Primers for multiple str typing of money white flower snake medicine, standard decoction and traditional chinese medicine formula granules, and application and identification method thereof

Abstract

The application discloses a primer for multiple STR typing of money white flower snake medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair and a third primer pair; wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO:1, and the sequence of the downstream primer thereof is shown as SEQ ID NO:2; the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO:3, and the sequence of the downstream primer thereof is shown as SEQ ID NO:4; the sequence of the upstream primer of the third primer pair is shown as SEQ ID NO:5, and the sequence of the downstream primer thereof is shown as SEQ ID NO:6. The application further discloses application of the primer and a discrimination method based on the primer. According to the application, money white flower snake, red chain snake and golden ring snake can be effectively discriminated simultaneously.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine identification, and particularly relates to a primer for multiple STR typing of Bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules, application thereof and an identification method. BACKGROUND

[0002] There are more than 200 species of snakes in China, of which more than 20 are common snakes, including Agkistrodon acutus, Zaocys dhumnades, Bungarus multicinctus, Bungarus fasciatus, Ptyas mucosus, Daboia russelii, Naja naja, Lycodon rufozonatus, Elaphe carinata, Ptyas korros, Gloydius brevicaudus, Elaphe taeniura, Elaphe moellendorffi and the like, most of which do not have clinical efficacy. Due to the similar morphologies of various snakes, it is difficult to identify medicinal materials, especially after the internal organs are removed and dried and smoked black during processing, the pattern characteristics and color on the skin are almost lost and difficult to identify, especially after being extracted with water to prepare standard decoction and further processed into formula granules, it is more difficult to effectively identify after losing the properties of medicinal materials.

[0003] Some studies have pointed out that PCR (polymerase chain reaction) method can be used to identify Bungarus multicinctus medicinal materials. However, the existing PCR method is difficult to apply due to the presence of a certain proportion of auxiliary materials in standard decoction and traditional Chinese medicine formula granules. In addition, standard decoction and traditional Chinese medicine formula granules often need to be heated for extraction during preparation, which leads to DNA degradation and difficulty in storing DNA fragments of more than 200 bp, resulting in low detection accuracy of the existing method. In addition, the existing PCR method can only identify a single species of snake, which is low in efficiency. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a primer for multiple STR typing of Bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Bungarus multicinctus, Lycodon rufozonatus and Bungarus fasciatus at the same time.

[0005] The technical problem to be solved by the present application is to provide a primer for multiple STR typing of Bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Bungarus multicinctus, Lycodon rufozonatus and Bungarus fasciatus at the same time.

[0006] The technical problem to be solved by the present application is to provide a primer for multiple STR typing of Bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Bungarus multicinctus, Lycodon rufozonatus and Bungarus fasciatus at the same time.

[0007] In order to solve the above technical problems, the present application provides a primer for multiple STR typing of money white flower medicine, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair and a third primer pair;

[0008] The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;

[0009] The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO: 3, and the sequence of the downstream primer is shown in SEQ ID NO: 4;

[0010] The sequence of the upstream primer of the third primer pair is shown in SEQ ID NO: 5, and the sequence of the downstream primer is shown in SEQ ID NO: 6.

[0011] Correspondingly, the present application also discloses the application of the above-mentioned primer in (1) or (2):

[0012] (1) identifying whether the to-be-tested sample is money white flower, red chain snake and / or gold ring snake;

[0013] (2) preparing a kit for identifying money white flower, red chain snake and / or gold ring snake;

[0014] As an improvement of the above technical solution, the money white flower is medicine, standard decoction or traditional Chinese medicine formula granules;

[0015] The red chain snake is medicine, standard decoction or traditional Chinese medicine formula granules;

[0016] The gold ring snake is medicine, standard decoction or traditional Chinese medicine formula granules.

[0017] Correspondingly, the present application also discloses the application of the above-mentioned primer in (1) or (2):

[0018] (1) identifying whether the to-be-tested sample contains money white flower, red chain snake and / or gold ring snake;

[0019] (2) preparing a kit for identifying whether the to-be-tested sample contains money white flower, red chain snake and / or gold ring snake.

