Primers for multiple str typing of the medicinal material of boa constrictor, standard decoction and traditional chinese medicine dispensing granules, and application and identification method thereof

Abstract

The application discloses a primer for multiple STR typing of Trimeresurus gramineus medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair, a third primer pair and a fourth primer pair, and the sequences of the primer pairs are shown as SEQ ID NO:1-8. The application further discloses application of the primer, a kit based on the primer and a discrimination method based on the primer. The application can effectively discriminate Trimeresurus gramineus, Elaphe carinata, Natrix tessellata and Oxyuranus macculatus 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 Zaocys dhumnades 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 Zaocys dhumnades medicinal materials can be identified by PCR (polymerase chain reaction) method. 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 Zaocys dhumnades medicinal material, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Zaocys dhumnades, Elaphe carinata, Ptyas korros and Elaphe moellendorffi 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 Zaocys dhumnades medicinal material, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Zaocys dhumnades, Elaphe carinata, Ptyas korros and Elaphe moellendorffi 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 Zaocys dhumnades medicinal material, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Zaocys dhumnades, Elaphe carinata, Ptyas korros and Elaphe moellendorffi at the same time.

[0007] In order to solve the above technical problems, the present application provides a primer for multiple STR typing of the medicinal material of Trimeresurus stejnegeri, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair, a third primer pair and a fourth 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] The sequence of the upstream primer of the fourth primer pair is shown in SEQ ID NO: 7, and the sequence of the downstream primer is shown in SEQ ID NO: 8.

[0012] Correspondingly, the present application also discloses the application of the above-mentioned primer in (1) or (2):

[0013] (1) identifying whether the to-be-tested sample is Trimeresurus stejnegeri, Elaphe bimaculata, Elaphe carinata and / or Elaphe rufescens;

[0014] (2) preparing a kit for identifying Trimeresurus stejnegeri, Elaphe bimaculata, Elaphe carinata and / or Elaphe rufescens.

[0015] As an improvement of the above technical solution, the Trimeresurus stejnegeri is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;

[0016] The Elaphe bimaculata is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;

[0017] The Elaphe carinata is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;

[0018] The Elaphe rufescens is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule.

[0019] Correspondingly, the present application also discloses the application of the above-mentioned primer in (1) or (2):

[0020] (1) identifying whether the to-be-tested sample contains Trimeresurus stejnegeri, Elaphe bimaculata, Elaphe carinata and / or Elaphe rufescens;

[0021] (2) preparing a kit for identifying whether the to-be-tested sample contains Trimeresurus stejnegeri, Elaphe bimaculata, Elaphe carinata and / or Elaphe rufescens.

[0022] Correspondingly, the present application also discloses a kit comprising the above-mentioned primer.

[0023] Accordingly, the present invention also discloses an identification method based on the above-mentioned primers, comprising:

[0024] Extract genomic DNA from the sample to be tested;

[0025] Using the genomic DNA as a template, PCR amplification was performed using the primers described above. If the amplification product contained a DNA characteristic peak of 130-135 bp, then the sample to be tested contained *Zaocys dhumnades* medicinal material, *Zaocys dhumnades* standard decoction, and / or *Zaocys dhumnades* traditional Chinese medicine formula granules.

[0026] If the amplification product contains a DNA characteristic peak of 180-185bp, then the sample to be tested contains Chinese cobra herbal medicine, Chinese cobra standard decoction and / or Chinese cobra herbal formula granules.

[0027] If the amplification product contains a DNA characteristic peak of 195-198bp, then the sample to be tested contains gray rat snake medicinal material, gray rat snake standard decoction and / or gray rat snake Chinese medicine formula granules.

[0028] If the amplification product contains a DNA characteristic peak of 245-250 bp, then the sample to be tested contains *Smilax china* medicinal material, *Smilax china* standard decoction, and / or *Smilax china* traditional Chinese medicine formula granules.

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

[0030] 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 and 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.

[0031] As an improvement to the above technical solution, the method for extracting genomic DNA from the sample to be tested is as follows:

[0032] 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.

