Method for identifying caulis spatholobi medicinal material and adulterants thereof based on ITS2 secondary structure
Through the method based on ITS2 secondary structure, DNA is extracted and the secondary structure of the ITS2 sequence is predicted, and a systematic evolution tree is constructed to identify chicken blood vines and their mixed products, which solves the problem of high difficulty and low accuracy in the existing technology. Achieve rapid and accurate identification results.
Patent Information
- Application Number
- CN202411678593.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-02-25
AI Technical Summary
It is difficult to quickly and accurately identify chicken blood vine medicinal materials and their mixed products in the prior art. Traditional methods have limitations, such as relying on professional equipment and experience, and DNA barcode methods have limitations in sample size and amplification success rate.
The secondary structure of ITS2 was used for identification. DNA was extracted and PCR amplified, and its secondary structure was predicted after obtaining the ITS2 sequence, and a phylogenetic tree was constructed to determine the species of the sample to be tested.
It has achieved rapid and accurate identification of chicken blood vine and its mixed products, overcomes the limitations of traditional methods, has high versatility and high amplification success rate, and can effectively distinguish chicken blood vine from species that are easily confused.
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Figure CN119506458A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of identification of medicinal material origin molecules, and more specifically to a method for identifying Millettia spatholobi medicinal materials and their mixed and counterfeit products based on ITS2 secondary structure. Background Art
[0002] Millettia suberectus is the dried vine stem of the legume plant Spatholobus suberectus. It is included in the "Pharmacopoeia of the People's Republic of China (2020 Edition)" and is mainly distributed in Guangxi, Guangdong, Fujian, Yunnan and other places in my country. Modern pharmacological and clinical studies have shown that its biologically active ingredients are mainly flavonoids, among which formononetin, genistein and isoflavones can prevent or treat cancer, and catechins can promote the proliferation of hematopoietic cells. Therefore, Millettia suberectus has become the basic raw material for many Chinese patent medicines, and the market demand for its wild resources is also continuing to increase. However, unlike other legumes, Spatholobus suberectus, the original plant of Millettia suberectus, has the characteristics of long growth cycle, low fruiting rate, and poor natural reproduction ability, resulting in insufficient supply of Millettia suberectus. In addition, the artificial cultivation system of Millettia reticulata has not yet been established, and excessive collection of wild resources has led to the extinction of Millettia reticulata in my country. The strong imbalance between supply and demand has caused the price of Millettia reticulata medicinal materials to rise year by year. Driven by high profits, unscrupulous merchants have adulterated and counterfeited Millettia reticulata during processing and preparation, and sold relatively cheap plants of the same family as authentic products. Due to the inconsistency of chemical composition and pharmacological effects, mixing real and fake medicinal materials will cause patients' condition to be delayed or even life-threatening.
[0003] Traditional methods for judging the authenticity of Chinese medicinal materials include property identification, microscopic identification and physical and chemical identification. Among them, property identification mainly relies on observing the shape, color, surface characteristics, cross-section, etc. of the Chinese medicine to judge the type and quality of the Chinese medicine. Although this method is simple and rapid, it has certain limitations when identifying multi-source medicinal materials, broken medicinal materials, powdered medicinal materials and Chinese patent medicines, and it is impossible to identify objectively and accurately; microscopic identification is a method of observing the tissue structure and powder characteristics of Chinese medicine under a microscope. It mainly identifies Millettia reticulata and its mixed products through the characteristics of cork cells, cortex and phloem stone cell groups, vascular bundles, wood vessels, etc., but this method relies on professional microscopic equipment and the rich identification experience of professionals, so it has the disadvantages of low identification efficiency, high labor costs, and great difficulty in operation; physical and chemical identification is Physical or chemical methods are used to conduct qualitative or quantitative analysis on the active ingredients or characteristic components contained in traditional Chinese medicine in order to identify the authenticity and quality of the medicinal materials. For example, Yang Ranran et al. established an HPLC fingerprint analysis method for Millettia reticulata medicinal materials by examining extraction methods (reflux method, ultrasonic extraction method, Soxhlet extraction method and immersion method), optimizing chromatographic conditions (methanol-water, acetonitrile-water, methanol-formic acid aqueous solution, acetonitrile-formic acid aqueous solution, methanol-phosphoric acid aqueous solution, acetonitrile phosphoric acid aqueous solution) and HPLC fingerprint analysis. The method was used to identify Millettia reticulata medicinal materials and their counterfeits and evaluate their quality. However, the physical and chemical identification method requires researchers to have rich chemical expertise and experience in optimizing experimental conditions. Otherwise, some subtle changes in chemical content will lead to errors in the identification results.