[0020] Correspondingly, the present application also discloses a kit, characterized in that it comprises the above-mentioned primer.

[0021] As an improvement of the above technical solution, it further comprises a PCR premix.

[0022] Correspondingly, the present application also discloses an identification method based on the above-mentioned primer, which comprises:

[0023] extracting genomic DNA of the sample to be detected;

[0024] If the amplification product contains the DNA characteristic peak of 158-160 bp, the sample to be detected contains Lycodon acutus medicinal material, Lycodon acutus standard decoction and / or Lycodon acutus traditional Chinese medicine dispensing granules;

[0025] If the amplification product contains the DNA characteristic peak of 205-210 bp, the sample to be detected contains Lycodon rufozonatus rufozonatus medicinal material, Lycodon rufozonatus rufozonatus standard decoction and / or Lycodon rufozonatus rufozonatus traditional Chinese medicine dispensing granules;

[0026] If the amplification product contains the DNA characteristic peak of 180-185 bp, the sample to be detected contains Lycodon rufozonatus rufozonatus medicinal material, Lycodon rufozonatus rufozonatus standard decoction and / or Lycodon rufozonatus rufozonatus traditional Chinese medicine dispensing granules.

[0027] As an improvement of the above technical solution, the method for extracting genomic DNA of the sample to be detected is as follows:

[0028] The sample to be detected is taken, and CTAB precipitation liquid and protease K precipitation extraction are carried out for 2-3 times; the precipitate is taken, CTAB extraction liquid and β-mercaptoethanol are added for extraction, and then chloroform-isoamyl alcohol is added for extraction for 2-3 times; the supernatant after extraction is taken, and isopropanol or isopropanol-sodium acetate is added for precipitation extraction; the precipitate is washed, incubated and dissolved with water, and the genomic DNA of the sample to be detected is obtained.

[0029] As an improvement of the above technical solution, the method for extracting genomic DNA of the sample to be detected is as follows:

[0030] Take 0.3-0.8g of the sample to be detected, grind into powder, and place in a centrifuge tube. Add 1-1.8mL of CTAB precipitation solution and 15-25μL of proteinase K, mix evenly, heat at 50-60℃ for 45-65min, cool to room temperature, centrifuge, and discard the supernatant. Add 800-1000μL of CTAB precipitation solution and 15-25μL of proteinase K, mix evenly, heat at 50-60℃ for 45-65min, cool to room temperature, centrifuge, and discard the supernatant. Take the precipitate, add 800-1000μL of CTAB extraction solution and 5-15μL of β-mercaptoethanol, mix evenly, heat at 60-70℃ for 100-150min, cool to room temperature, take the supernatant, add an equal volume of chloroform-isoamyl alcohol mixture, mix evenly by shaking, centrifuge, take 700-800μL of the supernatant, add an equal volume of chloroform-isoamyl alcohol mixture, mix evenly by shaking, centrifuge, take 400-500μL of the supernatant, add an equal volume of isopropanol or isopropanol-sodium acetate mixture, stand at -30--20℃ for 30-60min, centrifuge, discard the supernatant, wash the precipitate with ethanol for 2-4 times, discard the supernatant, incubate the precipitate at 35-38℃ for 20-40min, after the ethanol evaporates, add 30-50μL of sterilized water to dissolve, and the genomic DNA of the sample to be detected is obtained.

[0031] As an improvement of the above technical solution, the CTAB precipitation solution comprises CTAB, Tris-HCl, EDTA and water; wherein the concentration of CTAB is 1-3%(w / v), the concentration of Tris-HCl is 80-120mmol / L, and the concentration of EDTA is 10-30mmol / L.

[0032] The CTAB extraction solution comprises CTAB, Tris-HCl, EDTA, NaCl, PVP40 and water; wherein the concentration of CTAB is 1-3%(w / v), the concentration of Tris-HCl is 80-120mmol / L, the concentration of EDTA is 10-30mmol / L, the concentration of NaCl is 1-3mol / L, and the concentration of PVP40 is 10-30%(w / v).

[0033] As an improvement of the above technical solution, the template, primer, PCR premix solution and water are mixed evenly to obtain a PCR amplification system.

