CN116004838A - Multiple STR typing primer for Bungarus Parvus medicinal material, standard decoction and traditional Chinese medicine formula granule, application thereof and identification method - Google Patents

Abstract

The invention discloses a primer for multiple STR typing of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula particles, 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, the sequence of the downstream primer is shown as SEQ ID NO:2 is shown in the figure; the sequence of the upstream primer of the second primer pair is shown in SEQ ID NO:3, the sequence of the downstream primer is shown as SEQ ID NO:4 is shown in the figure; the sequence of the upstream primer of the third primer pair is shown in SEQ ID NO:5, the sequence of the downstream primer is shown as SEQ ID NO: shown at 6. The invention also discloses application of the primer and an identification method based on the primer. By implementing the invention, the Bungarus Parvus, the red-chain snake and the bungarus parvus can be effectively identified at the same time.

Description

Multiple STR typing primer for Bungarus Parvus medicinal material, standard decoction and traditional Chinese medicine formula granule, application thereof and identification method

Technical Field

The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer for multiple STR typing of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula particles, and application and an identification method thereof.

Background

More than two species of snakes in China exist, wherein more than 20 species are common snakes, including Agkistrodon Agkistrodon acutus, zaocys dhumnades, bungarus Parvus Bungarus multicinctus, bungarus fasciatus Bungarus fasciatus, ptyas mucosus, viper round-spotted viper Daboia russelii, naja Naja, red-chain snake Lycodon rufozonatus, elaphe carinata, ptyas korros, agkistrodon halys brevifolia Gloydius brevicaudus, elaphe taeniura, agkistrodon acutus Elaphe moellendorffi and the like, and most of the species have no clinical efficacy. Because of the approximate shape of various snakes, the identification of medicinal materials is difficult, especially after the viscera are cut off during processing, the drying and smoking treatment is carried out, the pattern features and the colors on the skin almost disappear, and the identification is difficult, especially after the medicinal materials are extracted by water to prepare standard decoction, the medicinal materials are further processed into formula particles, and the effective identification is more difficult after the properties of the medicinal materials are lost.

Some studies have indicated that the identification of Bungarus Parvus can be performed by PCR (polymerase chain reaction). However, standard decoction and traditional Chinese medicine formula particles often contain a certain proportion of auxiliary materials, so that the existing PCR method is difficult to be applied. Furthermore, in the preparation process of standard decoction and traditional Chinese medicine formula particles, heating extraction is often needed, so that DNA degradation is caused, DNA fragments exceeding 200bp are difficult to remain, and the detection precision of the existing method is low. In addition, the existing PCR method can only realize identification of single snakes, and has low efficiency.

Disclosure of Invention

The invention aims to solve the technical problem of providing a primer for multiple STR typing of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula particles, which can effectively identify Bungarus Parvus, alchetus and bungarus parvus simultaneously.

The invention also solves the technical problem of providing an application of the primer.

The invention also solves the technical problem of providing an identification method based on the primer.

In order to solve the technical problems, the invention provides a primer for multiple STR typing of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula particles, 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, the sequence of the downstream primer is shown as SEQ ID NO:2 is shown in the figure;

the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO:3, the sequence of the downstream primer is shown as SEQ ID NO:4 is shown in the figure;

the sequence of the upstream primer of the third primer pair is shown as SEQ ID NO:5, the sequence of the downstream primer is shown as SEQ ID NO: shown at 6.

Correspondingly, the invention also discloses application of the primer in (1) or (2):

(1) Identifying whether the sample to be tested is Bungarus Parvus, althaea aka and/or Bungarus Parvus;

(2) Preparing a kit for identifying the Bungarus Parvus, the red chain snake and/or the bungarus parvus;

as an improvement of the technical scheme, the Bungarus Parvus is medicinal materials, standard decoction or traditional Chinese medicine formula particles;

the red chain snake is medicinal material, standard decoction or traditional Chinese medicine formula granule;

the bunge snake is medicinal material, standard decoction or traditional Chinese medicine formula granule.

Correspondingly, the invention also discloses application of the primer in (1) or (2):

(1) Identifying whether the sample to be tested contains Bungarus Parvus, althaea aka and/or Bungarus Parvus;

(2) Preparing a kit for identifying whether a sample to be detected contains the Bungarus Parvus, the red chain snake and/or the bungarous.