[0004] DNA barcoding technology uses standard sequences to sequence plants. After obtaining the sequences, different plants can be classified and the distance of species relationship can be determined by constructing a phylogenetic tree. This method overcomes the limitations of traditional identification methods and enables materials with similar morphology and chemical composition to be quickly and accurately identified. At present, there are also DNA barcodes such as maturase K gene (matK) and psbA-trnH for the identification of Millettia spatholobi medicinal materials. However, these DNA barcodes identify Millettia spatholobi with a very small sample size (7-8 samples for all species) and do not draw more reliable conclusions based on a large number of samples. In addition, the sequences of matK and psbA-trnH are longer, and the failure rate of amplification and sequencing is higher, which has greater limitations in actual use. Therefore, it is of great significance to develop a new molecular identification method for Millettia spatholobi and its mixed products. Summary of the invention
[0005] In view of the above problems, the present invention applies the ITS2 secondary structure to the identification of Millettia spatholobi medicinal materials and their adulterants and verifies its effectiveness in identifying Millettia spatholobi.
[0006] In order to achieve the above object, the present invention adopts the following technical solution:
[0007] A method for identifying Millettia spatholobi medicinal materials and their mixed and counterfeit products based on ITS2 secondary structure, comprising the following steps:
[0008] (1) extracting genomic DNA of the sample to be tested, and using it as a template, performing PCR amplification using primers with the ITS2 gene of ribosomal DNA as a molecular marker, performing bidirectional sequencing on the PCR amplification product, and performing sequence splicing and correction on the sequencing results to obtain an accurate ITS2 spacer sequence as a comparison sequence, wherein the sequence of the upstream primer of the ITS2 gene is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;
[0009] (2) Perform BLAST comparison on all sequences obtained in step (1) in the NCBI database, determine the source species based on the homologous gene with the highest similarity score and the lowest E value, and cut the retrieved ITS2 sequences of Millettia spatholobi and its hybrids to obtain accurate ITS2 spacer sequences as reference sequences;
[0010] (3) predicting the secondary structures of the above-mentioned comparison sequences and reference sequences and constructing a phylogenetic tree based on the secondary structure of the ITS2 gene;
[0011] (4) The sample to be tested belongs to the species to which the ITS2 secondary structure of the sample to be tested belongs in the phylogenetic tree based on the ITS2 gene secondary structure in step (3).
[0012] Preferably, in step (1), the upstream primer sequence of the ITS2 gene shown in SEQ ID NO:1 is specifically 5'-ATGCGATACTTGGTGTGAAT-3', and the downstream primer sequence of the ITS2 gene shown in SEQ ID NO:2 is specifically 5'-GACGCTTCTCCAGACTACAAT-3'.
[0013] Preferably, the PCR amplification system described in step (1) is: 1 μL of DNA template with a concentration of 50 ng / μL, 12.5 μL of 2×TaqPCR master mix, 1 μL of each of the upstream and downstream primers with a concentration of 10 μmol / L, and 9.5 μL of ddH2O.
[0014] Preferably, the PCR amplification procedure in step (1) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s, for a total of 30 cycles; and finally extension at 72°C for 10 min and storage at 4°C.
[0015] Preferably, the splicing and correction in step (1) is specifically as follows: according to the annotation file, the primers and low-quality regions of the sequence completed by bidirectional sequencing are removed and cut out, and after obtaining the complete sequence, the 5.8S and 28S sequences are removed by using the HMMer annotation method based on the hidden Markov model of ITS2x software.
[0016] Preferably, the shearing in step (2) is to remove the 5.8S and 28S sequences using the HMMer annotation method based on the hidden Markov model using the ITS2x software to obtain an accurate ITS2 spacer sequence.
