Method, kit and server for identifying species of gentian medicinal material in mixture

Abstract

The invention discloses a method, a kit and a server for identifying species of a gentian medicinal material in a mixture. According to the invention, the optimized lysate formula significantly improves the quality of DNA extracted from a complex mixture. The reducibility of the lysate is enhanced by increasing the content of beta-mercaptoethanol, and the DNA is effectively prevented from being oxidized and damaged in the extraction process; the added protease K strengthens the digestive ability to protein, and reduces the wrapping and pollution of protein impurities to DNA (Deoxyribose Nucleic Acid); due to the addition of PVP, polyphenol substances in the mixture can be adsorbed, and degradation caused by combination of the polyphenol substances and DNA is avoided; the lysate can better cope with various interference components in a complex matrix due to the moderately improved CTAB concentration and salt concentration and stronger metal ion chelating ability, and meanwhile, the pH value of the solution is stabilized by the adjusted Tris-HCl concentration, so that the DNA is kept stable in the extraction process. Due to the improvements, the purity and the integrity of the extracted DNA are obviously improved, and a high-quality template is provided for subsequent species identification.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically a method, reagent kit, and server for identifying gentian species in mixtures. Background Technology

[0002] Gentian is the dried root and rhizome of *Gentiana manshurica* Kitag., *Gentiana scabra* Bge., *Gentiana triflora* PA reagent ll., or *Gentiana rigescens* Franch., all belonging to the Gentianaceae family. The first three are commonly referred to as "gentian," while the last is commonly referred to as "hard gentian."

[0003] Currently, there are over 100 commercially available traditional Chinese medicines containing gentian, such as Dang Gui Long Hui Wan and Long Dan Xie Gan Wan. Most use the content of gentiopicroside as a quality control indicator. Taking Long Dan Xie Gan Wan as an example, under the "Identification" section, the test is performed using thin-layer chromatography (General Rule 0502). A gentiopicroside reference standard is prepared by adding methanol to a solution containing 0.5 mg per ml. The test sample chromatogram should show spots of the same color at the corresponding positions as the reference standard chromatogram. Under the "Content Determination" section, the test is performed using high-performance liquid chromatography (General Rule 0512). The content of gentiopicroside (C16H20O9) in each 1g of the product should not be less than 0.80 mg. Gentianin is widely distributed in plants of the Gentian genus. In addition to the four original plants specified in the Chinese Pharmacopoeia, gentianin is also found in related plants such as Gentiana veitchiorum Hemsl., Gentiana lawrencei var. farreri (Balf. f.) TN Ho, and in non-related plants such as Swertia mussotii Franch., Incarvillea arguta (Royle) Royle.

[0004] Therefore, existing technologies based on gentiopicrin are insufficient for accurate identification of species in gentian medicinal materials in mixtures. When using traditional CTAB lysis buffer to treat complex mixtures during molecular identification, the reducing power, protein digestibility, and polyphenol adsorption capacity are insufficient. The detergent, salt concentration, and metal chelating capacity are also insufficient to cope with interference from complex matrices, and the pH stability is poor, resulting in low quality of extracted DNA. The commonly used 350bp library preparation is prone to failure due to DNA degradation, and the length of the fragments after splicing is insufficient, affecting the accuracy of barcode assembly. Summary of the Invention

[0005] The purpose of this invention is to provide a method, reagent kit, and server for identifying gentian species in mixtures in order to solve the problems mentioned above.

[0006] The technical solution adopted in this invention is as follows: A method for identifying gentian species in a mixture, comprising the following steps:

[0007] S1: Extract DNA from the mixture using our self-developed modified CTAB lysis buffer. The total amount of DNA extracted must be at least 1 microgram to provide a suitable template for subsequent library construction steps.

[0008] S2: PCR-free library construction was performed based on the DNA extracted in S1, with the library fragments controlled at 270bp. Then, high-throughput sequencing was used, and the sequencing data will be used for subsequent data quality control and analysis steps.

[0009] S3: The sequencing results obtained in S2 are checked for quality using FastQC, and then filtered and trimmed using Trimmomatic or FastP. The clean data after processing will then enter the splicing step.

