (2 g) in this embodiment. The concentration and integrity of the genomic DNA from the plant material and the prepared Danggui Buxue Wan were assessed using a NanoDrop 2000 UV / Vis spectrophotometer (Thermo Scientific, USA) and 1.2% agarose gel electrophoresis, respectively.

[0053] The reagents and kits used in the DNA extraction process, as well as the specific extraction methods, are as follows: Anhydrous ethanol (Guangdong Guanghua Technology Co., Ltd.); chloroform (Guangzhou Chemical Reagent Factory); isoamyl alcohol (Shanghai Maclean Biochemical Technology Co., Ltd.); dithiothreitol (DTT, Beijing Mengyimei Biotechnology Co., Ltd.), disodium ethylenediaminetetraacetate (EDTA-Na2, Beijing Zhongkangbo Biotechnology Co., Ltd.), Tris base (Lambolid (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 Maclean Biochemical Technology Co., Ltd.), cetyltrimethylammonium bromide (CTAB, Guangzhou Solarbio Biotechnology Co., Ltd.), sodium acetate trihydrate (NaAc·3H2O, Guangzhou Chemical Reagent Factory), etc., general-purpose DNA purification and recovery kit (Tiangen, DP214-03, centrifuge column type).

[0054] (1) DNA extraction from plant samples The specific steps are as follows: ① Weigh about 20 mg of the collected roots or leaves of Angelica sinensis, Astragalus membranaceus and their adulterants, wash the surface with tap water, disinfect with 75% alcohol and air dry. ② After cleaning, the sample was cut into small pieces with scissors and ground into fine powder in a mortar. 2 mL of preheated 4×CTAB was added (2 parts by weight of CTAB with a concentration of 1M DTT was added before preheating and mixed well) and ground. The mixture was then transferred to a 7 mL centrifuge tube and placed in a 65℃ water bath for 1.5 h, with intermittent shaking to ensure even mixing. ③ After the water bath, place the sample at room temperature or on ice to cool to room temperature, add an equal volume of chloroform-isoamyl alcohol, invert for about 5 minutes, place in a centrifuge, and centrifuge at 12000 rpm for 8 minutes; pipette the supernatant and transfer it to a 2 mL centrifuge tube. ④ Add an equal volume of chloroform-isoamyl alcohol, mix well and shake for 5-8 minutes, place in a centrifuge, centrifuge at 12000 rpm for 8 minutes, and use a pipette to transfer the supernatant to a new 2 mL centrifuge tube. ⑤ Add 0.7 times the volume of the supernatant to the centrifuge tube and pre-cooled isopropanol at -20℃. Place the tube in a -20℃ freezer for 2 hours to settle. Then, place the centrifuge tube in a centrifuge and centrifuge at 12,000 rpm for 8 minutes. Discard the waste liquid to obtain genomic DNA. ⑥ Add 1 mL of 70% ethanol to a centrifuge tube, invert the tube to wash the DNA, then centrifuge at 12000 rpm for 3 min, discard the waste liquid, and repeat the operation once. ⑦ Add 1 mL of anhydrous ethanol to the centrifuge tube, invert the tube to wash the DNA, then centrifuge at 12000 rpm for 3 min, discard the waste liquid, and repeat the operation once. ⑧ Dry the obtained DNA at room temperature, then add 100 μL ddH2O to a centrifuge tube to dissolve the DNA. Store the resulting DNA solution in a -20°C refrigerator for later use.