[0034] The PCR amplification system is amplified according to a preset amplification program to obtain an amplification product.

[0035] The amplification product is subjected to electrophoresis analysis, and the electropherogram is recorded. If the electropherogram contains a DNA characteristic peak of 158-159bp, the sample to be detected contains money white flower snake medicinal material, money white flower snake standard decoction and / or money white flower snake traditional Chinese medicine dispensing granules.

[0036] If the DNA characteristic peak of 206-207 bp is contained in the atlas, the sample to be detected contains the medicinal material of Trimeresurus stejnegeri, the standard decoction of Trimeresurus stejnegeri and / or the traditional Chinese medicine dispensing granules of Trimeresurus stejnegeri.

[0037] If the DNA characteristic peak of 182-183 bp is contained in the atlas, the sample to be detected contains the medicinal material of Trimeresurus gramineus, the standard decoction of Trimeresurus gramineus and / or the traditional Chinese medicine dispensing granules of Trimeresurus gramineus.

[0038] As an improvement of the above technical solution, the PCR amplification system is composed of the following substances:

[0039] PCR premix 12.5 μL; first primer pair upstream primer, downstream primer each 0.3 μL, second primer pair upstream primer, downstream primer each 0.3 μL, third primer pair upstream primer, downstream primer each 0.3 μL, template 1.5 μL, ddH2O 9.2 μL.

[0040] As an improvement of the above technical solution, the PCR amplification program is:

[0041] The amplification system is pre-denatured at 92-95℃ for 4-6 min, then cycled for 30-40 times under the preset program, and finally extended at 71-75℃ for 6-8 min;

[0042] The preset program is: denaturing the amplification system at 94-96℃ for 28-32 s, then annealing at 60-62℃ for 26-32 s, and then extending at 70-73℃ for 28-35 s.

[0043] As an improvement of the above technical solution, the PCR amplification program is:

[0044] The amplification system is pre-denatured at 95℃ for 5 min, then cycled for 35 times under the preset program, and finally extended at 72℃ for 7 min;

[0045] The preset program is: denaturing the amplification system at 95℃ for 30 s, then annealing at 61℃ for 30 s, and then extending at 72℃ for 30 s.

[0046] The implementation of the present application has the following beneficial effects:

[0047] 1. The primers of the present application can perform multiplex site-specific PCR reaction, effectively identify Lichanura maura, Trimeresurus stejnegeri and Trimeresurus gramineus and their mixed systems, and improve the identification efficiency.

[0048] 2. The primers and identification method in the present application can effectively overcome the problem of auxiliary material interference in the DNA extraction process, and solve the problem that standard decoction and traditional Chinese medicine dispensing granules are difficult to identify after losing form. BRIEF DESCRIPTION OF DRAWINGS

[0049] Fig. 1 is the electrophoresis detection result diagram of each sample in Example 3; wherein N is a blank sample (ddH2O), 1-2 is Lichuan Fangfeng medicinal material, 3 is Lichuan Fangfeng standard decoction (lyophilized powder);

[0050] Fig. 2 is the electrophoresis detection result diagram of each sample in Example 3; wherein 4 is Lichuan Fangfeng standard decoction (lyophilized powder), 5-6 is Lichuan Fangfeng traditional Chinese medicine formula granules, 7 is Chixian She medicinal material;

[0051] Fig. 3 is the electrophoresis detection result diagram of each sample in Example 3; wherein 8 is Chixian She medicinal material; 9-10 is Chixian standard decoction (lyophilized powder), 11 is Jinhuan She medicinal material;

[0052] Fig. 4 is the electrophoresis detection result diagram of each sample in Example 3; wherein 12 is Jinhuan She medicinal material; 13-14 is Jinhuan standard decoction (lyophilized powder). DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with the aid of drawings and specific embodiments.

[0054] The present application provides a primer for multiplex STR typing of Lichuan Fangfeng medicinal material, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair and a third primer pair, and the specific sequences are shown in the following table:

[0055]

[0056] The above primer can perform multiplex PCR reaction in the same PCR reaction system, and has no interference between each other and is easy to amplify.

