CN116004839A - Primer for multiple STR typing of Zaocys medicinal material, standard decoction and traditional Chinese medicine formula granule, application thereof and identification method - Google Patents
Primer for multiple STR typing of Zaocys medicinal material, standard decoction and traditional Chinese medicine formula granule, application thereof and identification method Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a primer for multiple STR typing of Zaocys medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises a first primer pair, a second primer pair, a third primer pair and a fourth primer pair; the sequence of the polypeptide is shown in SEQ ID NO:1 to 8. The invention also discloses application of the primer, a kit based on the primer and a method for identifying based on the primer. By implementing the invention, the black-tail snake, the king garter snake, the garter snake and the hundred flower garter snake can be effectively identified at the same time.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer for multiple STR typing of Zaocys 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 indicate that PCR (polymerase chain reaction) can be used to identify Zaocys drugs. 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 Zaocys, standard decoction and traditional Chinese medicine formula particles, which can effectively identify Zaocys, elaphe carinata, garter snake and Agkistrodon halys 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 Zaocys medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises a first primer pair, a second primer pair, a third primer pair and a fourth 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:6 is shown in the figure;
the sequence of the upstream primer of the fourth primer pair is shown as SEQ ID NO:7, the sequence of the downstream primer is shown as SEQ ID NO: shown at 8.
Correspondingly, the invention also discloses application of the primer in (1) or (2):
(1) Identifying whether the sample to be tested is zaocys dhumnade, brocade snake, king mallow and/or garter snake;
(2) Preparing a kit for identifying the zaocys dhumnade, the brocade snake, the king mallow and/or the garter snake.
As an improvement of the technical scheme, the zaocys dhumnade is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;
the brocade snake is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;
the malva sylvestris is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;
the garter 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 Zaocys, agkistrodon halys and/or Agkistrodon halys;
(2) Preparing a kit for identifying whether a sample to be detected contains zaocys dhumnade, brocade snake, king mallow and/or garter snake.
Correspondingly, the invention also discloses a kit which comprises the primer.
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 an amplification product contains a DNA characteristic peak of 130-135bp, a sample to be detected contains a Zaocys medicinal material, a Zaocys standard decoction and/or Zaocys traditional Chinese medicine formula particles;
if the amplified product contains 180-185bp DNA characteristic peak, the sample to be detected contains the Elaphe carinata medicinal material, elaphe carinata standard decoction and/or Elaphe carinata traditional Chinese medicine formula particle;
if the amplified product contains 195-198bp DNA characteristic peak, the sample to be detected contains a garter snake medicinal material, a garter snake standard decoction and/or garter snake traditional Chinese medicine formula particles;
if the amplified product contains 245-250bp DNA characteristic peak, the sample to be detected contains the brocade medicinal material, the brocade standard decoction and/or the brocade traditional Chinese medicine formula 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;
carrying out electrophoresis analysis on the amplified product, recording an electrophoresis pattern of the amplified product, and if the pattern contains 132-133bp DNA characteristic peaks, the sample to be detected contains Zaocys medicinal materials, zaocys standard decoction and/or Zaocys traditional Chinese medicine formula particles;
if the amplified product contains 180-181bp DNA characteristic peak, the sample to be detected contains the Elaphe carinata medicinal material, elaphe carinata standard decoction and/or Elaphe carinata traditional Chinese medicine formula granule;
if the amplified product contains 196-197bp DNA characteristic peak, the sample to be detected contains a garter snake medicinal material, a garter snake standard decoction and/or garter snake traditional Chinese medicine formula particles;
if the amplified product contains 247-248bp DNA characteristic peak, the sample to be detected contains the brocade medicinal material, the brocade standard decoction and/or the brocade traditional Chinese medicine formula granule.