[0055] (2) DNA extraction from self-made Angelica sinensis blood-nourishing pills DNA was extracted from the self-made Danggui Buxue Wan (Angelica Blood-Nourishing Pill) using the CTAB method combined with column purification. The extraction steps are as follows: (1) Grind the self-made Angelica Blood-Nourishing Pills into a fine powder and weigh out 2g; (2) Add 2g of fine powder to 10 mL of 4× CTAB extraction buffer (two-thousandths of the CTAB volume of 1 M DTT) preheated at 65℃, and place in a 50 mL centrifuge tube. Incubate at 65℃ for 1.5 h, shaking intermittently during the process. (3) After the water bath, the sample was cooled to room temperature, and an equal volume of chloroform-isoamyl alcohol mixed solution (volume ratio of 24:1) was added. The mixture was mixed for about 5 min and then centrifuged at 12,000 rpm for 8 min. The supernatant was then transferred to a 50 mL centrifuge tube using a pipette. (4) Repeat step (3) (5) Add 0.7 times the volume of the supernatant to the centrifuge tube and pre-cooled isopropanol at -20℃. Place the tube in a -20℃ refrigerator to settle for 2 h. Then, place the centrifuge tube in a centrifuge and centrifuge at 12000 rpm for 8 min. Discard the waste liquid to obtain genomic DNA. (6) Add 1 mL of 70% alcohol to the centrifuge tube to wash the DNA, then centrifuge at 12000 rpm for 3 min, discard the waste liquid, and repeat the operation once. (7) Add 1 mL of anhydrous ethanol to the centrifuge tube, invert the tube to wash the DNA, then centrifuge at 12000 rpm for 3 min, discard the waste liquid, and repeat the operation once. (8) After purifying DNA using a universal DNA purification and recovery kit (Tiangen, DP 214), add 100 uL ddH2O to dissolve and store at -20℃.

[0056] 2.2.2 Design of primers and probes for specific identification of Astragalus membranaceus In this embodiment, ITS sequence-specific primers for identifying Astragalus membranaceus were designed. Based on the target gene region amplified, specific probes were designed using the Primer 3 v.0.4.0 online website (https: / / bioinfo.ut.ee / primer3-0.4.0 / ). 6-FAM was used as a reporter gene and labeled at the 5' end of the probe, and BHQ-1 was used as a quenching gene and labeled at the 3' end. The primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd., China. The primer and probe sequences are shown in Table 3.

[0057] Table 3 Primer and probe sequences for specific identification of Astragalus membranaceus

[0058] 2.2.3 Preparation of plasmid standards In this embodiment, the genomic DNA of Astragalus membranaceus was amplified by conventional PCR using the primers (HQ-F / R) shown in Table 3. The amplified product (381 bp) was cloned into the pMD19-T plasmid (Takara, Dalian), which was then transfected into Escherichia coli and cultured. The plasmid was then extracted and purified using a plasmid purification kit (Omega, Bio-Tek, USA). The plasmid samples were prepared by Guangzhou Qianxun Biotechnology Co., Ltd. The plasmid concentration was determined using a NanoDrop 2000 (Thermo, USA). The theoretical copy number concentration of the plasmid standard was calculated using the following formula: N (copies / µL) = (6.02×10 23 )×(C ng / µL×10 -9 ) / (Plasmid DNA length×660g / mol) = 5.68×10 10 (C = 191.8 ng / µL; Plasmid DNA length = 3075 bp).

[0059] 2.2.4 ddPCR reaction system and procedure The total ddPCR reaction volume was 20 µL, including 10 µL of ddPCR premix, 1.8 µL each of forward and reverse primers (800 nM), 0.5 µL of probe (250 nM), 2 µL of DNA template, and the remainder made up to 20 µL with sterile ddH2O. The 20 µL mixture was added to a QX200 droplet generator (Bio-Rad, USA) along with 70 µL of droplet-generating oil to create water-in-oil droplets. The droplets were then transferred to a 96-well PCR plate, sealed with a PX1™ PCR plate, and the ddPCR reaction was performed using a C1000 PCR thermal cycler (Bio-Rad, USA). The reaction program was as follows: initial incubation at 95°C for 10 min, 40 cycles (94°C, 30 s; 55°C–65°C, 120 s), followed by incubation at 98°C for 10 min. After the PCR reaction was completed, the amplification signal was read using a QX 200™ droplet reader, and the results of positive and negative droplets were read using QuantaSoft v1.7.4 to obtain the DNA copy number concentration.

[0060] 2.2.5 Establishment of ddPCR detection method 2.2.5.1 Specificity verification of primers and probes To verify whether the designed primers and probes could achieve specific amplification of Astragalus membranaceus, DNA was extracted from three batches of genuine Astragalus membranaceus (ZY1, DT1, GM1) and four batches of adulterants (Caragana korshinskii: JJE1; Althaea rosea: SK1; Alfalfa: ZMX1; Astragalus membranaceus SWZ1) samples, and ddPCR amplification was performed using these samples as DNA templates. The ddPCR reaction system and procedure were the same as in "2.2.4".

