CN113981105A - Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof - Google Patents
Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof Download PDFInfo
- Publication number
- CN113981105A CN113981105A CN202111235328.8A CN202111235328A CN113981105A CN 113981105 A CN113981105 A CN 113981105A CN 202111235328 A CN202111235328 A CN 202111235328A CN 113981105 A CN113981105 A CN 113981105A
- Authority
- CN
- China
- Prior art keywords
- primer
- sample
- achyranthes
- detected
- ctab
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer for multiple PCR identification of achyranthes and cyathula root medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair and a second primer pair; wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; 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, respectively. The invention also discloses application of the primer, a kit based on the primer and an identification method based on the primer. The method can effectively identify the achyranthes and the cyathula.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer for multiple PCR identification of medicinal materials of achyranthes and cyathula root, standard decoction and traditional Chinese medicine formula granules and application thereof.
Background
Achyranthis radix is the dry root of Achyranthis radix Achyranthus videntata BI. of Amaranthaceae, and has effects of removing blood stasis, dredging channels, nourishing liver and kidney, strengthening tendons and bones, inducing diuresis, treating stranguria, and promoting blood circulation, and can be used for treating amenorrhea, dysmenorrhea, soreness of waist and knees, weakness of tendons and bones, stranguria syndrome, edema, headache, giddiness, toothache, aphtha, hematemesis, and epistaxis. Radix Cyathulae is the dry root of Cyathula officinalis Kuan of Amaranthaceae, has effects of removing blood stasis, dredging channels, promoting joint movement, inducing diuresis and treating stranguria, and can be used for treating amenorrhea, abdominal mass, retention of exocytosis, traumatic injury, rheumatalgia, flaccidity of foot, spasm of tendons, and hematuria and stranguria. Achyranthes and cyathula root have similar appearance but larger difference of efficacy, and the cyathula root has no efficacy of nourishing liver and kidney. The achyranthes and cyathula root dispensing granules are prepared by heating and extracting with water, effective quality control is difficult to carry out after the medicinal material properties of the achyranthes and cyathula root dispensing granules are lost by adopting a traditional identification method, the risk of quality control is high, and the problem of authenticity and quality evaluation of the extract after the appearance characteristics of the decoction pieces are lost is urgently needed to be solved.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a primer for multiple PCR identification of medicinal materials of achyranthes and cyathula root, standard decoction and traditional Chinese medicine formula granules, which can effectively identify achyranthes and cyathula root.
The technical problem to be solved by the present invention is to provide an application of the above primer.
The technical problem to be solved by the invention is to provide an identification method based on the primer.
In order to solve the technical problems, the invention provides a primer for multiple PCR identification of achyranthes and cyathula root medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair and a second primer pair;
wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
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, respectively.
Correspondingly, the invention also discloses an application of the primer in (1) or (2):
(1) identifying whether the sample to be detected is achyranthes and/or cyathula;
(2) preparing a kit for identifying achyranthes and/or cyathula root.
As an improvement of the technical scheme, the achyranthes bidentata is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule, and the radix cyathulae is a medicinal material, a standard decoction or a traditional Chinese medicine formula granule.
Correspondingly, the invention also discloses an application of the primer in (1) or (2):
(1) identifying whether the sample to be detected contains achyranthes and/or cyathula;
(2) preparing a kit for identifying whether the sample to be detected contains achyranthes and/or cyathula root.
Correspondingly, the invention also discloses a kit which comprises the primer.
As an improvement of the technical scheme, the method also comprises a PCR premix. .
Correspondingly, the invention also discloses an identification method based on the primer for the multiplex PCR identification of the achyranthes and cyathula root medicinal materials, the standard decoction and the traditional Chinese medicine formula granules, which comprises the following steps:
extracting the genome DNA of a sample to be detected;
performing PCR amplification by using the primer by using the genome DNA as a template, wherein if an amplification product contains a DNA band of 160-170bp, the sample to be detected contains medicinal cyathula root, the standard decoction of the medicinal cyathula root and/or the traditional Chinese medicine formula particles of the medicinal cyathula root;
if the amplification product contains 180-190bp DNA band, the sample to be detected contains achyranthes bidentata medicine, achyranthes bidentata standard decoction and/or achyranthes bidentata Chinese medicine formula particles.