Correspondingly, the invention also discloses a kit which is characterized by comprising the primer.

As an improvement of the technical scheme, the PCR premix solution is also included.

Correspondingly, the invention also discloses a primer-based identification method, which comprises the following steps:

extracting genome DNA of a sample to be detected;

carrying out PCR amplification by using the primer by taking the genome DNA as a template, and if the amplification product contains 158-160bp DNA characteristic peaks, the sample to be detected contains Bungarus Parvus medicinal materials, bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;

if the amplified product contains 205-210bp DNA characteristic peak, the sample to be detected contains red-chain snake medicinal material, red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;

if the amplified product contains 180-185p DNA characteristic peak, the sample to be detected contains bungaroid medicinal material, bungaroid standard decoction and/or bungaroid traditional Chinese medicine prescription granule.

As an improvement of the above technical scheme, the method for extracting genomic DNA of a sample to be detected is as follows:

adding CTAB precipitation solution and proteinase K precipitation to extract 2-3 times; adding CTAB extract and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamyl alcohol for extraction for 2-3 times; and adding isopropanol or isopropanol-sodium acetate into the supernatant after extraction to precipitate and extract, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.

As an improvement of the above technical scheme, the method for extracting genomic DNA of a sample to be detected is as follows:

taking 0.3-0.8 g of sample to be detected, grinding into powder, placing into a centrifuge tube, adding 1-1.8 mL of CTAB precipitate and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L of CTAB precipitate and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L of CTAB extract into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, adding an equal volume of chloroform-isoamyl alcohol mixed solution, vibrating and uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding an equal volume of chloroform-isoamyl alcohol mixed solution, vibrating and uniformly mixing, centrifuging, taking 400-500 mu L of supernatant, adding an equal volume of isopropanol or isopropanol-sodium acetate mixed solution, standing for 30-60 min at-30 to-20 ℃, centrifuging, discarding the supernatant, washing the precipitate with ethanol for 2-4 times, discarding the supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, adding 30-50 mu L of sterilized water for dissolving after the ethanol volatilizes, and obtaining the genome DNA of the sample to be detected.

As an improvement of the technical scheme, the CTAB precipitation liquid 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 extracting 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 80-120 mmol/L, and 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).

As an improvement of the technical scheme, a template, a primer, a PCR premix and water are uniformly mixed to obtain a PCR amplification system;

amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;

performing electrophoresis analysis on the amplified product, recording an electrophoresis pattern of the amplified product, and if the pattern contains 158-159bp DNA characteristic peaks, the sample to be detected contains Bungarus Parvus medicinal materials, bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;

if the map contains a DNA characteristic peak of 206-207bp, the sample to be detected contains red-chain snake medicinal materials, red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;

if the map contains 182-183bp DNA characteristic peaks, the sample to be detected contains bungarous coreana medicinal materials, bungarous coreana standard decoction and/or bungarous coreana traditional Chinese medicine formula particles.

As an improvement of the above technical scheme, the PCR amplification system consists of:

12.5 mu L of PCR premix; the first primer pair has an upstream primer and a downstream primer of 0.3. Mu.L each, the second primer pair has an upstream primer and a downstream primer of 0.3. Mu.L each, the third primer pair has an upstream primer and a downstream primer of 0.3. Mu.L each, the template has a 1.5. Mu.L, and ddH ₂ O 9.2μL。

As an improvement of the above technical scheme, the PCR amplification procedure is:

pre-denaturing the amplification system at 92-95 ℃ for 4-6 min, then circulating for 30-40 times under a preset program, and finally extending at 71-75 ℃ for 6-8 min;

wherein, the preset program is as follows: the amplification system is denatured at 94-96 ℃ for 28-32 s, then annealed at 60-62 ℃ for 26-32 s, and then extended at 70-73 ℃ for 28-35 s.

As an improvement of the above technical scheme, the PCR amplification procedure is:

pre-denaturing the amplification system at 95 ℃ for 5min, then cycling for 35 times under a preset program, and finally extending at 72 ℃ for 7min;

wherein, the preset program is as follows: the amplification system was denatured at 95℃for 30s, then annealed at 61℃for 30s, and extended at 72℃for 30s.