[0061] The results of ddPCR amplification are shown below. Figure 1 (Blue and gray droplets represent positive and no amplification signals, respectively; yellow vertical dashed lines separate each sample reaction well.) The figure shows that genuine Astragalus membranaceus (ZY1, DT1, GM1) can be detected with a large number of positive droplets, while adulterants (Caragana korshinskii: JJE1; Althaea rosea: SK1; Alfalfa: ZMX1; Astragalus membranaceus 'flat stem': SWZ1) show no positive droplets (see...) Figure 1 The results of ddPCR verification of primer and probe specificity were consistent with the previous results of conventional PCR verification, indicating that the designed primers and probes have high specificity for identifying genuine Astragalus membranaceus.

[0062] 2.2.5.2 Annealing Temperature Optimization The original concentration of the plasmid standard was 5.68 × 10⁻⁶. 10 Copies / µL diluted to a final concentration of 5.68 × 10⁻⁶. 5The DNA template for ddPCR amplification was obtained by using copies / µL of DNA. The gradient annealing temperature was set to 65℃~55℃. The optimal annealing temperature (or temperature range) for ddPCR was determined based on the clustering of positive and negative droplets.

[0063] The experimental results are shown in Figure 2 The plasmid standard DNA was successfully amplified within a temperature range of 55℃ to 65℃. Fluorescence signal separation between positive and negative droplets occurred at 64.5℃ and persisted until 59℃, after which a plateau phase was reached. The separation degree between positive and negative droplets was optimal between 55℃ and 59℃. In subsequent linear response and sensitivity tests of the ddPCR instrument, 59℃ was selected as the annealing temperature for ddPCR amplification.

[0064] 2.2.5.3 Linear response of ddPCR instrument Plasmid standard (initial concentration: 5.68 × 10⁻⁶) was prepared. 10 (copies / µL) diluted to 5.68×10 5 Starting from this level, perform 5-fold serial dilutions to obtain the following 8 concentrations of plasmid DNA: 5.68 × 10⁻⁶ copies / μL. 5 copies / μL, 1.136×10 5 copies / μL, 2.772×10 4 copies / μL, 4.544×10 3 copies / μL, 9.08×10 2 copies / μL, 1.92×10 2 DNA at concentrations of 36 copies / μL, 36 copies / μL, and 7 copies / μL were used as templates for ddPCR amplification. The ddPCR reaction system and procedure were as described above. The DNA copy number concentrations of the ddPCR amplification products were logarithmically transformed to the eight theoretical copy number concentrations of the DNA template, and then a scatter plot was plotted in SPSS v13.0 and linearly fitted to evaluate the linear response capability of the ddPCR instrument.

[0065] The experimental results are shown in Figure 3 ,in Figure 3 A represents the theoretical copy number concentration of DNA from eight plasmid standards and the actual DNA copy number measured after ddPCR amplification. Figure 3 B represents the linear relationship between the theoretical copy number of plasmid DNA (lg) and the measured copy number (lg). The graph shows that diluting the plasmid DNA to a concentration within the theoretical copy number range of 5.68 × 10⁻⁶ results in a linear relationship. 5Eight concentrations of DNA, ranging from 7 copies / μL to 6930 copies / μL, were amplified by ddPCR. The measured DNA copy number concentrations of the amplified products ranged from 0 copies / μL to 6930 copies / μL. Figure 3 A). A scatter plot of log10 plasmid DNA theoretical copy number concentration versus measured DNA copy number concentration was plotted and linearly fitted. The results showed a good linear relationship between the two. Y = 0.884 X -1.195, R 2 = 0.9937), Figure 3 B) indicates that it has a high linear response capability.

[0066] 2.2.5.4 ddPCR Sensitivity The initial concentration of Danggui Buxue Wan (ZP) DNA sample was prepared at 21.3 ng / µL and serially diluted 10-fold to obtain concentrations from 21.3 ng / µL to 2.0 × 10⁻⁶. -5 DNA samples at seven concentration gradients (ng / µL) were used as templates for ddPCR amplification at the optimal annealing temperature selected under section "2.2.5.2". The lower limit of DNA concentration (sensitivity) for ddPCR detection was determined based on the lower limit of DNA copy number of the amplified product.