As an improvement of the above technical scheme, the method for extracting the genome DNA of the sample to be detected comprises the following steps:
adding CTAB precipitation liquid and proteinase K precipitation into a sample to be detected, and extracting for 2-3 times; adding CTAB extracting solution and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamylol for extraction for 2-3 times; and (3) adding isopropanol or isopropanol-sodium acetate into the extracted supernatant for precipitation and extraction, 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 the genome DNA of the sample to be detected comprises the following steps:
taking 0.03-0.08 g of a sample to be detected, grinding the sample to be detected into powder, putting the powder into a centrifuge tube, adding 1-1.8 mL of CTAB precipitation solution 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 CTAB precipitation solution and 15-25 mu L 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 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 chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 400-500 mu L of supernatant, adding isopropanol or isopropanol-sodium acetate mixed solution with the same volume, standing at-30 to-20 ℃ for 30-60 min, centrifuging, discarding supernatant, washing the precipitate with ethanol for 2-4 times, discarding supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, volatilizing ethanol, adding 30-50 mu L of sterilized water for dissolving, and obtaining the genomic DNA of the sample to be detected.
As an improvement of the technical scheme, 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).
As an improvement of the technical scheme, a template, a primer, a PCR premix solution 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 electrophoretic analysis on the amplification product, recording an electrophoretic map of the amplification product, and if the map contains a 164bp DNA strip, determining that the sample to be detected contains medicinal cyathula root, standard decoction of the medicinal cyathula root and/or traditional Chinese medicine formula particles of the medicinal cyathula root;
if the map contains 187bp DNA band, the sample to be detected contains achyranthes bidentata medicinal material, achyranthes bidentata standard decoction and/or achyranthes bidentata Chinese medicinal formula particles.
As an improvement of the technical scheme, the PCR amplification system consists of the following substances:
PCR premix 12.5. mu.L; 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 template is 1 mu L, ddH2O 10.3μL。
As an improvement of the technical scheme, the PCR amplification procedure comprises the following steps:
pre-denaturing the amplification system at 92-95 ℃ for 4-6 min, then circulating for 35-45 times under a preset program, and finally extending at 71-75 ℃ for 4-6 min;
wherein the preset program is: the amplification system is denatured at 94-96 ℃ for 28-32 s, annealed at 60-62 ℃ for 26-32 s, and then extended at 70-73 ℃ for 28-35 s.
As an improvement of the technical scheme, the PCR amplification procedure comprises the following steps:
pre-denaturing the amplification system at 95 ℃ for 5min, then cycling 40 times under a preset program, and finally extending at 72 ℃ for 5 min;
wherein the preset program is: the amplification system was denatured at 95 ℃ for 30s, then annealed at 61 ℃ for 30s, and then extended at 72 ℃ for 30 s.
As an improvement of the above technical solution, the electrophoretic analysis method comprises: and diluting the amplification product, then spotting the diluted amplification product on 1.5-2% agarose gel, performing electrophoresis for 45-60 min under the condition of 145-160V voltage, and performing Gelred color development to obtain an electrophoresis pattern.
The implementation of the invention has the following beneficial effects:
the primer disclosed by the invention can be used for carrying out a multi-site specificity PCR reaction, effectively identifying the achyranthes and the cyathula root, and is high in efficiency and detection precision.
2, the primer and the identification method can effectively overcome the problem of auxiliary material interference in the DNA extraction process, and can solve the problem that standard decoction and traditional Chinese medicine formula granules are difficult to identify after losing forms.
Drawings
FIG. 1 is a diagram showing the results of the electrophoresis detection of the samples of Achyranthis radix and Cyathula root after multiple PCR amplifications in example 3; wherein M is DNA molecular weight standard, 1-10 is radix Achyranthis bidentatae, 11-20 is radix Cyathulae, and N is blank control (ddH)2O)；
FIG. 2 is a diagram showing the results of the electrophoresis detection of the samples of Achyranthis radix and Cyathula root after multiple PCR amplifications in example 3; wherein M is DNA molecular weight standard, 21-30 is Achyranthis radix standard decoction (lyophilized powder), 31-40 is radix Cyathulae standard decoction (lyophilized powder), and N is blank control (ddH)2O)；
FIG. 3 shows the samples of achyranthes bidentata and Cyathula officinalis in example 3A product multiple PCR amplified electrophoresis detection result chart; wherein M is DNA molecular weight standard, 41-50 is Achyranthis radix granule, 51-60 is radix Cyathulae granule, and N is blank control (ddH)2O)。
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and detailed description.