[0057] Based on the above characteristics, the primer in the present application can realize the following applications:

[0058] (1) Identifying whether the sample to be detected is Lichuan Fangfeng medicinal material, standard decoction and traditional Chinese medicine formula granules; or Chixian She medicinal material, standard decoction and traditional Chinese medicine formula granules; or Jinhuan She medicinal material, standard decoction and traditional Chinese medicine formula granules.

[0059] (2) Identifying whether the sample to be detected contains Lichuan Fangfeng and / or Chixian She and / or Jinhuan She; for example, it can be used for identifying traditional Chinese medicine compound containing Lichuan Fangfeng (such as rheumatism pain-relieving capsule).

[0060] (3) Preparing a kit for identifying Lichuan Fangfeng, Chixian She and / or Jinhuan She.

[0061] (4) a kit for identifying whether a sample to be detected contains Lichanura marmorata and / or Trimeresurus gramineus and / or Bungarus fasciatus.

[0062] Correspondingly, the application discloses a kit comprising the primers, and further comprises a PCR premix and water, wherein the PCR premix can be 2x Taq PCR Master Mix II (Tiangen Biotech (Beijing) Co., Ltd., KT201-12), but is not limited thereto.

[0063] The application further discloses an identification method based on the primers, comprising the following steps:

[0064] I. extracting genomic DNA of a sample to be detected;

[0065] The sample to be detected is provided in a solid form, and the standard soup is provided in a freeze-dried powder form (i.e., the freeze-dried powder is obtained by freeze-drying the standard soup).

[0066] Specifically, the extraction method is as follows:

[0067] (I) taking the sample to be detected, adding CTAB precipitation liquid and protease K precipitation extraction for 2-3 times to obtain a precipitate;

[0068] Specifically, 0.3-0.8 g of the sample to be detected is ground into powder, and then is placed in a centrifugal tube, 1-1.8 mL of CTAB precipitation liquid and 15-25 μL of protease K are added, and the mixture is uniformly mixed, heated at 50-60 ℃ for 45-65 min, cooled to room temperature, centrifuged, and the supernatant is discarded; 800-1000 μL of CTAB precipitation liquid and 15-25 μL of protease K are added, the mixture is uniformly mixed, heated at 50-60 ℃ for 45-65 min, cooled to room temperature, centrifuged, and the supernatant is discarded.

[0069] The CTAB precipitation liquid comprises 2% (w / v) CTAB, 100 mmol / L Tris-HCl (pH=8.0) and 20 mmol / L EDTA (pH=8.0).

[0070] (II) taking the precipitate, adding CTAB extraction liquid and β-mercaptoethanol extraction, and then adding chloroform-isoamyl alcohol extraction for 2-3 times to obtain supernatant after extraction;

[0071] Specifically, 800-1000 μL of CTAB extraction solution and 5-15 μL of β-mercaptoethanol are added to the precipitate, which is mixed uniformly and heated at 60-70 °C for 100-150 min. After cooling to room temperature, the supernatant is taken, and an equal volume of chloroform-isoamyl alcohol (24:1) mixed solution is added. After shaking and mixing, centrifugation is performed, and 700-800 μL of the supernatant is taken. An equal volume of chloroform-isoamyl alcohol (24:1) mixed solution is added, and shaking and mixing are performed after centrifugation. 400-500 μL of the supernatant is taken, and the genomic DNA of the sample to be detected is obtained.

[0072] It should be noted that in this step, after the CTAB extraction solution and β-mercaptoethanol extraction is completed, chloroform-isoamyl alcohol can be directly added, or the supernatant obtained after extraction can be taken and chloroform-isoamyl alcohol is added. Adding chloroform-isoamyl alcohol to the supernatant can improve the extraction accuracy.

[0073] The CTAB extraction solution is 1-3% (w / v) CTAB, 80-120 mmol / L Tris-HCl (pH=8.0), 10-30 mmol / L EDTA (pH=8.0), 1-3 mol / L NaCl, and 10-30% (w / v) PVP40.

[0074] (Three) The extraction solution is added with isopropanol or isopropanol-sodium acetate to precipitate the extraction, and the precipitate is washed, incubated, and then dissolved with water to obtain the genomic DNA of the sample to be detected.