[0067] The experimental results are shown in Figure 4 The template DNA concentration of Danggui Buxue Wan (ZP) was 2.1 ng / μL ~ 2.0 × 10⁻⁶. -3 At a concentration of ng / μL, the DNA copy number detected by ddPCR amplification products ranged from 5020 copies / μL to 14.1 copies / μL. When the DNA concentration of Danggui Buxue Wan (ZP) was 2.0 × 10⁻⁶ ng / μL... -4 At a concentration of ng / μL, no positive droplets were detected in the ddPCR amplification products, indicating no amplification signal. This suggests that the detection limit of ddPCR is 2.0 × 10⁻⁶. -3 ng / μL.

[0068] 2.2.6 Molecular quantification of Astragalus membranaceus in six batches of prepared Danggui Buxue Wan (Angelica Blood-Nourishing Pills) was performed using the established ddPCR method. The established ddPCR method was used to detect the target DNA copy number of Astragalus membranaceus in six batches of Danggui Buxue Wan samples with different Astragalus membranaceus feed ratios (triple replicates per batch). ddPCR amplification was performed under the optimal annealing temperature conditions screened in section "2.2.5.2" to detect the target DNA copy number. Linear regression analysis of the target DNA copy number of Astragalus membranaceus in the six batches of Danggui Buxue Wan and its feed ratio was performed using SPSS v13.0.

[0069] The target DNA copy number concentration of Astragalus membranaceus in six batches of Dang Gui Bu Xue Wan ranged from 551.00 copies / μL (Astragalus membranaceus content was 100%) to 0.00 copies / μL (Astragalus membranaceus content was 0%). The coefficient of variation of the target DNA copy number concentration of Astragalus membranaceus in each batch of Dang Gui Bu Xue Wan was between 0% and 20.47%, all less than 25% (see Table 4). The Astragalus membranaceus content (…) X ) and DNA copy number ( Y The scatter plot of the data was linearly fitted, and the regression equation was: Y = 5.56449 X ( R 2 = 0.9941), indicating that the DNA copy number concentration increased significantly with the increase of Astragalus membranaceus dosage in Danggui Buxue Wan (see...). Figure 5 This indicates that the copy number of Astragalus target DNA in Danggui Buxue Wan can reflect the amount of Astragalus added.

[0070] Table 4. Copy number of Astragalus target DNA in six batches of Danggui Buxue Wan with different Astragalus feeding ratios. n =3)

[0071] 2.2.7 Determination of astragaloside A content in six batches of prepared Danggui Buxue Wan by HPLC The content of astragaloside IV, a quality marker, in six batches of Danggui Buxue Pills with different Astragalus membranaceus feed ratios was determined by HPLC (Fan Jiaer, Liu Yinrong, Chao Zhi, et al. Establishment of fingerprint chromatogram and determination method of three indicator components in Danggui Buxue Pills [J]. China Pharmacy, 2022, 33(11):1343-1347+1354.). Each batch of Danggui Buxue Pills was tested three times. Astragaloside IV standard was purchased from Beijing Ruida Henghui Technology Development Co., Ltd. (batch number: 110781-202118, purity ≥96.9%). An Agilent 1260 series HPLC system (Agilent, USA) equipped with an Alltech 3300 ELSD (Alltech Associates, USA) was used. The mobile phase was HPLC grade, using acetonitrile (A)-0.2% formic acid aqueous solution (B) as the mobile phase for gradient elution: (0–10 min, 5→5% (A); 10–20 min, 5→25% (A); 20–30 min, 25→33% (A); 30–45 min, 33→60% (A); 45–50 min, 60→65% (A)). The sample injection volume was 20 µL, the ELSD detector drift tube temperature was 80 °C, the nitrogen flow rate was 1.5 L / min, and the gain was set to 4. Preparation of the standard solution: Accurately weigh 8.80 mg of astragaloside IV standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol to obtain a stock solution (concentration 880 µg / mL). Transfer 1.0 mL of the stock solution to a 10 mL volumetric flask and dilute to the mark with methanol to obtain the astragaloside IV standard solution (88.0 µg / mL). The preparation of the test sample solution was based on non-patent literature (Fan Jiaer, Liu Yinrong, Chao Zhi, et al. Establishment of fingerprint spectrum and content determination method of three index components of Danggui Buxue Pill [J]. China Pharmacy, 2022, 33(11):1343-1347+1354).