The invention provides a primer for multiple PCR identification of medicinal materials of achyranthes and cyathula root, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair and a second primer pair, and the specific sequences are shown in the following table:
the primers can carry out multiple PCR reactions in the same PCR reaction system, have no interference with each other and are easy to amplify.
Based on the above characteristics, the primers of the present invention can be applied to:
(1) identifying whether the sample to be detected is achyranthes bidentata medicinal material, standard decoction or traditional Chinese medicine formula granule; or radix Cyathulae herbs, standard decoction, and Chinese medicinal granule.
(2) Identifying whether the sample to be detected contains achyranthes and/or cyathula.
(3) Preparing a kit for identifying achyranthes and/or cyathula root.
(4) Preparing a kit for identifying whether the sample to be detected contains achyranthes and/or cyathula root.
Correspondingly, the invention discloses a kit, which comprises the primer, PCR premix and water. The PCR premix may be 2 Xpfu PCR Mix (Tiangen Biochemical technology, Inc. (Beijing) K.201), but is not limited thereto.
The invention also discloses a primer-based identification method, which comprises the following steps:
firstly, extracting genome DNA of a sample to be detected;
wherein, the sample to be detected is provided in a solid state form, and the standard decoction is provided in a freeze-dried powder form (i.e. freeze-drying the standard decoction to obtain the freeze-dried powder).
Specifically, the extraction method comprises the following steps:
taking a sample to be detected, adding CTAB precipitation solution and proteinase K precipitation, and extracting for 2-3 times to obtain a precipitate;
specifically, taking 0.03-0.08 g of a sample to be detected, grinding the sample to be detected into powder, putting the powder into a centrifuge tube, adding 1-1.8 mL of CTAB precipitation solution 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 CTAB precipitation solution and 15-25 mu L proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant to obtain the product.
The CTAB precipitation solution had a composition of 2% (w/v) CTAB, 100mmol/L Tris-HCl (pH 8.0), and 20mmol/L EDTA (pH 8.0).
(II) adding CTAB extracting solution 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 μ L of CTAB extracting solution and 5-15 μ L of beta-mercaptoethanol into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, taking supernatant, adding chloroform-isoamyl alcohol (24:1) mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking supernatant 700-800 μ L, adding chloroform-isoamyl alcohol (24:1) mixed solution with the same volume, shaking, uniformly mixing, centrifuging, and taking supernatant 400-500 μ L to obtain the compound.
In the step, after extraction with the CTAB extract and the β -mercaptoethanol is completed, chloroform-isoamyl alcohol may be directly added, or chloroform-isoamyl alcohol may be added to the supernatant obtained after extraction, and chloroform-isoamyl alcohol may be added to the supernatant to improve the extraction accuracy.
Wherein, CTAB extracting solution: 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, 10-30% (w/v) PVP 40.
And (III) adding isopropanol or isopropanol-sodium acetate into the extracting solution for precipitation and extraction, washing and incubating precipitates, and dissolving the precipitates with water to obtain the genomic DNA of the sample to be detected.
Specifically, the extracting solution is taken, isopropanol or isopropanol-3 mol/L sodium acetate mixed solution with the same volume is added, standing is carried out for 30-60 min at the temperature of minus 30-minus 20 ℃, supernatant is discarded after centrifugation, washing and precipitating are carried out for 2-4 times by using ethanol, the supernatant is discarded, the precipitate is incubated for 20-40 min at the temperature of 35-38 ℃, and after the ethanol is volatilized, 30-50 mu L of sterilized water is added to dissolve, so that the genome DNA of the sample to be detected is obtained.