[0075] Specifically, the extraction solution is taken, and an equal volume of isopropanol or isopropanol-3 mol / L sodium acetate mixed solution is added. It is placed at -30 to -20 °C for 30-60 min, and after centrifugation, the supernatant is discarded. The precipitate is washed with ethanol for 2-4 times, the supernatant is discarded, and the precipitate is incubated at 35-38 °C for 20-40 min. After the ethanol evaporates, 30-50 μL of sterilized water is added to dissolve it, and the genomic DNA of the sample to be detected is obtained.

[0076] It should be noted that when the applicant faced the extraction of genomic DNA from the standard decoction of Lachesis muta and Lachesis muta traditional Chinese medicine formula granules, the applicant tried the traditional CTAB method, chelating resin method, triton-100 method, SDS method, alkaline lysis method, DNeasy Blood&Tissue Kit method, and magnetic bead method, but all obtained insoluble precipitates. Therefore, the present inventors improved the CTAB precipitate, CTAB extraction solution, and extraction procedure on the basis of the traditional CTAB method, and obtained the above extraction method. The above extraction method can maximize the retention of genomic DNA information in the standard decoction of Lachesis muta and Lachesis muta traditional Chinese medicine formula granules, while overcoming the problem of interference of auxiliary materials.

[0077] II. Forming a PCR amplification system and performing amplification to obtain an amplification product;

[0078] Specifically, the template, the primer, the PCR premix, and the water are mixed uniformly to obtain a PCR amplification system.

[0079] More specifically, the total volume of the PCR amplification system is 25 μL, which comprises: 12.5 μL of PCR premix; 0.3 μL of the upstream primer and the downstream primer of the first primer pair, 0.3 μL of the upstream primer and the downstream primer of the second primer pair, 0.3 μL of the upstream primer and the downstream primer of the third primer pair, 1.5 μL of the template, and 9.2 μL of ddH2O.

[0080] The PCR premix can be 2×Taq PCR Master Mix II produced by Tiangen Biosciences (Beijing) Co., Ltd., but is not limited thereto.

[0081] The amplification procedure is as follows: pre-denaturation at 92-95℃ for 4-6 min; denaturation at 94-96℃ for 28-32 s, annealing at 60-62℃ for 26-32 s, and extension at 70-73℃ for 28-35 s, for 30-40 cycles; and finally extension at 71-75℃ for 6-8 min.

[0082] Preferably, the amplification procedure is as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 61℃ for 30 s, extension at 72℃ for 30 s, for 35 cycles, and finally extension at 72℃ for 7 min.

[0083] (3) analyzing the amplification product;

[0084] Specifically, the amplification product can be analyzed by electrophoresis or fluorescent staining, but is not limited thereto.

[0085] Preferably, the PCR amplification product is detected by ABI3730xl capillary electrophoresis, and the results are recorded.

[0086] The application will be described below with specific examples.

[0087] Example 1: primer design

[0088] Based on the NADH; Cytochrome Oxidase I; 12S rRNA; 16S rRNA; Cytochrome b sequence analysis of Chrysomya megacephala and common snakes in the Chinese Pharmacopoeia 2020 edition, the Cytochrome Oxidase I sequences of common snake species in the GeneBank database were homologously aligned using BioEdit software, and the specific SNP sites of Chrysomya megacephala, Trimeresurus stejnegeri, and Daboia russelii were analyzed after proofreading. The base sequence containing the SNP site was introduced into the Primer Premier 5 software for primer design.

[0089] By sequence comparison, it was found that: Lichanura muriel has T at the 581st Lichanura muriel specific site, while others have C; Oxybelis acutus has G at the 390th Oxybelis acutus specific site, while others have A, T or C; Bungarus flavoviridis has T at the 99th Bungarus flavoviridis specific site, while others have C or A. After determining the SNP site, the SNP site is close to the 3' end of the forward primer, the upstream primer position is moved, the primer score and GC content are adjusted, and the reverse primer position is further adjusted by means of Primer Premier 5 software, and the best combination is determined by the final primer score and product band score. In addition, during the design process, the damage to the DNA base sequence after high-temperature extraction also needs to be considered.