As an improvement of the above technical scheme, the PCR amplification system consists of:
5 mu L of PCR premix; the first primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the second primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the third primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the fourth primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the template has 2. Mu.L, and ddH 2 O 14μL。
As an improvement of the above technical scheme, the PCR amplification procedure is:
pre-denaturing the amplification system at 94-96 deg.c for 4-6 min, circulating for 30-40 times in the preset program and final extending at 71-75 deg.c for 6-10 min;
wherein, the preset program is as follows: the amplification system is denatured at 94-96 ℃ for 15-25 s, then annealed at 58-62 ℃ for 15-22 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 20s, then annealed at 60℃for 20s, and extended at 72℃for 30s.
The implementation of the invention has the following beneficial effects:
1, the primer of the invention can perform multiple site-specific PCR reaction, effectively identify zaocys dhumnade, king garter snake, garter snake and brocade snake, 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 of a zaocys dhumnade medicinal material and 3 of a zaocys dhumnade 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 Zaocys traditional Chinese medicine formula granule, 5-6 is Wang jin snake medicine material, 7 is Wang jin snake standard decoction (freeze-dried powder);
FIG. 3 is a graph showing the result of electrophoresis detection of each sample in example 3; wherein 8 is a standard decoction (freeze-dried powder) of the king brocade, 9 is a medicinal material of the hundred flower brocade, 10 is a standard decoction (freeze-dried powder) of the hundred flower brocade, 11 is a medicinal material of the garter snake, and 12 is a standard decoction (freeze-dried powder) of the garter snake.
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 Zaocys medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises a first primer pair, a second primer pair, a third primer pair and a fourth primer pair, wherein the specific sequences of the primer pair 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 zaocys dhumnade medicinal material, a standard decoction and traditional Chinese medicine formula particles; or the medicinal materials of the king mallow, the standard decoction and the traditional Chinese medicine formula granules; or garter snake, standard decoction and traditional Chinese medicine formula granules; or medicinal materials of the brocade snake, standard decoction and traditional Chinese medicine formula granules.
(2) And identifying whether the sample to be detected contains the traditional Chinese medicine compound of the zaocys dhumnade and/or the king mallow and/or the garter snake and/or the hundred-flower mallow.
(3) Preparing a kit for identifying the zaocys dhumnade and/or the malus carinata and/or the garter snake and/or the serpentis.
(4) Preparing a kit for identifying whether a sample to be detected contains zaocys dhumnade and/or Elaphe carinata and/or Agkistrodon halys and/or Agkistrodon acutus.
Correspondingly, the invention discloses a kit which comprises the primer, PCR premix and water. Specifically, the PCR premix may be Multiplex PCR 5 XMaster Mix (New England Biolabs (Beijin), M0284S), but is not limited thereto.
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 extract and 5-15 mu L of beta-mercaptoethanol into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, centrifuging and cooling to room temperature; adding the extracted supernatant into an equal volume of chloroform-isoamyl alcohol (24:1) mixed solution, shaking and uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding an equal volume of chloroform-isoamyl alcohol (24:1) mixed solution, shaking and uniformly mixing, centrifuging, and taking 400-500 mu L of supernatant to obtain the compound.
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 the extraction of genomic DNA from Zaocys standard decoction and Zaocys traditional Chinese medicine granule, 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. 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 Zaocys standard decoction and Zaocys traditional Chinese medicine granule 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: 5 mu L of PCR premix; the first primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the second primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the third primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the fourth primer pair has 0.5. Mu.L each of the upstream primer and the downstream primer, the template has 2. Mu.L, and ddH 2 O 14μL。
Wherein, the PCR premix can be Multiplex PCR 5 XMaster Mix (New England Biolabs (Beijin), M0284S).
The amplification procedure was: 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, and circulation for 30-40 times; finally, the extension is carried out for 6 to 10 minutes at the temperature of 71 to 75 ℃.