[0017] Preferably, step (3) comprises using RNAfold software to obtain the secondary structures of the comparison sequence and the reference sequence, and using LocARNA software to align the obtained secondary structures and construct a phylogenetic tree.
[0018] Through the above technical scheme, it can be known that the present invention applies the ITS2 secondary structure to the identification of Millettia spatholobi medicinal materials and their adulterants. By collecting Millettia spatholobi and its adulterants from different origins, the ITS2 sequence of the Millettia spatholobi species is constructed in combination with the adulterant sequence downloaded from GenBank, and the ITS2 secondary structure-based phylogenetic tree is used to determine that ITS2 is the optimal DNA barcode for Millettia spatholobi.
[0019] The present invention discloses a method for identifying Millettia spatholobi medicinal materials and their counterfeits based on ITS2 secondary structure. The DNA sequence of the sample to be tested is subjected to PCR amplification by nuclear ITS2 gene amplification primers to obtain the corresponding sequence, and then the secondary structure of the sample ITS2 sequence is obtained and a phylogenetic tree is constructed, and the Millettia spatholobi medicinal materials and their counterfeits are identified according to the topological analysis results. The ITS2 sequence primers of the present invention have good versatility, and their secondary structures are highly variable between species. Since the ITS2 sequence fragments are relatively small, the template requirements for participating in the PCR amplification reaction are lower, and the amplification success rate is higher. In addition, the present invention has strong repeatability, provides a new idea for the identification of Millettia spatholobi and its counterfeits, and is of great significance for solving the problem of adulteration of Millettia spatholobi medicinal materials in the medicinal material market. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1: Representative secondary structure maps of ITS2 of Millettia spatholobi and its counterfeits based on minimum free energy, including: A: Bauhinia championii, B: Callerya cinerea, C: Callerya nitida, D: Callerya dielsiana, E: Craspedolobium unijugum, F: Derris trifoliata, G: Entada phaseoloides, H: Kadsura heteroclite, I: Mucuna macrocarpa, J: Mucuna birdwoodiana, K: Wisteriopsis reticulata, L: Kadsurainterior, M: Padbruggea filipes, N: Mucuna sempervirens, O: Sargentodoxa cuneata, P: Schisandra propinqua, Q: Schisandra henryi, R: Spatholobus suberectus;
[0021] Figure 2 :The phylogenetic tree diagram of Millettia reticulata and its hybrids using ITS2 secondary structure. DETAILED DESCRIPTION
[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0023] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.
[0024] Materials and Methods
[0025] 1. Sample collection and specimen identification
[0026] Field sampling of Millettia spatholobi and plants that are easily confused with product sources was carried out in Baise, Laibin, Nanning, Qinzhou, Hechi, Guangxi Zhuang Autonomous Region, Xishuangbanna, Pu'er, Yunnan Province. The distance between individuals of the same population was greater than 50m. A total of 56 fresh leaves were collected, including Spatholobus suberectus (numbered Ss001 to Ss039), Mucuna sempervirens (numbered Ms001 to Ms006) and Craspedolobium unijugum (numbered Cu001 to Cu011), which were sealed in plastic bags with sufficient silica gel (to avoid DNA degradation) for later use. All plants were jointly identified by Huang Yunfeng, chief pharmacist, and Yan Kejian, deputy chief pharmacist, Guangxi Zhuang Autonomous Region Institute of Traditional Chinese Medicine according to traditional morphological methods. Spatholobus suerectus (specimen number: 00327831), Mucuna sempervirens (specimen number: 02014496) and Craspedolobium unijugum (specimen number: 02028691) can be found in the China Digital Herbarium (https: / / www.cvh.ac.cn / index.php).
[0027] 2. DNA extraction, amplification, sequencing and sequence assembly
[0028] (1) From each sample, 15 mg of silica gel-dried leaves were taken and genomic DNA was extracted using the FastPure Plant DNA Isolation Mini Kit (Vazyme, DC104-01) (for specific operations, see the instructions of the kit). The quality and concentration of the genomic DNA extracted from each sample were measured using a spectrophotometer (NanoDrop 1000, ThermoFisher). The DNA solution of each sample was diluted or concentrated to about 50 ng / μL for later use.