[0033] As an improvement to the above technical solution, the CTAB precipitate includes 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.

[0034] 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).

[0035] As an improvement to the above technical solution, the template, primers, PCR premix, and water are mixed evenly to obtain the PCR amplification system;

[0036] The PCR amplification system was amplified according to a preset amplification program to obtain amplification products;

[0037] The amplification products were analyzed by electrophoresis, and their electrophoretic patterns were recorded. If the pattern contained a DNA characteristic peak of 132-133 bp, the sample to be tested contained Chinese black snake medicinal material, Chinese black snake standard decoction and / or Chinese black snake traditional Chinese medicine formula granules.

[0038] If the amplification product contains a DNA characteristic peak of 180-181bp, then the sample to be tested contains Chinese cobra herbal medicine, Chinese cobra standard decoction and / or Chinese cobra herbal formula granules.

[0039] If the amplification product contains a DNA characteristic peak of 196-197bp, then the sample to be tested contains gray rat snake medicinal material, gray rat snake standard decoction and / or gray rat snake Chinese medicine formula granules.

[0040] If the amplification product contains a DNA characteristic peak of 247-248 bp, then the sample to be tested contains *Smilax china* medicinal material, *Smilax china* standard decoction, and / or *Smilax china* traditional Chinese medicine formula granules.

[0041] As an improvement to the above technical solution, the PCR amplification system is composed of the following substances:

[0042] 5 μL of PCR premix; 0.5 μL each of the upstream and downstream primers of the first primer pair; 0.5 μL each of the upstream and downstream primers of the second primer pair; 0.5 μL each of the upstream and downstream primers of the third primer pair; 0.5 μL each of the upstream and downstream primers of the fourth primer pair; 2 μL of template; 14 μL of ddH2O.

[0043] As an improvement to the above technical solution, the PCR amplification procedure is as follows:

[0044] The amplification system was pre-denatured at 94–96 °C for 4–6 min, then cycled 30–40 times under a preset program, and finally extended at 71–75 °C for 6–10 min.

[0045] The preset procedure is as follows: denature the amplification system at 94-96℃ for 15-25s, then anneal at 58-62℃ for 15-22s, and then extend at 70-73℃ for 28-35s.

[0046] As an improvement to the above technical solution, the PCR amplification procedure is as follows:

[0047] 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.

[0048] The preset procedure is as follows: denature the amplification system at 95°C for 20 seconds, then anneal at 60°C for 20 seconds, and then extend it at 72°C for 30 seconds.

[0049] Implementing this invention has the following beneficial effects:

[0050] 1. The primers of this invention can perform multiple site-specific PCR reactions, effectively identifying the rat snake, king snake, gray rat snake, and hundred-flower rat snake, thus improving the identification efficiency.

[0051] 2. The primers and identification methods in this invention can effectively overcome the problem of interference from excipients during DNA extraction, and can also solve the problem of difficulty in identifying standard decoctions and traditional Chinese medicine granules after they have lost their morphology. Attached Figure Description

[0052] Fig. 1 The images show the electrophoretic detection results of each sample in Example 3; where N is the blank sample (ddH2O), 1-2 are the medicinal materials of Zaocys dhumnades, and 3 is the standard decoction of Zaocys dhumnades (lyophilized powder);

[0053] Fig. 2 These are the electrophoresis results of each sample in Example 3; among them, 4 is the Chinese herbal formula granules of Zaocys dhumnades, 5-6 are the medicinal materials of Ratna tigrinosa, and 7 is the standard decoction of Ratna tigrinosa (freeze-dried powder).

[0054] Fig. 3 These are the electrophoresis results of each sample in Example 3; among them, 8 is the standard decoction of King Rat Snake (lyophilized powder), 9 is the medicinal material of Rat Snake, 10 is the standard decoction of Rat Snake (lyophilized powder), 11 is the medicinal material of Gray Rat Snake, and 12 is the standard decoction of Gray Rat Snake (lyophilized powder). Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] This invention provides primers for multiplex STR typing of *Zaocys dhumnades* medicinal material, standard decoction, and traditional Chinese medicine granules. The primers include a first primer pair, a second primer pair, a third primer pair, and a fourth primer pair, the specific sequences of which are shown in the table below:

[0057]

[0058] The primers mentioned above can be used in multiplex PCR reactions in the same PCR reaction system without interfering with each other, making them easy to amplify.