It should be noted that, when the applicant faces to the extraction of genomic DNA from the achyranthes/cyathula root standard decoction and the achyranthes/cyathula root traditional Chinese medicine formula particles, the traditional CTAB method, chelating resin method, triton-100 method, SDS method, alkaline lysis method, DNeasy Blood & Tissue Kit method and magnetic bead method are tried, but all insoluble precipitates are obtained. Therefore, the present inventors have obtained the above extraction method by improving a CTAB precipitation solution, a CTAB extraction solution, and an extraction procedure based on the conventional CTAB method. The extraction method retains genome DNA information of Achyranthis radix/radix Cyathulae standard decoction and Achyranthis radix/radix Cyathulae Chinese medicinal granule as much as possible, and can overcome the problem of adjuvant interference.
Forming a PCR amplification system, and carrying out amplification to obtain an amplification product;
specifically, uniformly mixing a template, a primer, a PCR premix solution and water to obtain a PCR amplification system;
more specifically, the total volume of the PCR amplification system was 25. mu.L, which included: PCR premix 12.5. mu.L; 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 template is 1 mu L, ddH2O 10.3μL。
The PCR premix may be 2 Xpfu PCR Mix (Tiangen Biochemical technology, Inc. (Beijing) K.201), 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 circulating for 35-45 times; finally, extending for 4-6 min at 71-75 ℃.
Preferably, the amplification procedure is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 61 deg.C for 30s, and extension at 72 deg.C for 30s, and circulating for 40 times, and finally extension at 72 deg.C for 5 min.
(3) Analyzing the amplification product;
specifically, the amplification product can be analyzed by electrophoresis or fluorescent staining, but is not limited thereto.
Preferably, the amplification product is analyzed by electrophoresis, which specifically comprises the following steps:
taking an amplification product, diluting the amplification product, spotting the diluted amplification product on 1.5-2% agarose gel, performing electrophoresis for 45-60 min under the condition of 145-160V voltage, and performing Gelred color development to obtain an electrophoresis pattern.
If the map contains 164bp DNA bands, the sample to be detected contains medicinal cyathula root, cyathula root standard decoction and/or cyathula root traditional Chinese medicine formula particles; if the map contains 187bp DNA band, the sample to be detected contains achyranthes bidentata medicinal material, achyranthes bidentata standard decoction and/or achyranthes bidentata Chinese medicinal formula particles.
The present invention will be described below with reference to specific examples.
Example 1 primer design
Based on the analysis of sequences of ITS, psbA-trnH, matK, trnL-trnF and the like of achyranthes medicinal plants in 2020 edition of Chinese pharmacopoeia, the ITS sequences of common achyranthes medicinal plants in a GeneBank database are subjected to homologous comparison by using BioEdit software, specific SNP sites of medicinal cyathula roots are analyzed after the comparison, and base sequences containing the SNP sites are introduced into Primer Premier 5 software for Primer design.
The sequence comparison shows that the specific site of the radix cyathulae is C at the 70 th site, the others are T, the specific site of the radix cyathulae is A at the 204 th site, the others are C or G, after the SNP site is determined, the SNP site is respectively close to the 3' ends of the forward and reverse primers, the Primer score and the 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 the final Primer score and the product band score. In the design process, the damage effect on the DNA base sequence after high-temperature extraction is also considered.