[0010] S4: Use FLASH or PEAR to stitch together the quality control data processed in S3 to obtain the merged.fastq file, which will be used in the subsequent gentian reads extraction step;

[0011] S5: Collect ITS2 and psbA-trnH sequences of Gentiana species to construct K31-K77 K-MER libraries. Based on 95% sequence similarity, construct an ultra-lightweight reference database. Extract Gentiana sequencing reads from merged.fastq obtained in S4 to reduce computational resources for subsequent analysis. These extracted reads will be used in the hybrid assembly step.

[0012] S6: The sequencing reads extracted from S5 were mixed and assembled using MEGAHIT or MetaSPAdes software. The assembly results will be used for annotation of the core DNA barcode region.

[0013] S7: Using a core DNA barcode region annotation tool based on the HMM strategy, the core DNA barcode region is annotated from the assembly results of S6. The annotation results will then proceed to the OTU clustering step.

[0014] S8: OTU clustering is performed using 97%–99% similarity to remove sequence redundancy and obtain unique OTUs. These OTUs will be used in the credibility calculation step.

[0015] S9: Use the reads mapping method to map the gentian reads extracted in S5 to the unique OTUs obtained in S8, calculate the confidence of each OTU, remove low-confidence OTUs based on coverage and sequencing depth, and use the screened OTUs for species identification steps.

[0016] S10: Using the BLAST method, genetic distance method, and phylogenetic tree method, the OTUs screened from S9 were compared with the standard ITS2 sequence of gentian to determine whether they were gentian species, thus completing the final identification.

[0017] In a preferred embodiment, in step S1, the lysis buffer formulation includes: 200 mM Tris-HCl, 150 mM EDTA, 2.0 M NaCl, 2.5% CTAB, 3% Tween-20, 2% SDS, and 2% PVP-40, wherein 1.5% β-mercaptoethanol, 0.2 mg / mL proteinase K, and 10 μg / mL RNase A need to be added before use.

[0018] In a preferred embodiment, the DNA extraction step in step S1 is as follows:

[0019] S1-1. Take 5g of the sample containing gentian root Chinese medicine into a 50 mL sterile centrifuge tube, add 40 mL of PA reagent, place it in a constant temperature water bath shaker, shake at 180 RPM and 65℃ for 10 min until the sample is completely mixed, centrifuge at 12000 RPM for 20 min, discard the supernatant to obtain the precipitate.

[0020] S1-2. Add 4 mL of PA reagent to the above 50 mL centrifuge tube, vortex for 30 s to resuspend each particle, transfer to a 5 mL sterile centrifuge tube, centrifuge at 12000 RPM for 5 min, and discard the supernatant.

[0021] The PA reagents include: 25 mM EDTA at pH 8.0, 100 mM Tris-HCl at pH 8.0, 200 mM NaCl, and 2% PVP-40.

[0022] S1-3. Add 4 mL of PA reagent to the above 5 mL centrifuge tube, vortex for 30 s to resuspend each particle, then transfer to a 5 mL sterile centrifuge tube, centrifuge at 12000 RPM for 5 min, and discard the supernatant.

[0023] S1-4. Prepare the lysis solution according to the above formula, and add β-mercaptoethanol [final concentration 1.5%], proteinase K [final concentration 0.2 mg / mL] and RNase [final concentration 10 μg / mL] before use.

[0024] S1-5. Add 3 mL of lysis buffer to the centrifuge tube above, resuspend the particles, add 2 steel beads, and grind using a ball mill at 30 Hz for 2 min. After removing the steel beads, store the mixture at -20 ℃ until DNA extraction.

[0025] Incubate at 6.65℃ for 2 hours; centrifuge at 12000 RPM for 5 minutes, and transfer the supernatant to two new 2 mL centrifuge tubes, A and B, approximately 800 μL per tube;

[0026] S1-7. Add an equal volume (approximately 800 μL) of ice-cold isopropanol and 1 / 10 volume of CB [3M sodium acetate] to each tube, along with 10 μL of magnetic beads, and vortex mix. Incubate at -20°C for 30 min.

[0027] S1-8. Magnetic separation, remove supernatant, add 600μL of 80% ethanol to each tube for washing, vortex mix for 30s, and magnetically separate to remove supernatant;

[0028] S1-9. Add 600 μL of 75% ethanol to each tube for washing, vortex mix for 30 s, and remove the supernatant by magnetic separation;

[0029] S1-10. Open the lid and air dry in the ultra-clean workbench; add 100μL of double-distilled water to tube A and mix by suction, let stand at room temperature for 7 minutes, and then perform magnetic separation.