[0059] Based on the above characteristics, the primers in this invention can achieve the following applications:

[0060] (1) Identify whether the sample to be tested is the medicinal material, standard decoction, or Chinese medicine formula granule of the black-striped snake; or the medicinal material, standard decoction, or Chinese medicine formula granule of the king snake; or the medicinal material, standard decoction, or Chinese medicine formula granule of the gray rat snake; or the medicinal material, standard decoction, or Chinese medicine formula granule of the hundred-flower snake.

[0061] (2) Identify whether the sample to be tested contains traditional Chinese medicine compound containing black-striped snake and / or king snake and / or gray rat snake and / or hundred-flower snake.

[0062] (3) Prepare a kit for identifying the rat snake and / or king snake and / or gray rat snake and / or flower rat snake.

[0063] (4) Prepare a kit for identifying whether the sample to be tested contains rat snake and / or king snake and / or gray rat snake and / or flower snake.

[0064] Accordingly, this invention discloses a kit comprising the aforementioned primers, as well as PCR premix and water. Specifically, the PCR premix may be Multiplex PCR 5×Master Mix (New England Biolabs (Beijing), M0284S), but is not limited to this.

[0065] This invention also discloses an identification method based on the above primers, comprising:

[0066] 1. Extract genomic DNA from the sample to be tested;

[0067] The samples to be tested are provided in solid form, while the standard decoction is provided in the form of lyophilized powder (i.e., the standard decoction is freeze-dried to obtain lyophilized powder).

[0068] Specifically, the extraction method is as follows:

[0069] (a) Take the sample to be tested, add CTAB precipitation solution and proteinase K precipitation extraction 2 to 3 times to obtain precipitate;

[0070] Specifically, 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 to obtain the final product.

[0071] The CTAB precipitate consisted of 2% (w / v) CTAB, 100 mmol / L Tris-HCl (pH=8.0), and 20 mmol / L EDTA (pH=8.0).

[0072] (ii) Take the precipitate and add CTAB extraction solution and β-mercaptoethanol for extraction; then add chloroform-isoamyl alcohol for extraction 2 to 3 times to obtain the supernatant after extraction;

[0073] Specifically, add 800–1000 μL of CTAB extraction solution and 5–15 μL of β-mercaptoethanol to the precipitate, mix well, heat at 60–70 °C for 100–150 min, centrifuge, and cool to room temperature; take the supernatant after extraction, add an equal volume of chloroform-isoamyl alcohol (24:1) mixture, vortex to mix, centrifuge, take 700–800 μL of supernatant, add another equal volume of chloroform-isoamyl alcohol (24:1) mixture, vortex to mix, centrifuge, and take 400–500 μL of supernatant to obtain the final product.

[0074] It should be noted that in this step, after extraction with CTAB extractant and β-mercaptoethanol, chloroform-isoamyl alcohol can be added directly, or the supernatant obtained after extraction can be taken and then chloroform-isoamyl alcohol can be added. Taking the supernatant and then adding chloroform-isoamyl alcohol can improve the extraction accuracy.

[0075] The CTAB extraction solution consisted of 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.

[0076] (iii) Take the extract and add isopropanol or isopropanol-sodium acetate to precipitate and extract. After washing and incubation, the precipitate is dissolved in water to obtain the genomic DNA of the sample to be tested.

[0077] Specifically, take the extract, add an equal volume of isopropanol or isopropanol-3mol / L sodium acetate mixture, let stand at -30 to -20℃ for 30 to 60 minutes, centrifuge and discard the supernatant, then wash the precipitate with ethanol 2 to 4 times, discard the supernatant, incubate the precipitate at 35 to 38℃ for 20 to 40 minutes, and after the ethanol evaporates, add 30 to 50 μL of sterile water to dissolve it, thus obtaining the genomic DNA of the sample to be tested.