Preferably, the amplification procedure is: pre-denaturation at 95℃for 5min, denaturation at 95℃for 20s, annealing at 60℃for 20s, 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
Zaocys and common snake NADH in 2020 edition of Chinese pharmacopoeia; 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 Zaocys, elaphe carinata, agkistrodon acutus and Amyda sinensis are analyzed after the comparison, and a base sequence containing the SNP sites is introduced into Primer Premier 5 software to carry out Primer design. The Zaocys is found by sequence comparison: at position 325 the Zaocys specific site is T and the others are A or C. All-grass of Paris-: at the 110 th position, the brocade snake is G, and the other is A; the 124 th site of the brocade snake is G, the others are C; the 336 th site of the brocade snake is C, and the others are T. King snake): at 580 th, the king snake is T, the others are C. Garter snake: the 345 th garter snake is G, the other snakes are A, C or T. 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:
WSS.F(6-Fam)：5’-CCCCTAATAATCGGAGCG-3’；
WSS.R：5’-TACTGTTCACCCAGTGCC-3’；
WJS.F(6-Fam)：5’-TCCATTCTAGGAGCAATTAACTTTA-3’；
WJS.R：5’-GTATTTAGGTTTCGGTCGGTTAA-3’；
HSS.F(6-Fam)：5’-CCCCTAATAATTGGAGCACC-3’；
HSS.R：5’-GAAAAAATGGCTAAGTCTACCGA-3’。
BHJS.F(6-Fam)：5’-ATGACCAGGTTTTTAATGTTCTG-3’；
BHJS.R：5’-ACGGGGGGTAGGCTGTTC-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 8.
(3) PCR amplification
PCR amplification system: multiplex PCR 5 XMaster Mix (New England Biolabs (Beijing), M0284S) 5. Mu.L, 10. Mu. Mol/L WSS.F (6-Fam) primer 0.5. Mu.L, 10. Mu. Mol/L WSS.R primer 0.5. Mu.L, 10. Mu. Mol/L WJS.F (6-Fam) primer 0.5. Mu.L, 10. Mu. Mol/L WJS.R primer 0.5. Mu.L, 10. Mu. Mol/L HSS.F (6-Fam) primer 0.5. Mu.L, 10. Mu. Mol/L HSS.R primer 0.5. Mu.L, 10. Mu. Mol/L BHJS.F (6-Fam) primer 0.5. Mu.L, 10. Mu. Mol/L BHJS.R primer 0.5. Mu.L, 100 ng/mu.L DNA template 2. Mu.L, 14. 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 20s, annealing at 60℃for 20s, elongation 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 a DNA characteristic peak of 132-133bp, the sample to be detected contains Zaocys medicinal materials, zaocys standard decoction and/or Zaocys traditional Chinese medicine formula particles; if the amplified product contains 180-181bp DNA characteristic peak, the sample to be detected contains the Elaphe carinata medicinal material, elaphe carinata standard decoction and/or Elaphe carinata traditional Chinese medicine formula granule; if the amplified product contains 196-197bp DNA characteristic peak, the sample to be detected contains a garter snake medicinal material, a garter snake standard decoction and/or garter snake traditional Chinese medicine formula particles; if the amplified product contains 247-248bp DNA characteristic peak, the sample to be detected contains the brocade medicinal material, the brocade standard decoction and/or the brocade traditional Chinese medicine formula granule.
Example 3 authentication method verification
Samples of Zaocys, elaphe carinata, elaphe grifola and Elaphe carinata were selected (see Table 1 in particular).
Samples were identified using the identification method established in example 2.
Table 1 sample table
The identification results are shown in figures 1-3, and the identification results show that the characteristic peaks of 132-133bp exist in the Zaocys medicinal material, zaocys standard decoction (freeze-dried powder) and Zaocys traditional Chinese medicine formula particles; while none of the other snake samples gave this characteristic peak. From the figure, the characteristics peak of 180-181bp exists in the medicinal material of the king mallow and the standard decoction (freeze-dried powder) of the king mallow; while none of the other snake samples gave this characteristic peak. From the graph, the characteristic peaks of 196-197bp exist in the standard decoction (freeze-dried powder) of the garter snake medicinal material and the garter snake; from the graph, characteristic peaks of 247-248bp exist in all the medicinal materials of the brocade snake and the standard decoction (freeze-dried powder) of the brocade snake; 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.