[0030] S1-11. Take the supernatant from tube A and transfer it to tube B. Aspirate and mix well. Let it stand at room temperature for 7 minutes. Magnetically separate the supernatant and transfer it to a new centrifuge tube. Store at -20℃.

[0031] In a preferred embodiment, in step S1, the self-developed modified CTAB lysis buffer enhances reducing power by increasing β-mercaptoethanol content, strengthens protein digestion with proteinase K, adsorbs polyphenols with PVP, and moderately increases CTAB and NaCl salt concentrations, effectively addressing interference from complex matrices in the mixture. During extraction, the sample is first pretreated with PA reagent to remove impurities, then the cells are ground and broken using a ball mill, and finally purified with magnetic beads to obtain high-quality DNA, ensuring smooth subsequent library construction.

[0032] In a preferred embodiment, in step S3, the base quality, GC content and other indicators of the sequencing data are comprehensively evaluated using the FastQC tool, and then low-quality bases and adapter sequences are removed using the Trimmomatic or FastP tool to obtain clean sequencing data, providing a reliable foundation for subsequent sequence assembly and analysis.

[0033] In a preferred embodiment, step S4 uses FLASH or PEAR software to splice the paired-end sequencing data, integrating the sequences from both ends of the same DNA fragment into a longer single sequence, forming a merged.fastq file. This increases the amount of information in the sequence and is more conducive to the subsequent screening and analysis of Gentian genus reads.

[0034] In a preferred embodiment, in step S5, ITS2 and psbA-trnH sequences of Gentiana species are collected, a K-MER library of K31 to K77 is constructed, and an ultra-lightweight reference database is established based on 95% sequence similarity. Sequencing reads belonging to Gentiana are accurately extracted from the spliced ​​sequences, and subsequent analysis is performed only on these reads, which can significantly reduce the consumption of computing resources and improve analysis efficiency.

[0035] In a preferred embodiment, in step S6, the extracted Gentian genus sequencing reads are mixed and assembled using MEGAHIT or MetaSPAdes software, splicing short sequences into longer continuous fragments, providing a complete sequence basis for subsequent annotation of core DNA barcode regions.

[0036] In a preferred embodiment, a kit for identifying gentian species in a mixture is provided. The kit is assembled with CTAB lysis buffer and other conventional reagents including: double-distilled water, phenol:chloroform:isoamyl alcohol = 25:24:1, chloroform:isoamyl alcohol = 24:1, isopropanol, sodium acetate, magnetic nanobeads, and detergent.

[0037] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0038] 1. In this invention, the optimized lysis buffer formulation significantly improves the quality of DNA extraction from complex mixtures. The increased β-mercaptoethanol content enhances the reducing power of the lysis buffer, effectively preventing DNA oxidative damage during extraction; the added proteinase K strengthens the digestibility of proteins, reducing protein contamination and encapsulation of DNA; the addition of PVP adsorbs polyphenols in the mixture, preventing them from binding to DNA and causing degradation; the moderately increased CTAB and salt concentrations, along with stronger metal ion chelating ability, allow the lysis buffer to better cope with various interfering components in complex matrices, while the adjusted Tris-HCl concentration stabilizes the solution pH, ensuring DNA stability during extraction. These improvements significantly enhance the purity and integrity of the extracted DNA, providing a high-quality template for subsequent species identification.

[0039] 2. In this invention, a 270bp library fragment length is used instead of the traditional 350bp, effectively solving the problem caused by DNA degradation in mixtures. DNA in complex mixtures often degrades due to processing or storage conditions, resulting in shorter fragments. Using a 350bp fragment often leads to library construction failure because a sufficiently long effective fragment cannot be obtained. The 270bp fragment length is more suitable for degraded DNA, significantly improving the success rate of library construction. Simultaneously, through PE sequence splicing, the originally short sequencing fragments can be integrated into longer sequences, making the subsequent assembly of DNA barcode regions more precise, reducing sequence splicing errors, and thus making species identification results more accurate and reliable, improving the stability and accuracy of the entire identification method. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the process principle of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Reference Figure 1 ,