[0078] It should be noted that when faced with the challenge of extracting genomic DNA from the standard decoction and granules of *Zaocys dhumnades*, the applicant attempted traditional methods such as CTAB, chelation resin, Triton-100, SDS, alkaline lysis, DNeasyBlood & Tissue Kit, and magnetic bead methods, but all yielded insoluble precipitates. Therefore, based on the traditional CTAB method, the inventors improved the CTAB precipitate, CTAB extractant, and extraction procedure, resulting in the extraction method described above. This extraction method retains as much genomic DNA information as possible from the standard decoction and granules of *Zaocys dhumnades*, while also overcoming the problem of excipient interference.

[0079] 2. Form a PCR amplification system and perform amplification to obtain amplification products;

[0080] Specifically, the template, primers, PCR premix, and water are mixed evenly to obtain the PCR amplification system;

[0081] More specifically, the total volume of the PCR amplification system is 25 μL, which includes: 5 μL of PCR premix; 0.5 μL each of the upstream and downstream primers of the first primer pair; 0.5 μL each of the upstream and downstream primers of the second primer pair; 0.5 μL each of the upstream and downstream primers of the third primer pair; 0.5 μL each of the upstream and downstream primers of the fourth primer pair; 2 μL of template; and 14 μL of ddH2O.

[0082] The PCR premix solution can be Multiplex PCR 5×Master Mix (New England Biolabs (Beijing), M0284S).

[0083] The amplification program was as follows: pre-denaturation at 94–96℃ for 4–6 min; denaturation at 94–96℃ for 15–25 s, annealing at 58–62℃ for 15–22 s, extension at 70–73℃ for 28–35 s, for 30–40 cycles; and finally extension at 71–75℃ for 6–10 min.

[0084] Preferably, the amplification program is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 20 s, 60℃ annealing for 20 s, 72℃ extension for 30 s, 35 cycles, and finally extension at 72℃ for 7 min.

[0085] (3) Analyze the amplification products;

[0086] Specifically, electrophoresis or fluorescence staining can be used to analyze the amplification products, but this is not the only method.

[0087] Preferably, the PCR amplification products are detected by capillary electrophoresis using an ABI 3730xl tube, and the results are recorded.

[0088] The present invention will now be described with reference to specific embodiments.

[0089] Example 1 Primer Design

[0090] Based on the NADH, Cytochrome Oxidase I, 12S rRNA, 16S rRNA, and Cytochrome b sequence analysis of *Zaocys dhumnades* and common snakes in the 2020 edition of the Chinese Pharmacopoeia, homology comparison of Cytochrome Oxidase I sequences of common snake species in the GeneBank database was performed using BioEdit software. After correction, specific SNP sites for *Zaocys dhumnades*, *Ratna chinensis*, *Ratna fasciata*, and *Ratna fasciata* were analyzed. The base sequences containing SNP sites were imported into Primer Premier 5 software for primer design. Sequence comparison revealed that for *Zaocys dhumnades*: the specific site at position 325 is T, while others are A or C. For *Ratna fasciata*: the specific site at position 110 is G, while others are A; at position 124 is G, while others are C; at position 336 is C, while others are T. For *Ratna fasciata*: the specific site at position 580 is T, while others are C. For rat snakes: the 345th position is G, while other snakes are A, C, or T. After determining this SNP site, the SNP site was positioned close to the 3' end of the forward primer. The primer score and GC content were adjusted by moving the upstream primer, and the reverse primer position was further adjusted using Primer Premier 5 software. The optimal combination was determined by the final primer score and product band score. Furthermore, the destructive effect of high-temperature extraction on the DNA base sequence must be considered during the design process. Taking all factors into account, the obtained primer combinations are as follows:

[0091] WSS.F(6-Fam):5'-CCCCTAATAATCGGAGCG-3';

[0092] WSS.R: 5'-TACTGTTCACCCAGTGCC-3';

[0093] WJS.F(6-Fam):5'-TCCATTCTAGGAGCAATTAACTTTA-3';

[0094] WJS.R: 5'-GTATTTAGGTTTCGGTCGGTTAA-3';

[0095] HSS.F(6-Fam):5'-CCCCTAATAATTGGAGCACC-3';

[0096] HSS.R: 5'-GAAAAAATGGCTAAGTCTACCGA-3'.