The implementation of the invention has the following beneficial effects:

the primer of the invention can perform multiple site-specific PCR reaction, effectively identify the Bungarus Parvus, the red-chain snake, the bungarus and the mixed pseudo system of the three, and improve the identification efficiency.

The primer and the identification method can effectively solve the problem of auxiliary material interference in the DNA extraction process, and can solve the difficult problem that standard decoction and traditional Chinese medicine formula particles are difficult to identify after losing form.

Drawings

FIG. 1 is a graph showing the results of electrophoresis detection of each sample in example 3; wherein N is a blank sample (ddH ₂ O), 1-2 is a Bungarus Parvus medicinal material, and 3 is Bungarus Parvus standard decoction (freeze-dried powder);

FIG. 2 is a graph showing the result of electrophoresis detection of each sample in example 3; wherein 4 is a standard decoction (freeze-dried powder) of the Bungarus Parvus, 5-6 is a traditional Chinese medicine prescription granule of the Bungarus Parvus, and 7 is a medicinal material of the Bungarus Parvus;

FIG. 3 is a graph showing the result of electrophoresis detection of each sample in example 3; wherein 8 is red-chain snake medicine; 9 to 10 are red chain snake standard decoction (freeze-dried powder), and 11 are bunge snake medicinal materials;

FIG. 4 is a graph showing the result of electrophoresis detection of each sample in example 3; wherein, 12 bunge snake medicinal materials; 13-14 are golden ring standard decoction (freeze-dried powder).

Detailed Description

The present invention will be described in further detail with reference to the drawings and the detailed description, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.

The invention provides a primer for multiple STR typing of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises a first primer pair, a second primer pair and a third primer pair, wherein the specific sequences of the primers are shown in the following table:

the primers can perform multiplex PCR reaction in the same PCR reaction system, have no interference with each other and are easy to amplify.

Based on the above characteristics, the primer of the present invention can be used in the following applications:

(1) Identifying whether the sample to be detected is a medicinal material of Bungarus Parvus, standard decoction and traditional Chinese medicine formula granule; or red chain snake, standard decoction and traditional Chinese medicine formula granules; or bunge pit viper, standard decoction and traditional Chinese medicine formula granules.

(2) Identifying whether the sample to be detected contains Bungarus Parvus and/or Bungarus Parvus; for example, can be used for identifying traditional Chinese medicine compounds containing Bungarus Parvus (such as rheumatism pain relieving capsules).

(3) Preparing a kit for identifying the Bungarus Parvus, the red chain snake and/or the golden flower snake.

(4) Preparing a kit for identifying whether the sample to be detected contains the Bungarus Parvus and/or the Althaea akamansis and/or the Bungarus Parvus.

Correspondingly, the invention discloses a kit which comprises the primer, PCR premix and water, wherein the PCR premix can be selected from 2X Taq PCR Master Mix II (Tiangen Biochemical technology (Beijing) Co., ltd., KT 201-12), but is not limited to the above.

The invention also discloses an identification method based on the primer, which comprises the following steps:

1. extracting genome DNA of a sample to be detected;

wherein, the sample to be detected is provided in a solid state, and the standard decoction is provided in a freeze-dried powder form (namely, the freeze-dried powder is obtained after the standard decoction is freeze-dried).

Specifically, the extraction method comprises the following steps:

firstly, taking a sample to be detected, adding CTAB precipitation solution and proteinase K to precipitate and extract for 2-3 times to obtain a precipitate;

specifically, 0.3-0.8 g of sample to be detected is taken, ground into powder, placed into a centrifuge tube, added with 1-1.8 mL of CTAB precipitate and 15-25 mu L of proteinase K, uniformly mixed, heated for 45-65 min at 50-60 ℃, cooled to room temperature, centrifuged, and the supernatant is removed; adding 800-1000 mu L of CTAB precipitate and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant.

Wherein, the composition of CTAB precipitation liquid is 2% (w/v) CTAB,100mmol/L Tris-HCl (pH=8.0), 20mmol/L EDTA (pH=8.0).