[0043] Example 1: Preparation of reagents to complete the extraction of DNA from traditional Chinese medicine containing gentian:

[0044] DNA extraction was performed using the lysis buffer formulation described in this invention patent. The lysis buffer formulation includes:

[0045] 200 mM Tris-HCl (pH 8.0); 150 mM EDTA (pH 8.0); 2.0 M NaCl; 2.5% CTAB; 3% Tween-20; 2% SDS; 1.5% β-mercaptoethanol (add before use); 2% PVP-40 (MW 40000); 0.2 mg / mL proteinase K (add before use); 10 μg / mL RNase A (add before use);

[0046] The DNA extraction steps are as follows:

[0047] 1. Take 5g of the sample containing gentian root Chinese medicine into a 50mL sterile centrifuge tube, add 40mL of PA reagent, place it in a constant temperature water bath shaker, shake at 180RPM and 65℃ for 10min until the sample is completely mixed, centrifuge at 12000RPM for 20min, discard the supernatant to obtain the precipitate.

[0048] The PA reagents include: 25 mM EDTA at pH 8.0, 100 mM Tris-HCl at pH 8.0, 200 mM NaCl, and 2% PVP-40.

[0049] 2. Add 4 mL of PA reagent to the above 50 mL centrifuge tube, vortex for 30 s to resuspend each particle, transfer to a 5 mL sterile centrifuge tube, centrifuge at 12000 RPM for 5 min, and discard the supernatant.

[0050] 3. Add 4 mL of PA reagent to the above 5 mL centrifuge tube, vortex for 30 seconds to resuspend each particle, then transfer to a 5 mL sterile centrifuge tube, centrifuge at 12000 RPM for 5 minutes, and discard the supernatant.

[0051] 4. Prepare the lysis solution according to the above formula, and add β-mercaptoethanol [final concentration 1.5%], proteinase K [final concentration 0.2 mg / mL] and RNase [final concentration 10 μg / mL] before use.

[0052] 5. Add 3 mL of lysis buffer to the centrifuge tube (step 3) to resuspend the particles. Add 2 steel balls and grind using a ball mill at 30 Hz for 2 min. After removing the steel balls, store the mixture at -20 ℃ until DNA extraction.

[0053] Incubate at 6.65℃ for 2 hours; centrifuge at 12000 RPM for 5 minutes, and transfer the supernatant to two new 2 mL centrifuge tubes, A and B, approximately 800 μL in each tube;

[0054] 7. Add an equal volume (approximately 800 μL) of ice-cold isopropanol and 1 / 10 volume of CB [3M sodium acetate] to each tube, along with 10 μL of magnetic beads, and vortex mix. Incubate at -20°C for 30 min.

[0055] 8. Magnetic separation, remove supernatant, add 600μL of 80% ethanol to each tube for washing, vortex mix for 30s, and magnetically separate to remove supernatant;

[0056] 9. Add 600 μL of 75% ethanol to each tube for washing, vortex mix for 30 s, and remove the supernatant by magnetic separation;

[0057] 10. Open the lid and let it air dry in the clean bench; add 100μL of double-distilled water to tube A and mix by suction, let it stand at room temperature for 7 minutes, and then perform magnetic separation.

[0058] 11. Take the supernatant from tube A and transfer it to tube B. Aspirate and mix well. Let it stand at room temperature for 7 minutes. Magnetically separate the supernatant and transfer it to a new centrifuge tube. Store at -20℃.

[0059] DNA was extracted from six samples using the method described above, and the results are as follows:

[0060] Sample ID Qubit ng / ul 25D28517 53.4 25D28518 72.99 25D28519 93.32 25D28520 100.55 25D28521 82.54 25D28522 50.4

[0061] Example 2: Identification of Gentian Root Extract in Dang Gui Long Hui Wan

[0062] [Materials Description]:

[0063] Prescription: Angelica sinensis (processed with wine) 100g, Gentiana scabra (processed with wine) 100g, Aloe vera 50g, Indigo naturalis 50g, Gardenia jasminoides 100g, Coptis chinensis (processed with wine) 100g, Scutellaria baicalensis (processed with wine) 100g, Phellodendron chinense (processed with salt) 100g, Rheum palmatum (processed with wine) 50g, Aucklandia lappa 25g, Artificial musk 5g.