[0097] BHJS.F(6-Fam):5'-ATGACCAGGTTTTTAATGTTCTG-3';

[0098] BHJS.R: 5'-ACGGGGGGTAGGCTGTTC-3'.

[0099] Example 2: Establishment of Identification Method

[0100] (1) DNA extraction and concentration adjustment

[0101] Take 0.3g of dried sample, grind it into powder, place it in a 2mL centrifuge tube, add 1.5mL of CTAB precipitation solution preheated at 56℃ and 20μL of proteinase K, mix well, heat in a water bath at 56℃ for 60min, cool to room temperature, centrifuge at 10000r / min for 5min, discard the supernatant, add 900μL of CTAB precipitation solution and 20μL of proteinase K, and operate in the same way. Add 900 μL of CTAB extraction solution and 10 μL of β-mercaptoethanol to the centrifuge tube, mix well, heat in a 65°C water bath for 120 min, centrifuge, and collect the supernatant after cooling to room temperature; add an equal volume of chloroform-isoamyl alcohol (24:1), vortex for 3 min, mix well; centrifuge at 12000 r / min, 4°C for 10 min, collect 750 μL of the supernatant and add it to a new 2 mL centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (24:1), vortex for 3 min, mix well, centrifuge at 12000 r / min, 4°C for 10 min; collect 450 μL of the supernatant and add an equal volume of isopropanol, and let stand at -20°C for 30–60 min. Remove the centrifuge tube and centrifuge at 12000 r / min for 5 min. Discard the supernatant and wash the precipitate twice with 75% ethanol and anhydrous ethanol respectively. Discard the supernatant and incubate the precipitate at 37℃ for 30 min. After the ethanol evaporates, add 30 μL of sterile water to dissolve it.

[0102] The CTAB precipitate consisted of the following components: 2% (w / v) CTAB (Shanghai Yu Bo Biotech Co., Ltd.), 100 mM Tris-HCl (pH=8.0) (Beijing Solarbio), and 20 mM EDTA (pH=8.0) (Shanghai Yu Bo Biotech Co., Ltd.).

[0103] The CTAB extraction solution consisted of the following: 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.).

[0104] Take the above DNA sample, measure the DNA concentration using a BioSpec-nano micro-ultraviolet spectrophotometer, and record OD260 / OD230 and OD260 / OD280. Adjust the concentration to 100 ng / μL to obtain the DNA template.

[0105] (2) Design primers

[0106] Primer sequences are shown in SEQ ID NO: 1-8.

[0107] (3) PCR amplification

[0108] PCR amplification system: 5 μL Multiplex PCR 5×Master Mix (New England Biolabs (Beijing), M0284S), 0.5 μL 10 μmol / L WSS.F (6-Fam) primers, 0.5 μL 10 μmol / L WSS.R primers, 0.5 μL 10 μmol / L WJS.F (6-Fam) primers, 0.5 μL 10 μmol / L WJS.R primers, 0.5 μL 10 μmol / L HSS.F (6-Fam) primers, 0.5 μL 10 μmol / L HSS.R primers, 0.5 μL 10 μmol / L BHJS.F (6-Fam) primers, 0.5 μL 10 μmol / L BHJS.R primers, 2 μL 100 ng / μL DNA template, and 14 μL deionized water. The above liquid is shaken to mix well, then centrifuged briefly to obtain the PCR amplification system.

[0109] PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 20 s, 60℃ annealing for 20 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 7 min.

[0110] (4) Electrophoresis detection

[0111] The amplification products were collected and detected by capillary electrophoresis using an ABI 3730xl, and the results were recorded.