Adding CTAB extract and beta-mercaptoethanol into the precipitate for extraction; then adding chloroform-isoamyl alcohol for extraction for 2-3 times to obtain supernatant after extraction;

specifically, adding 800-1000 mu L of CTAB extracting solution and 5-15 mu L of beta-mercaptoethanol into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, taking supernatant, adding an equal volume of chloroform-isoamyl alcohol (24:1) mixed solution, vibrating and uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding an equal volume of chloroform-isoamyl alcohol (24:1) mixed solution, vibrating and uniformly mixing, centrifuging, and taking 400-500 mu L of supernatant to obtain the product.

In this step, after extraction with CTAB extract and β -mercaptoethanol is completed, chloroform-isoamyl alcohol may be directly added, or the supernatant obtained after extraction may be further added with chloroform-isoamyl alcohol, and the supernatant may be further added with chloroform-isoamyl alcohol to improve the extraction accuracy.

Wherein, CTAB extract: 1 to 3% (w/v) CTAB,80 to 120mmol/L Tris-HCl (pH=8.0), 10 to 30mmol/L EDTA (pH=8.0), 1 to 3mol/LNaCl,10 to 30% (w/v) PVP40.

And thirdly, adding isopropanol or isopropanol-sodium acetate into the extracting solution for precipitation extraction, washing and incubating the precipitate, and then dissolving the precipitate with water to obtain the genome DNA of the sample to be detected.

Specifically, the extracting solution is taken, isopropanol or isopropanol-3 mol/L sodium acetate mixed solution with equal volume is added, standing is carried out for 30-60 min at minus 30-minus 20 ℃, supernatant is removed after centrifugation, ethanol is used for washing sediment for 2-4 times, supernatant is removed, sediment is incubated for 20-40 min at 35-38 ℃, after ethanol is volatilized, 30-50 mu L of sterilizing water is added for dissolution, and then genome DNA of a sample to be detected is obtained.

In addition, the applicant tried the traditional CTAB method, chelating resin method, triton-100 method, SDS method, basic cleavage method, DNeasy Blood & Tissue Kit method and magnetic bead method, but all obtained insoluble precipitate when facing the extraction of genome DNA in the standard decoction of Bungarus Parvus and traditional Chinese medicine prescription granule of Bungarus Parvus. Therefore, the inventor improves CTAB precipitation liquid, CTAB extracting liquid and extracting procedure based on the traditional CTAB method, and the extracting method is obtained. The extraction method can maintain genomic DNA information in the standard decoction of Bungarus Parvus and the traditional Chinese medicine granule of Bungarus Parvus as much as possible, and can overcome the problem of adjuvant interference.

2. Forming a PCR amplification system, and amplifying to obtain an amplification product;

specifically, uniformly mixing a template, a primer, a PCR premix and water to obtain a PCR amplification system;

more specifically, the total volume of the PCR amplification system was 25. Mu.L, which includes: 12.5 mu L of PCR premix; the upstream primer and the downstream primer of the first primer pair are respectively 0.3 mu L, the upstream primer and the downstream primer of the second primer pair are respectively 0.3 mu L, the upstream primer and the downstream primer of the third primer pair are respectively 0.3 mu L, the template is 1.5 mu L and ddH ₂ O 9.2μL。

Wherein, the PCR premix can be 2X Taq PCR Master Mix II with model KT201-12 produced by Tiangen Biochemical technology (Beijing) Co., ltd, but is not limited thereto.

The amplification procedure was: 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, extension at 70-73 ℃ for 28-35 s, and circulation for 30-40 times; finally, the extension is carried out for 6 to 8 minutes at the temperature of 71 to 75 ℃.

Preferably, the amplification procedure is: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 61℃for 30s, extension at 72℃for 30s, cycling for 35 times, and extension at 72℃for 7min.

(3) Analyzing the amplified product;

specifically, the amplification product may be analyzed by electrophoresis or fluorescent staining, but is not limited thereto.

Preferably, the PCR amplification products are detected by ABI3730xl capillary electrophoresis and the results recorded.

The invention is illustrated below by means of specific examples.