[0029] (2) Using ITS2-2F (5'-ATGCGATACTTGGTGTGAAT-3', SEQ ID No.1) and ITS2-3R: (5'-GACGCTTCTCCAGACTACAAT-3', SEQ ID No.2) as primers and the DNA solution (50 ng / μL) of the above samples as templates, PCR amplification was performed respectively. The PCR amplification system was: 1 μL template DNA (50 ng / μL), 12.5 μL 2× TaqPCR master mix (Vazyme, P112-01), 1 μL of 10 μmol / L ITS2-2F / ITS2-3R primers, and 9.5 μL ddH2O. The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s, for a total of 30 cycles; and finally extension at 72°C for 10 min and storage at 4°C.
[0030] (3) All PCR products were detected by agarose gel electrophoresis, and the gel was photographed by a fully automatic multifunctional imaging system (Tanon, 5200Multi). The DNA was purified using Gel DNA Extraction Mini Kit (Vazyme, DC301-01), and the reaction mixture was bidirectionally sequenced on an ABI 3130xl automatic sequencer (Applied Biosystems).
[0031] (4) The sequencing peak graph was spliced and aligned using Codon Code Aligner 8.0.2 software. The primers and low-quality regions of the sequenced ITS2 sequence were removed and cut according to the annotation file to obtain the complete sequence. The 5.8S and 28S sequences were removed using the HMMer annotation method based on the hidden Markov model using ITS2x software to obtain the accurate ITS2 interval sequence, which is the comparison sequence. The specific sequence information is shown in Table 1.
[0032] Table 1 ITS2 interval sequence information of field samples of Millettia spatholobi and plants that are easily confused with product sources
[0033]
[0034]
[0035]
[0036] 3. Characteristic comparison and genetic analysis
[0037] (1) The ITS2 sequences of P. densiflora, P. truncatula and P. crotonii shown in Table 1 were submitted to the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / , accession number: PP465924-PP465979, retrieval time: March 12, 2024). All obtained sequences were aligned to the National Center for Biotechnology Information (NCBI) database using BLAST, and the source species were determined based on the homologous gene with the highest similarity score and the lowest E value. The search found Calleryadielsiana (specimen number: 02015435), Sargentodoxa cuneata (specimen number: 02108794), Mucuna birdwoodiana (specimen number: 02098429), Callerya cinerea (specimen number: 01924183), Kadsura interior (specimen number: 02015552), Kadsura heteroclite (specimen number: 02231169), Wisteriopsis reticulata (specimen number: 01988791), Derris trifoliata, specimen number: 01924020), Entadaphaseoloides, specimen number: 02074730), Mucuna macrocarpa, specimen number: 01781725, Padbruggeafilipes, specimen number: 1289072, Bauhinia championii, specimen number: 01965870, Callerya nitida, specimen number: 01965870, Schisandra propinqua, specimen number: 02231152, Schisandra henryi, specimen number: 02231143. A total of 236 ITS2 sequences from the above 15 false species and Bauhinia, Entadaphaseoloides, and Mucuna macrocarpa were downloaded from GenBank. The ITS2x software was used to remove the 5.8S and 28S sequences using the HMMer annotation method based on the hidden Markov model to obtain the accurate ITS2 interval sequence, which is the reference sequence. The specific information is shown in Table 2.
[0038] Table 2 ITS2 interval sequences of 15 counterfeit species and Cryophyllum densiflorum, Glehnia littoralis, and Croton tiglium in the NCBI database
[0039]
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048] The secondary structure of ITS2 of Millettia reticulata and its counterfeits was obtained using RNAfold software. The specific parameters were as follows: the folding algorithm was the minimum free energy method and isolated base pairs were avoided. Under any conditions, both sides of the helix had dangling energy. The energy parameter selected RNA, and the modified base selection forced the known reverse transcriptase blocking bases to remain unpaired. Representative secondary structure maps were selected for comparative analysis. Figure 1A-1R As shown in Figure 1, there are obvious differences in the length and number of arms and the size and number of loops in the ITS2 secondary structure between Millettia reticulata and other species.