[0112] If the amplification spectrum contains a DNA characteristic peak of 132-133 bp, the sample to be tested contains *Zaocys dhumnades* medicinal material, *Zaocys dhumnades* standard decoction, and / or *Zaocys dhumnades* traditional Chinese medicine formula granules; if the amplification product contains a DNA characteristic peak of 180-181 bp, the sample to be tested contains *Echinochloa crus-galli* medicinal material, *Echinochloa crus-galli* standard decoction, and / or *Echinochloa crus-galli* traditional Chinese medicine formula granules; if the amplification product contains a DNA characteristic peak of 196-197 bp, the sample to be tested contains *Gnaphalium affine* medicinal material, *Gnaphalium affine* standard decoction, and / or *Gnaphalium affine* traditional Chinese medicine formula granules; if the amplification product contains a DNA characteristic peak of 247-248 bp, the sample to be tested contains *Echinochloa crus-galli* medicinal material, *Echinochloa crus-galli* standard decoction, and / or *Echinochloa crus-galli* traditional Chinese medicine formula granules.

[0113] Example 3: Verification of the identification method

[0114] Samples of black-banded rat snake, king rat snake, gray rat snake, and white rat snake were selected (see Table 1 for details).

[0115] The identification method established in Example 2 was used to identify the sample.

[0116] Table 1 Sample Table

[0117]

[0118] The results of the identification are as follows Figs. 1-3 As shown in the figure, the characteristic peak of 132-133 bp is present in the *Zaocys dhumnades* medicinal material, *Zaocys dhumnades* standard decoction (lyophilized powder), and *Zaocys dhumnades* traditional Chinese medicine formula granules; while other snake samples did not show this characteristic peak. The characteristic peak of 180-181 bp is present in the *Elaphe carinata* medicinal material and *Elaphe carinata* standard decoction (lyophilized powder); while other snake samples did not show this characteristic peak. The characteristic peak of 196-197 bp is present in the *Gnaphalium affine* medicinal material and *Gnaphalium affine* standard decoction (lyophilized powder); and the characteristic peak of 247-248 bp is present in the *Elaphe carinata* medicinal material and *Elaphe carinata* standard decoction (lyophilized powder); while other snake samples did not show this characteristic peak. These results indicate that the identification method of this invention has an accuracy rate of 100%.

[0119] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention. sequence list <120> Primers for multiple STR typing of Zaocys dhumnades medicinal material, standard decoctions, and traditional Chinese medicine granules, their applications, and identification methods. <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 cccctaataa tcggagcg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 tactgttcac ccagtgcc 18 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <400> 3 tccattctag gagcaattaa cttta 25 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 gtatttaggt ttcggtcggt taa 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 cccctaataa ttggagcacc 20 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 gaaaaaatgg ctaagtctac cga 23 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7 atgaccaggt ttttaatgtt ctg 23 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <400> 8 acggggggta ggctgttc 18

Claims

1. A reagent kit for detecting *Zaocys dhumnades* medicinal materials, standard decoctions, and traditional Chinese medicine granules, characterized in that, It includes the first primer pair, the second primer pair, the third primer pair, and the fourth primer pair; The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of its 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 sequence of the upstream primer of the fourth primer pair is shown in SEQ ID NO: 7, and the sequence of its downstream primer is shown in SEQ ID NO:

8.

2. The application of the kit as described in claim 1 in (1) or (2): (1) Identify whether the sample to be tested is a rat snake, a common rat snake, a king snake and / or a gray rat snake; (2) Prepare a kit for identifying rat snake, raccoon snake, king snake and / or rat snake; in, The black-striped snake mentioned is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule; The Hundred-Flower Rat Snake is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule. The king cobra mentioned is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule; The gray rat snake is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule.

3. The application of the kit as described in claim 1 in (1) or (2): (1) Identify whether the sample to be tested contains black-banded snake, variegated snake, king snake and / or gray rat snake; (2) Prepare a kit for identifying whether a sample to be tested contains black-banded rat snake, white-flowered rat snake, king rat snake and / or gray rat snake; in, The black-striped snake mentioned is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule; The Hundred-Flower Rat Snake is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule. The king cobra mentioned is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule; The gray rat snake is a medicinal material, a standard decoction, or a traditional Chinese medicine formula granule.