Example 1 primer design

The method comprises the steps of using the Chinese pharmacopoeia 2020 edition of longhairy snake and common snake NADH; cytochrome Oxidase I;12S rRNA;16S rRNA; based on Cytochrome b sequence analysis, the Cytochrome Oxidase I sequences of common snake species in a GenBank database are subjected to homologous comparison by using BioEdit software, specific SNP sites of the Bungarus Parvus, the red-chain snake and the bungarus parvus are analyzed after the comparison, and the base sequence containing the SNP sites is introduced into Primer Premier 5 software for Primer design.

The comparison of the sequences shows that the Bungarus Parvus: at position 581, the Bungarus Parvus specific site is T, while the others are C; red chain snake: at position 390 the red-chain snake specific site is G, while the others are A, T or C; golden snake: at position 99 the bungaroid specificity site is T, while the others are C or A. After the SNP locus is determined, the SNP locus is made to approach the 3' end of the forward Primer, the Primer score and GC content are adjusted by moving the upstream Primer position, the reverse Primer position is further adjusted by means of Primer Premier 5 software, and the optimal combination is determined by final Primer score and product band score. In addition, in the design process, the destructive effect on the DNA base sequence after high-temperature extraction is also considered.

Combining the above factors, the primer combinations obtained were as follows:

JQBH.F(6-Fam)：5’-GGAGCAATCAATTTTATTACAACAT-3’；

JQBHS.R：5’-GATCGGTTAAAAGTATTGTAACTGC-3’；

CLS.F(6-Fam)：5’-ACCGTGTACCCGCCACTG-3’；

CLS.R：5’-AAGCATGATAGCAGTAATTAGCACA-3’

JHS.F(6-Fam)：5’-TCTAGGTAGCGATCAAATCTTTAAC-3’；

JHS.R：5’-GAAGAAGTGCTGGTGGGAGT-3’。

example 2 authentication method establishment

(1) DNA extraction and concentration modulation

Taking 0.3g of a dried sample, grinding into powder, placing into a 2mL centrifuge tube, adding 1.5mL of CTAB precipitate preheated at 56 ℃ and 20 mu L of proteinase K, uniformly mixing, heating in a water bath at 56 ℃ for 60min, cooling to room temperature, centrifuging at 10000r/min for 5min, discarding the supernatant, and then adding 900 mu L of CTAB precipitate and 20 mu L of proteinase K, and performing the same method. Sequentially adding 900 μl of CTAB extract and 10 μl of beta-mercaptoethanol into the centrifuge tube, mixing, heating in water bath at 65deg.C for 120min, centrifuging, cooling to room temperature, and collecting supernatant; adding equal volume of chloroform-isoamyl alcohol (24:1), shaking for 3min, and mixing; centrifuging at 12000r/min and 4deg.C for 10min, collecting 750 μl supernatant, adding into new 2mL centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24:1), shaking for 3min, mixing, centrifuging at 12000r/min and 4deg.C for 10min; then 450 mu L of supernatant is taken and put into a 1.5mL centrifuge tube, added with isopropyl alcohol with equal volume and kept stand for 30-60 min at the temperature of minus 20 ℃. Taking out the centrifuge tube, centrifuging at 12000r/min for 5min, discarding supernatant, washing precipitate with 75% ethanol and anhydrous ethanol respectively twice, discarding supernatant, incubating precipitate at 37deg.C for 30min, volatilizing ethanol, and adding sterilized water 30 μL for dissolving.

Wherein, CTAB precipitation liquid comprises the following components: 2% (w/v) CTAB (shanghai source leaf biotechnology limited), 100mM Tris-HCl (ph=8.0) (beijin Solarbio), 20mM EDTA (ph=8.0) (Shanghai Yu Bo Biotech co., ltd).

The CTAB extract consists of the following components: 2% (w/v) CTAB (Shanghai source leaf 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 (West Long science Co., ltd.), 20% PVP40 (Shanghai source leaf biotechnology Co., ltd.).

Taking the DNA sample, measuring the DNA concentration by adopting a BioSpec-nano micro ultraviolet spectrophotometer, simultaneously recording OD260/OD230 and OD260/OD280, and adjusting the concentration to 100 ng/. Mu.L to obtain the DNA template.

(2) Designing primers

The primer sequence is shown in SEQ ID NO:1 to 6.