Sequence listing
<120> primer for multiple STR typing of Zaocys, standard decoction and Chinese medicinal granule, and application and identification method thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cccctaataa tcggagcg 18
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tactgttcac ccagtgcc 18
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tccattctag gagcaattaa cttta 25
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gtatttaggt ttcggtcggt taa 23
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gaaaaaatgg ctaagtctac cga 23
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgaccaggt ttttaatgtt ctg 23
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
acggggggta ggctgttc 18
Claims (13)
1. The primer for multiple STR typing of Zaocys medicinal materials, standard decoction and traditional Chinese medicine formula particles is characterized by comprising a first primer pair, a second primer pair, a third primer pair and a fourth 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:6 is shown in the figure;
the sequence of the upstream primer of the fourth primer pair is shown as SEQ ID NO:7, the sequence of the downstream primer is shown as SEQ ID NO: shown at 8.
2. The use of the primer of claim 1 in (1) or (2):
(1) Identifying whether the sample to be tested is zaocys dhumnade, brocade snake, king mallow and/or garter snake;
(2) Preparing a kit for identifying the zaocys dhumnade, the brocade snake, the king mallow and/or the garter snake.
3. The use as claimed in claim 2, wherein the zaocys dhumnade is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;
the brocade snake is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;
the malva sylvestris is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule;
the garter 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 Zaocys, agkistrodon halys and/or Agkistrodon halys;
(2) Preparing a kit for identifying whether a sample to be detected contains zaocys dhumnade, brocade snake, king mallow and/or garter snake.
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 with the genome DNA as a template, wherein if the amplified product contains a DNA characteristic peak of 130-135bp, the sample to be detected contains Zaocys medicinal material, zaocys standard decoction and/or Zaocys traditional Chinese medicine formula particles;
if the amplified product contains 180-185bp DNA characteristic peak, the sample to be detected contains the Elaphe carinata medicinal material, elaphe carinata standard decoction and/or Elaphe carinata traditional Chinese medicine formula particle;
if the amplified product contains 195-198bp DNA characteristic peak, the sample to be detected contains a garter snake medicinal material, a garter snake standard decoction and/or garter snake traditional Chinese medicine formula particles;
if the amplified product contains 245-250bp DNA characteristic peak, the sample to be detected contains the brocade medicinal material, the brocade standard decoction and/or the brocade traditional Chinese medicine formula 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;
carrying out electrophoresis analysis on the amplified product, recording an electrophoresis pattern of the amplified product, and if the pattern contains 132-133bp DNA characteristic peaks, the sample to be detected contains Zaocys medicinal materials, zaocys standard decoction and/or Zaocys traditional Chinese medicine formula particles;
if the amplified product contains 180-181bp DNA characteristic peak, the sample to be detected contains the Elaphe carinata medicinal material, elaphe carinata standard decoction and/or Elaphe carinata traditional Chinese medicine formula granule;
if the amplified product contains 196-197bp DNA characteristic peak, the sample to be detected contains a garter snake medicinal material, a garter snake standard decoction and/or garter snake traditional Chinese medicine formula particles;
if the amplified product contains 247-248bp DNA characteristic peak, the sample to be detected contains the brocade medicinal material, the brocade standard decoction and/or the brocade traditional Chinese medicine formula granule.
11. The identification method of claim 10, wherein the PCR amplification system consists of:
5. Mu.L of PCR premix, 0.5. Mu.L of each of the upstream primer and the downstream primer of the first primer pair, 0.5. Mu.L of each of the upstream primer and the downstream primer of the second primer pair, 0.5. Mu.L of each of the upstream primer and the downstream primer of the third primer pair, 0.5. Mu.L of each of the upstream primer and the downstream primer of the fourth primer pair, 2. Mu.L of the template and ddH 2 O 14μL。
12. The identification method of claim 10, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 94-96 deg.c for 4-6 min, circulating for 30-40 times in the preset program and final extending at 71-75 deg.c for 6-10 min;
wherein, the preset program is as follows: the amplification system is denatured at 94-96 ℃ for 15-25 s, then annealed at 58-62 ℃ for 15-22 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 20s, then annealed at 60℃for 20s, and extended at 72℃for 30s.
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