[0072] The content of astragaloside IV in six batches of Danggui Buxue Wan decreased with the decrease of the proportion of Astragalus membranaceus added, ranging from 0.487 mg / g to 0.000 mg / g. A linear fit of the scatter plot of Astragalus membranaceus addition (X) and astragaloside IV (Y) (Y = 0.0045X + 0.015, R² = 0.9824) indicates that the content of astragaloside IV can reflect the amount of Astragalus membranaceus added to Danggui Buxue Wan.

[0073] Table 5. Content of Astragaloside IV in Six Batches of Danggui Buxue Pills (n=3)

[0074] 2.2.8 Correlation analysis between molecular quantification and chemical quantification results Normal distribution tests were performed on the copy number of Astragalus target DNA and the content of astragaloside IV in six batches of Danggui Buxue Wan (Angelica Blood-Nourishing Pill) to determine a suitable correlation analysis method (Pearson or Spearman) between molecular quantification and chemical quantification. After determining the analytical method, correlation analysis was conducted between the two methods. To evaluate the consistency between the measurement results of ddPCR and HPLC, the intraclass correlation coefficient (ICC) between the copy number of log-transformed Astragalus target DNA and the content of astragaloside IV in the six batches of Danggui Buxue Wan was calculated. The ICC value ranged from 0 to 1; generally, ICC ≤ 0.4 indicated poor reliability, while ICC ≥ 0.75 indicated high reliability.

[0075] The Shapiro-Wilk normality test results showed that the content of astragaloside IV and the concentration of astragalus target DNA copy number in the six batches of Danggui Buxue Wan were all normally distributed. P >0.05 (see Table 6 and Figure 7 Therefore, Pearson correlation analysis was performed on the two, and the results showed that astragaloside A content was significantly positively correlated with target DNA copy number concentration. r = 0.986, P <0.001), which also indicates that the DNA copy number can reflect the content of astragaloside IV, a quality marker in Danggui Buxue Wan.

[0076] Table 6. Shapiro-Wilk test and Pearson correlation analysis of astragaloside IV content and astragalus target DNA copy number concentration in six batches of Danggui Buxue Wan.

[0077] The intergroup correlation coefficient (ICC) between the copy number of Astragalus target DNA and the content of astragaloside IV in six batches of Danggui Buxue Pills was 0.8160 (95% confidence interval: 0.5896–0.9581). The ICC was greater than 0.75, indicating that the quantitative detection results of Astragalus in Danggui Buxue Pills by ddPCR and HPLC methods have high consistency.

[0078] In summary, the specific primers and probes in this invention not only demonstrate high specificity in agarose gel electrophoresis detection when using conventional PCR to amplify Astragalus membranaceus and its adulterants (Astragalus membranaceus shows a single, clear electrophoretic band, while the adulterants show no amplification band), but also, when combined with fluorescent probes and used in ddPCR to amplify genuine Astragalus membranaceus and its adulterants, only genuine Astragalus membranaceus amplifies positive droplets, further demonstrating the high specificity of the designed primers and probes, enabling qualitative identification of Astragalus membranaceus. Furthermore, this invention utilizes DNA of different dilutions (21.3 ng / µL ~ 2.0 × 10⁻⁶) prepared from Angelica sinensis blood-tonifying pills. -5The sensitivity of ddPCR was tested using samples of DNA at the pg level (ng / µL), and the results showed that it could detect DNA samples at the pg level, with a detection limit of 2 × 10⁻⁶. - 3 The concentration is ng / µL, or 14.1 copies / µL. The ddPCR detection method established in this invention can achieve absolute molecular quantification of Astragalus target DNA in Danggui Buxue Pills with different Astragalus feed ratios, and can reflect the feed ratio of Astragalus raw materials. Simultaneously, the results of quantification of the quality marker astragaloside IV in Danggui Buxue Pills using the HPCL method show that the molecular quantification results are highly correlated with the chemical quantification results, and both can reflect the amount of raw materials fed (or the amount of adulteration). Therefore, the ddPCR method established in this invention can achieve qualitative and quantitative detection of raw materials in Danggui Buxue Pills in one step.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A specific primer pair for detecting Astragalus membranaceus in traditional Chinese medicine preparations, characterized in that, include: For example, the upstream primer shown in SEQ ID NO.1: GCACCACGACCTCCCTTTG; The downstream primer shown in SEQ ID NO.2 is: GCCATCATTCGCCCTAAA.