Combining the above factors, the obtained primer combinations are as follows:
NX-F：5’-AGCAGAATGACCAGCGAAC-3’；
NX-R：5’-CCAGGAAATCCGAGTAAGG-3’；
CNX-F：5’-GTTTTCAAGGGTCCTACGAGC-3’；
CNX-R：5’-GTGGGGAAAGGATGATGG-3’。
example 2 identification method establishment
(1) DNA extraction and concentration regulation
Taking 0.05g of a dried sample, grinding the dried sample into powder, placing the powder in a 2mL centrifuge tube, adding 1.5mL of CTAB precipitation solution preheated at 56 ℃ and 20 mu L of proteinase K, uniformly mixing, heating in a 56 ℃ water bath for 60min, cooling to room temperature, centrifuging for 5min at 10000r/min, removing supernatant, adding 900 mu L of CTAB precipitation solution and 20 mu L of proteinase K, and operating in the same way. Sequentially adding 900 mu L of CTAB extracting solution and 10 mu L of beta-mercaptoethanol into the centrifugal tube, uniformly mixing, heating in water bath at 65 ℃ for 120min, centrifuging, and taking supernatant after cooling to room temperature; adding chloroform-isoamyl alcohol (24:1) with the same volume, shaking for 3min, and mixing; centrifuging at 12000r/min at 4 deg.C for 10min, collecting supernatant 750 μ L, adding into new 2mL centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24:1), shaking for 3min, mixing, centrifuging at 12000r/min at 4 deg.C for 10 min; and taking 450 mu L of the supernatant, putting the supernatant into a 1.5mL centrifuge tube, adding isopropanol with the same volume, and standing for 30-60 min at-20 ℃. Taking out the centrifuge tube, centrifuging at 12000r/min for 5min, discarding supernatant, washing the precipitate with 75% ethanol and anhydrous ethanol twice, discarding supernatant, incubating the precipitate at 37 deg.C for 30min, volatilizing ethanol, and adding 30 μ L sterilized water to dissolve.
Wherein, the CTAB precipitation solution comprises the following components: 2% (w/v) CTAB (Shanghai leaf biotechnology limited), 100mM Tris-HCl (pH 8.0) (Beijing Solarbio), 20mM EDTA (pH 8.0) (Shanghai Yu Bo Biotech co., Ltd).
The CTAB extract consisted of: 2% (w/v) CTAB (Shanghai-leaf biotechnology, 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 longa science, Ltd), 20% PVP40 (Shanghai-leaf biotechnology, Ltd).
And (3) taking the DNA sample, determining the DNA concentration by using a BioSpec-nano micro ultraviolet spectrophotometer, simultaneously recording OD260/OD230 and OD260/OD280, and adjusting the concentration to 100 ng/mu L to obtain a DNA template.
(2) Design of primers
The primer sequence is shown as SEQ ID NO: 1 to 6.
(3) PCR amplification
PCR amplification System: 2 Xpfu PCR Mix (Tiangen Biochemical technology (Beijing) Co., Ltd., KP201) 12.5. mu.L, 10. mu. mol/L NX-F primer 0.3. mu.L, 10. mu. mol/L NX-R primer 0.3. mu.L, 10. mu. mol/L CNX-F primer 0.3. mu.L, 10. mu. mol/L CNX-R primer 0.3. mu.L, 100 ng/. mu.L DNA template 1. mu.L, deionized water 10.3. mu.L. And oscillating and uniformly mixing the liquid, and performing instantaneous centrifugation to obtain the PCR amplification system.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; finally, extension is carried out for 5min at 72 ℃.
(4) Electrophoretic detection
And adding 5 mu L of 6 × loading buffer into the amplification product, uniformly mixing, spotting 6 mu L of the mixed product on 2% agarose gel, performing electrophoresis for 45min under the condition of 150V voltage, developing color by Gelred, and observing and recording the result by a gel imager.
If the map contains 164bp DNA bands, the sample to be detected contains medicinal cyathula root, cyathula root standard decoction and/or cyathula root traditional Chinese medicine formula particles; if the map contains 187bp DNA band, the sample to be detected contains achyranthes bidentata medicinal material, achyranthes bidentata standard decoction and/or achyranthes bidentata Chinese medicinal formula particles.
Example 3 authentication method verification
Taking 60 batches of achyranthes and cyathula root samples of different producing areas and different sample forms. The samples were identified using the identification method established in example 2.
The identification results are shown in fig. 1 and fig. 2, and it can be seen from the figures that 187bp bands exist in achyranthes root medicinal material, achyranthes root standard decoction (freeze-dried powder) and achyranthes root traditional Chinese medicine formula particles; the Cyathula officinalis sample did not have the band. As can be seen from the figure, 164bp bands exist in the medicinal material of radix cyathulae, the standard decoction (lyophilized powder) of radix cyathulae and the traditional Chinese medicine formula granules of radix cyathulae; none of the achyranthes samples gave the band. The result shows that the accuracy of the identification method in the invention reaches 100%.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Sequence listing
<110> Guangdong-side pharmaceutical Co., Ltd
<120> primers for multiplex PCR identification of achyranthes and cyathula root medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccaggaaatc cgagtaagg 19
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gttttcaagg gtcctacgag c 21
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Claims (15)
1. A primer for multi-PCR identification of achyranthes and cyathula root medicinal materials, standard decoction and traditional Chinese medicine formula granules is characterized by comprising a first primer pair and a second primer pair;
wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
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, respectively.