[0049] The obtained ITS2 secondary structures were aligned and a phylogenetic tree was constructed using the LocARNA software. The alignment scoring parameters were set as follows: structure weight of 200, openness score of insertion or deletion sequence of small fragments of -800, insertion or deletion sequence score of small fragments of -50, match score of 50, and mismatch score of 0. The RNA folding parameters were set as follows: the maximum base pair span of local folding was 150, the temperature was 37°C, and the energy parameter model selected the Turner model (2024). The heuristic method for speed / accuracy trade-off was set as follows: the minimum pairing probability was 0.0005, the minimum probability of constructing a guide tree was 0.005, the maximum difference in matching arc size was 30, and the maximum difference in alignment edges was 60. Other parameters were set to prohibit isolated pairs. The phylogenetic tree is shown in Figure 2. Figure 2 As shown in the figure, at the secondary structure level, the phylogenetic tree constructed based on the ITS2 secondary structure can cluster the same species of Millettia reticulata in the same branch, and different species from Millettia reticulata in different branches and can distinguish them from each other.
[0050] In summary, there were obvious topological differences between Millettia spatholobi and 17 easily confused species including Glechoma longituba and Croton tiglium, which demonstrated the effectiveness of ITS2 in identifying Millettia spatholobi.
[0051] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0052] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Application of ITS2 secondary structure in the identification of Millettia reticulata medicinal materials and their adulterants.
2. A method for identifying Millettia spatholobi medicinal materials and their mixed and counterfeit products based on ITS2 secondary structure, characterized in that: The following steps are involved: (1) extracting genomic DNA of the sample to be tested, and using it as a template, performing PCR amplification using primers with the ITS2 gene of ribosomal DNA as a molecular marker, performing bidirectional sequencing on the PCR amplification product, and splicing and correcting the sequencing results to obtain an accurate ITS2 spacer sequence as a comparison sequence, wherein the sequence of the upstream primer of the ITS2 gene is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2; (2) Perform BLAST comparison on all sequences obtained in step (1) in the NCBI database, determine the source species based on the homologous gene with the highest similarity score and the lowest E value, and cut the retrieved ITS2 sequences of Millettia spatholobi and its hybrids to obtain accurate ITS2 spacer sequences as reference sequences; (3) predicting the secondary structures of the above-mentioned comparison sequences and reference sequences and constructing a phylogenetic tree based on the secondary structure of the ITS2 gene; (4) The sample to be tested belongs to the species to which the ITS2 secondary structure of the sample to be tested belongs in the phylogenetic tree based on the ITS2 gene secondary structure in step (3).
3. The method for identifying Millettia spatholobi medicinal materials and their mixed and counterfeit products based on ITS2 secondary structure according to claim 2, characterized in that: The PCR amplification system described in step (1) is: 1 μL of DNA template with a concentration of 50 ng / μL, 12.5 μL of 2×Taq PCR master mix, 1 μL of each of the upstream and downstream primers with a concentration of 10 μmol / L, and 9.5 μL of ddH2O.
4. The method for identifying Millettia spatholobi medicinal materials and their mixed and counterfeit products based on ITS2 secondary structure according to claim 2, characterized in that: The PCR amplification procedure described in step (1) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s, for a total of 30 cycles; and finally extension at 72°C for 10 min and storage at 4°C.
5. The method for identifying Millettia spatholobi medicinal materials and their counterfeits based on ITS2 secondary structure according to claim 2, characterized in that: The splicing and correction in step (1) are specifically as follows: according to the annotation file, the primers and low-quality regions of the sequence completed by bidirectional sequencing are removed and cut out, and after obtaining the complete sequence, the 5.8S and 28S sequences are removed by using the HMMer annotation method based on the hidden Markov model of ITS2x software.
6. The method for identifying Millettia spatholobi medicinal materials and their counterfeits based on ITS2 secondary structure according to claim 2, characterized in that: The shearing described in step (2) is to remove the 5.8S and 28S sequences using the HMMer annotation method based on the hidden Markov model of the ITS2x software to obtain an accurate ITS2 interval sequence.
7. The method for identifying Millettia spatholobi medicinal materials and their mixed and counterfeit products based on ITS2 secondary structure according to claim 2, characterized in that: Step (3) includes using RNAfold software to obtain the secondary structures of the comparison sequence and the reference sequence, and using LocARNA software to align the obtained secondary structures and construct a phylogenetic tree.