(3) PCR amplification

PCR amplification system: 2X Taq PCR Master Mix II (Tiangen Biochemical Co., ltd., KT 201-12) 12.5. Mu.L, 10. Mu. Mol/L JQBHS.F (6-Fam) primer 0.3. Mu.L, 10. Mu. Mol/L JQBHS.R primer 0.3. Mu.L, 10. Mu. Mol/L CLS.F (6-Fam) primer 0.3. Mu.L, 10. Mu. Mol/L CLS.R primer 0.3. Mu.L, 10. Mu. Mol/L JHS.F (6-Fam) primer 0.3. Mu.L, 10. Mu. Mol/L JHS.R primer 0.3. Mu.L, 100 ng/mu.L DNA template 1.5. Mu.L, deionized water 9.2. Mu.L. And (5) uniformly mixing the liquid in an oscillating way, and performing instantaneous centrifugation to obtain the PCR amplification system.

PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 61℃for 30s, extension at 72℃for 30s for 35 cycles; finally, the extension is carried out at 72 ℃ for 7min.

(4) Electrophoresis detection

Amplification products were taken, detected by ABI3730xl capillary electrophoresis, and the results recorded.

If the map contains 158-159bp DNA characteristic peaks, the sample to be detected contains Bungarus Parvus medicinal materials, bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles; if the map contains a DNA characteristic peak of 206-207bp, the sample to be detected contains red-chain snake medicinal materials, red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles; if the map contains 182-183bp DNA characteristic peaks, the sample to be detected contains bungarous coreana medicinal materials, bungarous coreana standard decoction and/or bungarous coreana traditional Chinese medicine formula particles.

Example 3 authentication method verification

Samples of Bungarus Parvus, alaska and Bungarus Parvus (specifically shown in Table 1) were taken.

Samples were identified using the identification method established in example 2.

Table 1 sample table

The identification results are shown in figures 1-4, and it can be seen from the figures that the characteristic peaks of 158-159bp exist in the Bungarus Parvus medicinal material, bungarus Parvus standard decoction (freeze-dried powder) and Bungarus Parvus traditional Chinese medicine formula particles; while none of the other snake samples gave this characteristic peak. From the graph, the characteristic peaks of 206-207bp exist in the red-chain snake medicinal material and the red-chain snake standard decoction (freeze-dried powder); while none of the other snake samples gave this characteristic peak. As can be seen from the figure, the golden snake medicinal material and golden snake standard decoction (freeze-dried powder) have 182-183bp characteristic peaks; while none of the other snake samples gave this characteristic peak. The result shows that the accuracy of the identification method in the invention reaches 100%.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.

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Sequence listing

<120> primer for multiple STR typing of Bungarus Parvus medicinal material, standard decoction and traditional Chinese medicine formula granule, and application and identification method thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ggagcaatca attttattac aacat 25

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gatcggttaa aagtattgta actgc 25

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

accgtgtacc cgccactg 18

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

aagcatgata gcagtaatta gcaca 25

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

tctaggtagc gatcaaatct ttaac 25

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gaagaagtgc tggtgggagt 20

Claims (13)

1. The primer for multiple STR typing of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula particles is characterized by comprising 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, the sequence of the downstream primer is shown as SEQ ID NO:2 is shown in the figure;

the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO:3, the sequence of the downstream primer is shown as SEQ ID NO:4 is shown in the figure;

the sequence of the upstream primer of the third primer pair is shown as SEQ ID NO:5, the sequence of the downstream primer is shown as SEQ ID NO: shown at 6.

2. The use of the primer of claim 1 in (1) or (2):

(1) Identifying whether the sample to be tested is Bungarus Parvus, althaea aka and/or Bungarus Parvus;

(2) Preparing a kit for identifying the Bungarus Parvus, the red chain snake and/or the bungarus parvus.

3. The use according to claim 2, wherein said Bungarus Parvus is a medicinal material, a standard decoction or a traditional Chinese medicine formulation;

the red chain snake is medicinal material, standard decoction or traditional Chinese medicine formula granule;

the bunge snake is medicinal material, standard decoction or traditional Chinese medicine formula granule.

4. The use of the primer of claim 1 in (1) or (2):

(1) Identifying whether the sample to be tested contains Bungarus Parvus, althaea aka and/or Bungarus Parvus;

(2) Preparing a kit for identifying whether a sample to be detected contains the Bungarus Parvus, the red chain snake and/or the bungarous.