4. An identification method based on the kit described in claim 1, characterized in that, include: Extract genomic DNA from the sample to be tested; Using the genomic DNA as a template, PCR amplification is performed using the kit described in claim 1. If the amplification product contains a DNA characteristic peak of 130-135 bp, then the sample to be tested contains *Zaocys dhumnades* medicinal material, *Zaocys dhumnades* standard decoction, and / or *Zaocys dhumnades* traditional Chinese medicine formula granules. If the amplification product contains a DNA characteristic peak of 180-185bp, then the sample to be tested contains Chinese cobra herbal medicine, Chinese cobra standard decoction and / or Chinese cobra herbal formula granules. If the amplification product contains a DNA characteristic peak of 195-198bp, then the sample to be tested contains gray rat snake medicinal material, gray rat snake standard decoction and / or gray rat snake Chinese medicine formula granules. If the amplification product contains a DNA characteristic peak of 245-250bp, then the sample to be tested contains *Smilax china* medicinal material, *Smilax china* standard decoction, and / or *Smilax china* traditional Chinese medicine formula granules.

5. The identification method as described in claim 4, characterized in that, The method for extracting genomic DNA from the sample to be tested is as follows: 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 and 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.

6. The identification method as described in claim 4, 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℃ 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℃ 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 and 5-15 μL of... Mix β-mercaptoethanol thoroughly and heat at 60-70℃ for 100-150 min. After cooling to room temperature, take the supernatant and add an equal volume of chloroform-isoamyl alcohol mixture. Shake well and centrifuge. Take 700-800 μL of the supernatant and add another equal volume of chloroform-isoamyl alcohol mixture. Shake well and centrifuge. Take 400-500 μL of the supernatant and add an equal volume of isopropanol or isopropanol-sodium acetate mixture. Let stand at -30 to -20℃ for 30-60 min. Centrifuge and discard the supernatant. Wash the precipitate with ethanol 2-4 times and discard the supernatant. Incubate the precipitate at 35-38℃ for 20-40 min. After the ethanol evaporates, add 30-50 μL of sterile water to dissolve it, thus obtaining the genomic DNA of the sample to be tested.

7. The identification method as described in claim 5 or 6, characterized in that, 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).

8. The identification method as described in claim 4, 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 132-133 bp, the sample to be tested contained Chinese black snake medicinal material, Chinese black snake standard decoction and / or Chinese black snake traditional Chinese medicine formula granules. If the amplification product contains a DNA characteristic peak of 180-181bp, then the sample to be tested contains Chinese cobra herbal medicine, Chinese cobra standard decoction and / or Chinese cobra herbal formula granules. If the amplification product contains a DNA characteristic peak of 196-197bp, then the sample to be tested contains gray rat snake medicinal material, gray rat snake standard decoction and / or gray rat snake Chinese medicine formula granules. If the amplification product contains a DNA characteristic peak of 247-248 bp, then the sample to be tested contains *Smilax china* medicinal material, *Smilax china* standard decoction, and / or *Smilax china* traditional Chinese medicine formula granules.

9. The identification method as described in claim 8, characterized in that, The PCR amplification system consists of the following substances: 5 μL of PCR premix, 0.5 μL each of the upstream and downstream primers of the first primer pair, 0.5 μL each of the upstream and downstream primers of the second primer pair, 0.5 μL each of the upstream and downstream primers of the third primer pair, 0.5 μL each of the upstream and downstream primers of the fourth primer pair, 2 μL of template, and 14 μL of ddH2O.

10. The identification method as described in claim 8, characterized in that, The PCR amplification procedure is as follows: The amplification system was pre-denatured at 94-96℃ for 4-6 min, then cycled 30-40 times under a preset program, and finally extended at 71-75℃ for 6-10 min. The preset procedure is as follows: denature the amplification system at 94~96℃ for 15~25s, then anneal at 58~62℃ for 15~22s, and then extend at 70~73℃ for 28~35s.

11. The identification method as described in claim 10, 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 20 seconds, then anneal at 60°C for 20 seconds, and then extend it at 72°C for 30 seconds.

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