[0090] Based on the above factors, the obtained primer combination is as follows:

[0091] JQBH.F (6-Fam): 5'-GGAGCAATCAATTTTATTACAACAT-3';

[0092] JQBHS.R: 5'-GATCGGTTAAAAGTATTGTAACTGC-3';

[0093] CLS.F (6-Fam): 5'-ACCGTGTACCCGCCACTG-3';

[0094] CLS.R: 5'-AAGCATGATAGCAGTAATTAGCACA-3'

[0095] JHS.F (6-Fam): 5'-TCTAGGTAGCGATCAAATCTTTAAC-3';

[0096] JHS.R: 5'-GAAGAAGTGCTGGTGGGAGT-3'.

[0097] Example 2 Identification method establishment

[0098] (1) DNA extraction and concentration adjustment

[0099] Take dry sample 0.3g, grind into powder, place in 2mL centrifuge tube, add 1.5mL preheated at 56℃ CTAB precipitate, 20μL protease K, mix, 56℃ water bath heating for 60min, cool to room temperature, centrifuge at 10000r / min for 5min, discard supernatant, add 900μL CTAB precipitate, 20μL protease K, and operate in the same way. Add 900μL CTAB extract, 10μL β-mercaptoethanol to the centrifuge tube in turn, mix, heat at 65℃ water bath for 120min, centrifuge, take the supernatant after cooling to room temperature; add equal volume of chloroform-isoamyl alcohol (24:1), shake for 3min, mix; centrifuge at 12000r / min, 4℃ for 10min, take 750μL supernatant and add to a new 2mL centrifuge tube, add equal volume of chloroform-isoamyl alcohol (24:1), shake for 3min, mix, centrifuge at 12000r / min, 4℃ for 10min; take 450μL supernatant in a 1.5mL centrifuge tube, add equal volume of isopropanol, stand at -20℃ for 30-60min. Take out the centrifuge tube and centrifuge at 12000r / min for 5min, discard the supernatant, wash the precipitate with 75% ethanol and anhydrous ethanol respectively twice, discard the supernatant, incubate the precipitate at 37℃ for 30min, add 30μL sterilized water to dissolve after evaporation of ethanol.

[0100] The CTAB precipitate is composed of 2%(w / v) CTAB (Shanghai Yuanye Biotechnology Co., Ltd.), 100mM Tris-HCl (pH=8.0) (Beijing Solarbio), and 20mM EDTA (pH=8.0) (Shanghai Yu Bo Biotech Co., Ltd.).

[0101] The CTAB extract is composed of 2%(w / v) CTAB (Shanghai Yuanye Biotechnology Co., Ltd.), 100mM Tris-HCl (pH=8.0) (Beijing Solarbio), 20mM EDTA (pH=8.0) (Shanghai Yu Bo Biotech Co., Ltd.), 2.5mol / L NaCl (Xilong Scientific Co., Ltd.), and 20% PVP40 (Shanghai Yuanye Biotechnology Co., Ltd.).

[0102] Take the above DNA sample, use BioSpec-nano micro ultraviolet spectrophotometer to determine the DNA concentration, record OD260 / OD230 and OD260 / OD280 at the same time, and adjust the concentration to 100ng / μL to obtain the DNA template.

[0103] (2) Design primers

[0104] The primer sequences are shown as SEQ ID NO: 1-6.

[0105] (3) PCR amplification

[0106] PCR amplification system: 2x Taq PCR Master Mix II (Tiangen Biochemical Technology (Beijing) Co., Ltd., KT201-12) 12.5 μL, 10 μmol / L JQBHS.F (6-Fam) primer 0.3 μL, 10 μmol / L JQBHS.R primer 0.3 μL, 10 μmol / L CLS.F (6-Fam) primer 0.3 μL, 10 μmol / L CLS.R primer 0.3 μL, 10 μmol / L JHS.F (6-Fam) primer 0.3 μL, 10 μmol / L JHS.R primer 0.3 μL, 100 ng / μL DNA template 1.5 μL, deionized water 9.2 μL. The above liquids are mixed uniformly by oscillation, and then centrifuged instantly to obtain the PCR amplification system.