5. A kit comprising the primer of claim 1.

6. An identification method based on the primer according to claim 1, comprising:

extracting genome DNA of a sample to be detected;

carrying out PCR amplification by using the primer as claimed in claim 1 and taking the genome DNA as a template, wherein if the amplification product contains 158-160bp DNA characteristic peaks, the sample to be detected contains Bungarus Parvus medicinal materials, bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;

if the amplified product contains 205-210bp DNA characteristic peak, the sample to be detected contains red-chain snake medicinal material, red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;

if the amplified product contains 180-185p DNA characteristic peak, the sample to be detected contains bungaroid medicinal material, bungaroid standard decoction and/or bungaroid traditional Chinese medicine prescription granule.

7. The method of identification as claimed in claim 6, wherein the method of extracting genomic DNA of the sample to be detected is as follows:

adding CTAB precipitation solution and proteinase K precipitation to extract 2-3 times; adding CTAB extract and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamyl alcohol for extraction for 2-3 times; and adding isopropanol or isopropanol-sodium acetate into the supernatant after extraction to precipitate and extract, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.

8. The method of identification as claimed in claim 6, wherein the method of extracting genomic DNA of the sample to be detected is as follows:

taking 0.3-0.8 g of sample to be detected, grinding into powder, placing into a centrifuge tube, adding 1-1.8 mL of CTAB precipitate and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L of CTAB precipitate and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L of CTAB extract into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, adding an equal volume of chloroform-isoamyl alcohol mixed solution, vibrating and uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding an equal volume of chloroform-isoamyl alcohol mixed solution, vibrating and uniformly mixing, centrifuging, taking 400-500 mu L of supernatant, adding an equal volume of isopropanol or isopropanol-sodium acetate mixed solution, standing for 30-60 min at-30 to-20 ℃, centrifuging, discarding the supernatant, washing the precipitate with ethanol for 2-4 times, discarding the supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, adding 30-50 mu L of sterilized water for dissolving after the ethanol volatilizes, and obtaining the genome DNA of the sample to be detected.

9. The identification method of claim 7 or 8, wherein 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 80-120 mmol/L, and the concentration of EDTA is 10-30 mmol/L;

the CTAB extracting 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 80-120 mmol/L, and 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).

10. The identification method according to claim 6, wherein the template, the primer, the PCR premix and the water are uniformly mixed to obtain a PCR amplification system;

amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;

performing electrophoresis analysis on the amplified product, recording an electrophoresis pattern of the amplified product, and if the pattern contains 158-159bp DNA characteristic peaks, the sample to be detected contains Bungarus Parvus medicinal materials, bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;

if the map contains a DNA characteristic peak of 206-207bp, the sample to be detected contains red-chain snake medicinal materials, red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;

if the map contains 182-183bp DNA characteristic peaks, the sample to be detected contains bungarous coreana medicinal materials, bungarous coreana standard decoction and/or bungarous coreana traditional Chinese medicine formula particles.

11. The identification method of claim 10, wherein the PCR amplification system consists of:

12.5 mu L of PCR premix; the first primer pair has an upstream primer and a downstream primer of 0.3. Mu.L each, the second primer pair has an upstream primer and a downstream primer of 0.3. Mu.L each, the third primer pair has an upstream primer and a downstream primer of 0.3. Mu.L each, the template has a 1.5. Mu.L, and ddH ₂ O 9.2μL。

12. The identification method of claim 10, wherein the PCR amplification procedure is:

pre-denaturing the amplification system at 92-95 ℃ for 4-6 min, then circulating for 30-40 times under a preset program, and finally extending at 71-75 ℃ for 6-8 min;

wherein, the preset program is as follows: the amplification system is denatured at 94-96 ℃ for 28-32 s, then annealed at 60-62 ℃ for 26-32 s, and then extended at 70-73 ℃ for 28-35 s.

13. The identification method of claim 12, wherein the PCR amplification procedure is:

pre-denaturing the amplification system at 95 ℃ for 5min, then cycling for 35 times under a preset program, and finally extending at 72 ℃ for 7min;

wherein, the preset program is as follows: the amplification system was denatured at 95℃for 30s, then annealed at 61℃for 30s, and extended at 72℃for 30s.

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