[0064] Preparation: Except for artificial musk, the other ten ingredients are pulverized into fine powder, ground together with musk, sieved and mixed evenly, then made into pills with water and dried at low temperature.

[0065] [Reagent Formulation]

[0066] The lysis buffer formulation and auxiliary reagents are the same as those in Study Example 1.

[0067]

Operating Steps

[0068] DNA extraction: Refer to steps 1-15 of Study Example 1;

[0069] Library construction: PCR-free method was used to construct the library (fragment length 270bp);

[0070] Sequencing: Illumina NovaSeq platform;

[0071] Data processing includes:

[0072] a. FastQC quality inspection → Trimmomatic filtration → FLASH assembly → Gentian read extraction → MEGAHIT hybrid assembly → HMM barcode annotation → 97%–99% OTU clustering → read mapping to remove low-confidence OTUs → Species identification using BLAST / genetic distance / phylogenetic tree methods.

[0073]

Key Parameters

[0074] Document excerpt: 270bp

[0075] Sequencing platform: Illumina NovaSeq

[0076] Sequencing data volume: 1.17Gb

[0077] OTU cluster similarity: 97%–99%

[0078] Barcode area: ITS2

[0079] [Data Results]:

[0080] DNA extraction (qubit assay): concentration 62.8 ng / μL, total amount 5.24 μg;

[0081] Quality rating: Level III (minor fragment contamination, suitable for database construction);

[0082] Species identification: A 234bp OTU of the Gentian genus ITS2 was obtained, and the species was identified as Gentiana rigescens.

[0083] Example 3: Species identification using Gentianae Radix et Rhizoma Decoction (Reagent kit + server)

[0084] [Materials Description]

[0085] Prescription: Gentian root 120g, Bupleurum root 120g, Scutellaria root 60g, Gardenia fruit (fried) 60g, Alisma rhizome 120g, Akebia stem 60g, Salt-processed Plantago seed 60g, Wine-processed Angelica root 60g, Rehmannia root 120g, Prepared licorice root 60g

[0086] Preparation: Grind the ten ingredients into a fine powder, sift and mix well, then make into pills with water and dry.

[0087] [Reagent Formulation]

[0088] Reagents in the kit: PA reagent, CTAB lysis buffer (LA), isopropanol (GD), sodium acetate (GN), magnetic nanobeads (NB), 80% ethanol (PW1), 75% ethanol (PW2), double-distilled water (LE);

[0089] Add the following to LA before use: β-mercaptoethanol (final concentration 1.5%), proteinase K (final concentration 0.2 mg / mL), and RNase (final concentration 10 μg / mL).

[0090]

Operating Steps

[0091] DNA extraction:

[0092] a. Take 5g of sample and add 40mL of PA reagent → shake at 180RPM / 65℃ for 10min → centrifuge at 12000RPM for 20min and discard the supernatant;

[0093] b. Wash twice with PA reagent (4 mL / time, vortex for 30 s → centrifuge at 12000 RPM for 5 min and discard the supernatant);

[0094] c. Add 3 mL of premixed LA → ball mill at 30 Hz for 2 min → store at -20 ℃;

[0095] d. Incubate at 65℃ for 2 hours → Centrifuge and aliquot the supernatant into 2mL tubes;

[0096] e. Add an equal volume of GD, 1 / 10 volume of GN, and 10 μL of NB → place at -20℃ for 30 min;

[0097] f. Wash twice with ethanol (600 μL each for PW1 and PW2, vortex for 30 s) → air dry → dissolve in LE → combine supernatants and store.

[0098] Sequencing: Illumina NovaSeq platform;

[0099] Server processing: Upload data → Click buttons 1-8 (corresponding to steps 3-10) to complete automated analysis.

[0100]

Key Parameters

[0101] Extracted parameters: Same as in research example 1;

[0102] Sequencing platform: Illumina NovaSeq;

[0103] Sequencing data volume: 1.87 Gb;

[0104] Server buttons: 1-8 correspond to steps 3-10

[0105] [Data Results]

[0106] DNA extraction (qubit assay): concentration 52.6 ng / μL, total amount 4.47 μg;

[0107] Quality rating: Level III;

[0108] Species identification: A 233bp OTU of the Gentian genus ITS2 sequence was obtained, identifying the species as Gentiana ascabra.