[0107] PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles; finally 72℃ extension for 7 min.

[0108] (4) Electrophoresis detection

[0109] The amplification product is detected by ABI3730xl capillary electrophoresis, and the results are recorded.

[0110] If the spectrum contains a DNA characteristic peak of 158-159 bp, the sample to be detected contains Lichanura muda medicinal materials, Lichanura muda standard decoction and / or Lichanura muda traditional Chinese medicine formula granules; if the spectrum contains a DNA characteristic peak of 206-207 bp, the sample to be detected contains Lichanura muda medicinal materials, Lichanura muda standard decoction and / or Lichanura muda traditional Chinese medicine formula granules; if the spectrum contains a DNA characteristic peak of 182-183 bp, the sample to be detected contains Lichanura muda medicinal materials, Lichanura muda standard decoction and / or Lichanura muda traditional Chinese medicine formula granules.

[0111] Example 3 verification of identification method

[0112] Select samples of Lichanura muda, Lichanura muda and Lichanura muda (specifically as shown in Table 1).

[0113] The identification method established in Example 2 is used to identify the samples.

[0114] Table 1 Sample Table

[0115]

[0116] The identification results are as follows Figs. 1-4As shown in the figure, the money white flower snake medicine, money white flower snake standard decoction (freeze-dried powder), money white flower snake traditional Chinese medicine formula granules all exist 158-159bp characteristic peak; while other snake samples are not obtained the characteristic peak. As shown in the figure, the red chain snake medicine, red chain snake standard decoction (freeze-dried powder) all exist 206-207bp characteristic peak; while other snake samples are not obtained the characteristic peak. As shown in the figure, the gold ring snake medicine, gold ring snake standard decoction (freeze-dried powder) all exist 182-183bp characteristic peak; while other snake samples are not obtained the characteristic peak. The results show that the accuracy of the identification method in the application reaches 100%.

[0117] The above is the preferred embodiment of the application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements are also considered to be within the scope of the present application.

Claims

1. A method for identifying the medicinal material *Snakehead sinensis*, standard decoctions, and traditional Chinese medicine granules, characterized in that... include: Take the sample to be tested, add CTAB precipitation solution and proteinase K precipitation extraction 2-3 times; take the precipitate and add CTAB extraction solution and β-mercaptoethanol extraction, then add chloroform-isoamyl alcohol extraction 2-3 times; take the supernatant after extraction, add isopropanol or isopropanol-sodium acetate precipitation extraction, wash the precipitate, incubate and dissolve it in water to obtain the genomic DNA of the sample to be tested; Using the genomic DNA as a template, PCR amplification was performed using the first primer pair, the second primer pair, and the third primer pair. If the amplification product contains a DNA characteristic peak of 158-160 bp, then the sample to be tested contains the medicinal material of Snakehead alopecuroides, the standard decoction of Snakehead alopecuroides, and / or the traditional Chinese medicine formula granules of Snakehead alopecuroides. If the amplification product contains a DNA characteristic peak of 205-210bp, then the sample to be tested contains red-chain snake medicinal material, red-chain snake standard decoction and / or red-chain snake Chinese medicine formula granules. If the amplification product contains a DNA characteristic peak of 180-185p, then the sample to be tested contains *Gynostemma pentaphyllum* medicinal material, *Gynostemma pentaphyllum* standard decoction, and / or *Gynostemma pentaphyllum* traditional Chinese medicine formula granules. The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2; The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO: 3, and the sequence of its downstream primer is shown in SEQ ID NO: 4; The sequence of the upstream primer of the third primer pair is shown in SEQ ID NO: 5, and the sequence of its downstream primer is shown in SEQ ID NO: 6; The CTAB precipitate comprises CTAB, Tris-HCl, EDTA and water; wherein the concentration of CTAB is 1-3% (w / v), the concentration of Tris-HCl is 80-120 mmol / L, and the concentration of EDTA is 10-30 mmol / L. The CTAB extract comprises CTAB, Tris-HCl, EDTA, NaCl, PVP40, and water; wherein the concentration of CTAB is 1–3% (w / v), the concentration of Tris-HCl is 80–120 mmol / L, the concentration of EDTA is 10–30 mmol / L, the concentration of NaCl is 1–3 mol / L, and the concentration of PVP40 is 10–30% (w / v).