2. A probe for detecting Astragalus membranaceus in traditional Chinese medicine preparations, characterized in that, The sequence of the probe is shown in SEQ ID NO:3: SEQ ID NO: 3 AACTAAAATTCGATCAATGTGCCCCGTC.

3. A method for qualitative detection of Astragalus membranaceus components in traditional Chinese medicine preparations, characterized in that, The method is based on the specific primer pair described in claim 1 and the probe described in claim 2 to detect the Astragalus component in traditional Chinese medicine.

4. The method according to claim 3, characterized in that, The specific probe is modified with a fluorescent group 6-FAM at its 5' end and a fluorescent quenching group BHQ1 at its 3' end.

5. The method according to claim 3, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted genomic DNA of the sample to be tested as a template, perform ddPCR amplification reaction based on the specific primer pair and the probe; (3) Detect ddPCR amplification products.

6. The method according to claim 5, characterized in that, In step (1), the method for extracting genomic DNA from the sample to be tested includes the following steps: ① Grind the sample to be tested; then add it to 4×CTAB extraction buffer preheated at 65℃; before preheating the 4×CTAB extraction buffer, add 0.2% volume of 1 M dithiothreitol (DTT) solution; after mixing, incubate in a 65℃ water bath for 1.5~2 h; ② After cooling, add an equal volume of chloroform-isoamyl alcohol mixed solution, mix well, and centrifuge; the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution is 24:

1. ③ Take the supernatant, add 0.7~1 volume of isopropanol pre-cooled at -20℃, precipitate at -20℃ for 1~2 h, centrifuge and discard the waste liquid to obtain crude DNA; repeat this step twice. ④ 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. Preferably, in step ①, every 2 g of the sample to be tested is ground and added to 10~15 mL of 4×CTAB extraction buffer.

7. The method according to claim 5, characterized in that, In step (2), the annealing temperature of the ddPCR amplification reaction is 55~65℃, preferably 59℃.

8. A method for quantitatively detecting Astragalus membranaceus components in traditional Chinese medicine preparations, characterized in that, Includes the following steps: DNA was extracted from traditional Chinese medicine preparations containing different proportions of Astragalus membranaceus (Huang Qi) with known feed ratios. ddPCR amplification was performed using the specific primer pair described in claim 1 and the probe described in claim 2 to determine the copy number range corresponding to different proportions of Astragalus membranaceus. DNA was extracted from the traditional Chinese medicine preparation to be tested and ddPCR amplification was performed using the specific primer pair described in claim 1 and the probe described in claim 2 to calculate the target DNA copy number of Astragalus membranaceus. The content of Astragalus membranaceus in the traditional Chinese medicine preparation to be tested was confirmed by comparison.

9. The method according to claim 8, characterized in that, The method for extracting DNA from traditional Chinese medicine preparations or the method for extracting DNA from traditional Chinese medicine preparations to be tested includes the following steps: ① Grind the sample to be tested; then add it to 4×CTAB extraction buffer preheated at 65℃; before preheating the 4×CTAB extraction buffer, add 0.2% volume of 1 M dithiothreitol (DTT) solution; after mixing, incubate in a 65℃ water bath for 1.5~2 h; ② After cooling, add an equal volume of chloroform-isoamyl alcohol mixed solution, mix well, and centrifuge; the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution is 24:

1. ③ Take the supernatant, add 0.7~1 volume of isopropanol pre-cooled at -20℃, precipitate at -20℃ for 1 h, centrifuge and discard the waste liquid to obtain crude DNA; repeat this step twice. ④ 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. Preferably, in step ①, every 2 g of the sample to be tested is ground and added to 10~15 mL of 4×CTAB extraction buffer.

10. The method according to claim 8, characterized in that, The annealing temperature for the ddPCR amplification reaction is 55~65℃, preferably 59℃.

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