[0109] Each example is presented in a structured manner, strictly following the document content, covering all required modules, and ensuring that the information is accurate and clearly hierarchical.

[0110] From the above, we can conclude that:

[0111] In this invention, the optimized lysis buffer formulation significantly improves the quality of DNA extraction from complex mixtures. The increased β-mercaptoethanol content enhances the reducing power of the lysis buffer, effectively preventing DNA oxidative damage during extraction; the added proteinase K strengthens protein digestion, reducing protein contamination and encapsulation of DNA; the addition of PVP adsorbs polyphenols in the mixture, preventing them from binding to DNA and causing degradation; the moderately increased CTAB and salt concentrations, along with stronger metal ion chelating ability, allow the lysis buffer to better handle various interfering components in complex matrices, while the adjusted Tris-HCl concentration stabilizes the solution pH, ensuring DNA stability during extraction. These improvements significantly enhance the purity and integrity of the extracted DNA, providing a high-quality template for subsequent species identification.

[0112] In this invention, a 270bp library fragment length is used instead of the traditional 350bp, effectively solving the problem caused by DNA degradation in mixtures. DNA in complex mixtures often degrades due to processing or storage conditions, resulting in shorter fragments. Using a 350bp fragment often leads to library construction failure because a sufficiently long effective fragment cannot be obtained. The 270bp fragment length is more suitable for degraded DNA, significantly improving the success rate of library construction. Simultaneously, by assembling PE sequences, the originally short sequencing fragments can be integrated into longer sequences, making the subsequent assembly of DNA barcode regions more precise, reducing sequence assembly errors, and thus making species identification results more accurate and reliable, improving the stability and accuracy of the entire identification method.

[0113] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0114] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for species identification of gentian medicinal materials in a mixture, characterized in that: The method includes the following steps: S1: Extract DNA from the mixture using a modified CTAB lysis buffer. The total amount of DNA extracted must be at least 1 microgram. S2: PCR-free library construction was performed based on DNA extracted from S1, with the library fragments controlled at 270bp, and then sequencing was performed using a high-throughput platform; S3: The sequencing results obtained in S2 are checked for quality using FastQC, and then filtered and pruned using Trimmomatic or FastP. S4: Use FLASH or PEAR to stitch together the quality control data processed in S3 to obtain the merged.fastq file; S5: Collect ITS2 and psbA-trnH sequences of Gentiana species to construct K31-K77 K-MER libraries, construct an ultra-lightweight reference database based on 95% sequence similarity, and extract sequencing reads of Gentiana species from merged.fastq obtained in S4. S6: The sequencing reads extracted from S5 were mixed and assembled using MEGAHIT or MetaSPAdes software; S7: Use the core DNA barcode region annotation tool based on the HMM strategy to annotate the core DNA barcode region from the assembly results of S6; S8: OTU clustering is performed using 97%–99% similarity, and sequence redundancy is removed to obtain unique OTUs; S9: Use the reads mapping method to map the gentian reads extracted in S5 to the unique OTUs obtained in S8, calculate the confidence level of each OTU, and remove low-confidence OTUs based on coverage and sequencing depth. S10: Using the BLAST method, genetic distance method, and phylogenetic tree method, the OTUs screened in S9 were compared with the standard ITS2 sequence and psbA-trnH sequence of Gentiana to determine whether they were Gentiana species, thus completing the final identification.

2. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: In step S1, the lysis buffer formulation includes: 200mM Tris-HCl, 150mM EDTA, 2.0M NaCl, 2.5% CTAB, 3% Tween-20, 2% SDS, and 2% PVP-40, wherein 1.5% β-mercaptoethanol, 0.2mg / mL proteinase K, and 10μg / mL RNase A need to be added before use.

3. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: In step S1, the DNA extraction step is as follows: S1-1. Take 5g of the sample containing gentian Chinese patent medicine into a 50 mL sterile centrifuge tube, add 40mL of PA reagent, place it in a constant temperature water bath shaker, shake at 180RPM and 65℃ for 10min until the sample is completely mixed, centrifuge at 12000RPM for 20min, discard the supernatant to obtain the precipitate. S1-2. Add 4 mL of PA reagent to the above 50 mL centrifuge tube, vortex for 30 s to resuspend each particle, transfer to a 5 mL sterile centrifuge tube, centrifuge at 12000 RPM for 5 min, and discard the supernatant. The PA reagents include: 25 mM EDTA with a pH of 8.0, 100 mM Tris-HCl with a pH of 8.0, 200 mM NaCl, and 2% PVP-40. S1-3. Add 4 mL of PA reagent to the above 5 mL centrifuge tube, vortex for 30 s to resuspend each particle, then transfer to a 5 mL sterile centrifuge tube, centrifuge at 12000 RPM for 5 min, and discard the supernatant. S1-4. Prepare the lysis solution according to the above formula, and add β-mercaptoethanol [final concentration 1.5%], proteinase K [final concentration 0.2 mg / mL] and RNase [final concentration 10 μg / mL] before use; S1-5. Add 3 mL of lysis buffer to the centrifuge tube above, resuspend the particles, add 2 steel balls, and grind using a ball mill at 30 Hz for 2 min; after removing the steel balls, store the mixture at -20 ℃ until DNA extraction is performed; Incubate at 6.65℃ for 2 hours; centrifuge at 12000 RPM for 5 minutes, and transfer the supernatant to two new 2 mL centrifuge tubes, A and B, approximately 800 μL per tube; S1-7. Add an equal volume (approximately 800 μL) of ice-cold isopropanol and 1 / 10 volume of CB [3M sodium acetate] to each tube, along with 10 μL of magnetic beads, and vortex mix. Incubate at -20°C for 30 min. S1-8. Magnetic separation, remove supernatant, add 600μL of 80% ethanol to each tube for washing, vortex mix for 30s, and magnetically separate to remove supernatant; S1-9. Add 600 μL of 75% ethanol to each tube for washing, vortex mix for 30 s, and remove the supernatant by magnetic separation; S1-10. Open the lid and air dry in the ultra-clean workbench; add 100μL of double-distilled water to tube A and mix by suction, let stand at room temperature for 7 minutes, and then perform magnetic separation. S1-11. Take the supernatant from tube A and transfer it to tube B. Aspirate and mix well. Let it stand at room temperature for 7 minutes. Magnetically separate the supernatant and transfer it to a new centrifuge tube. Store at -20℃.

4. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: In step S1, the self-developed modified CTAB lysis buffer enhances reducing power by increasing β-mercaptoethanol content, adds proteinase K to strengthen protein digestion, adds PVP to adsorb polyphenols, and also moderately increases the CTAB concentration and NaCl salt concentration, which can effectively cope with the interference of complex matrices in the mixture. During extraction, the sample is first pretreated with PA reagent to remove impurities, then the cells are ground and broken by ball milling, and finally high-quality DNA is obtained by magnetic bead purification.

5. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: In step S3, the base quality, GC content and other indicators of the sequencing data are comprehensively evaluated using the FastQC tool, and then low-quality bases and adapter sequences are removed using the Trimmomatic or FastP tool to obtain clean sequencing data.

6. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: Step S4 uses FLASH or PEAR software to splice the paired-end sequencing data, integrating the sequences from both ends of the same DNA fragment into a longer single sequence, forming a merged.fastq file.

7. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: In step S5, ITS2 and psbA-trnH sequences of Gentiana species are collected, K-MER libraries from K31 to K77 are constructed, and an ultra-lightweight reference database is established based on 95% sequence similarity. Sequencing reads belonging to Gentiana are accurately extracted from the spliced ​​sequences.

8. The method for species identification of gentian medicinal materials in a mixture as described in claim 1, characterized in that: In step S6, the extracted Gentian genus sequencing reads are mixed and assembled using MEGAHIT or MetaSPAdes software, splicing short sequences into longer continuous fragments to provide a complete sequence basis for subsequent annotation of core DNA barcode regions.

9. A kit for species identification of gentian medicinal materials in a mixture, characterized in that: The kit uses the CTAB lysis buffer and other conventional reagents used in step S1 as described in claim 1, including: double-distilled water, phenol:chloroform:isoamyl alcohol = 25:24:1, chloroform:isoamyl alcohol = 24:1, isopropanol, sodium acetate, magnetic nanobeads, and detergent to assemble the kit.

10. A server for species identification of gentian medicinal materials in a mixture, characterized in that: The server performs all of the steps S3 to S10 as described in claim 1.

Images