2. The identification method as described in claim 1, characterized in that, The method for extracting genomic DNA from the sample to be tested is as follows: Take 0.3–0.8 g of the sample to be tested, grind it into powder, place it in a centrifuge tube, add 1–1.8 mL of CTAB precipitation solution and 15–25 μL of proteinase K, mix well, heat at 50–60 °C for 45–65 min, cool to room temperature, centrifuge, and discard the supernatant; add 800–1000 μL of CTAB precipitation solution and 15–25 μL of proteinase K, mix well, heat at 50–60 °C for 45–65 min, cool to room temperature, centrifuge, and discard the supernatant; take the precipitate and add 800–1000 μL of... CTAB extraction solution, 5-15 μL β-mercaptoethanol, mixed thoroughly, heated at 60-70℃ for 100-150 min, cooled to room temperature, and the supernatant was collected. An equal volume of chloroform-isoamyl alcohol mixture was added, vortexed and centrifuged, and 700-800 μL of the supernatant was collected. Another equal volume of chloroform-isoamyl alcohol mixture was added, vortexed and centrifuged, and 400-500 μL of the supernatant was collected. An equal volume of isopropanol or isopropanol-sodium acetate mixture was added, and the mixture was allowed to stand at -30 to -20℃ for 30-60 min. After centrifugation, the supernatant was discarded, and the precipitate was washed with ethanol 2-4 times. The supernatant was discarded, and the precipitate was incubated at 35-38℃ for 20-40 min. After the ethanol evaporated, 30-50 μL of sterile water was added to dissolve it, thus obtaining the genomic DNA of the sample to be tested.

3. The identification method as described in claim 1, characterized in that, Mix the template, primers, PCR premix, and water thoroughly to obtain the PCR amplification system; The PCR amplification system was amplified according to a preset amplification program to obtain amplification products; The amplification products were analyzed by electrophoresis, and their electrophoretic patterns were recorded. If the pattern contained a DNA characteristic peak of 158-159 bp, the sample to be tested contained the medicinal material of Snakehead aegypti, the standard decoction of Snakehead aegypti, and / or the traditional Chinese medicine formula granules of Snakehead aegypti. If the spectrum contains a DNA characteristic peak of 206-207bp, then the sample to be tested contains red-banded snake medicinal material, red-banded snake standard decoction and / or red-banded snake Chinese medicine formula granules. If the spectrum contains a DNA characteristic peak of 182-183bp, the sample to be tested contains *Gynostemma pentaphyllum* medicinal material, *Gynostemma pentaphyllum* standard decoction, and / or *Gynostemma pentaphyllum* traditional Chinese medicine formula granules.

4. The identification method as described in claim 3, characterized in that, The PCR amplification system consists of the following substances: PCR premix 12.5 μL; 0.3 μL each of the upstream and downstream primers of the first primer pair, 0.3 μL each of the upstream and downstream primers of the second primer pair, 0.3 μL each of the upstream and downstream primers of the third primer pair, 1.5 μL template, 9.2 μL ddH2O.

5. The identification method as described in claim 3, characterized in that, The PCR amplification procedure is as follows: The amplification system was pre-denatured at 92–95°C for 4–6 min, then cycled 30–40 times under a preset program, and finally extended at 71–75°C for 6–8 min. The preset procedure is as follows: denature the amplification system at 94-96℃ for 28-32s, then anneal at 60-62℃ for 26-32s, and then extend at 70-73℃ for 28-35s.

6. The identification method as described in claim 5, characterized in that, The PCR amplification procedure is as follows: The amplification system was pre-denatured at 95°C for 5 min, then cycled 35 times under a preset program, and finally extended at 72°C for 7 min. The preset procedure is as follows: denature the amplification system at 95°C for 30 seconds, then anneal at 61°C for 30 seconds, and then extend at 72°C for 30 seconds.

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