2. The use of the primer according to claim 1 in (1) or (2):
(1) identifying whether the sample to be detected is achyranthes and/or cyathula;
(2) preparing a kit for identifying achyranthes and/or cyathula root.
3. The use as claimed in claim 2, wherein Achyranthis radix is a medicinal material, a standard decoction or a Chinese medicinal granule, and Cyathula root is a medicinal material, a standard decoction or a Chinese medicinal granule.
4. The use of the primer according to claim 1 in (1) or (2):
(1) identifying whether the sample to be detected contains achyranthes and/or cyathula;
(2) preparing a kit for identifying whether the sample to be detected contains achyranthes and/or cyathula root.
5. A kit comprising the primer of claim 1.
6. The kit of claim 5, further comprising a PCR premix.
7. The method for identifying the primers for the multiple PCR identification of the achyranthes and cyathula root medicinal materials, the standard decoction and the traditional Chinese medicine formula granules according to claim 1 is characterized by comprising the following steps:
extracting the genome DNA of a sample to be detected;
performing PCR amplification by using the genomic DNA as a template and the primer as claimed in claim 1, wherein if the amplification product contains a DNA band of 160-170bp, the sample to be detected contains radix Cyathulae medicinal material, radix Cyathulae standard decoction and/or radix Cyathulae Chinese medicinal formula particles;
if the amplification product contains 180-190bp DNA band, the sample to be detected contains achyranthes bidentata medicine, achyranthes bidentata standard decoction and/or achyranthes bidentata Chinese medicine formula particles.
8. The identification method according to claim 7, wherein the genomic DNA of the sample to be tested is extracted by the following method:
adding CTAB precipitation liquid and proteinase K precipitation into a sample to be detected, and extracting for 2-3 times; adding CTAB extracting solution and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamylol for extraction for 2-3 times; and (3) adding isopropanol or isopropanol-sodium acetate into the extracted supernatant for precipitation and extraction, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.
9. The identification method according to claim 7, wherein the genomic DNA of the sample to be tested is extracted by the following method:
taking 0.03-0.08 g of a sample to be detected, grinding the sample to be detected into powder, putting the powder into a centrifuge tube, adding 1-1.8 mL of CTAB precipitation solution 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 CTAB precipitation solution and 15-25 mu L 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 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 chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 400-500 mu L of supernatant, adding isopropanol or isopropanol-sodium acetate mixed solution with the same volume, standing at-30 to-20 ℃ for 30-60 min, centrifuging, discarding supernatant, washing the precipitate with ethanol for 2-4 times, discarding supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, volatilizing ethanol, adding 30-50 mu L of sterilized water for dissolving, and obtaining the genomic DNA of the sample to be detected.
10. The method of claim 8 or 9, 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).
11. The identification method according to claim 7, wherein the template, the primer, the PCR premix and water are mixed uniformly to obtain a PCR amplification system;
amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;
carrying out electrophoretic analysis on the amplification product, recording an electrophoretic map of the amplification product, and if the map contains a 164bp DNA strip, determining that the sample to be detected contains medicinal cyathula root, standard decoction of the medicinal cyathula root and/or traditional Chinese medicine formula particles of the medicinal cyathula root;
if the map contains 187bp DNA band, the sample to be detected contains achyranthes bidentata medicinal material, achyranthes bidentata standard decoction and/or achyranthes bidentata Chinese medicinal formula particles.
12. The method of claim 11, wherein the PCR amplification system consists of:
PCR premix 12.5. mu.L; 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 template is 1 mu L, ddH2O 10.3μL。
13. The identification method of claim 11, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 92-95 ℃ for 4-6 min, then circulating for 35-45 times under a preset program, and finally extending at 71-75 ℃ for 4-6 min;
wherein the preset program is: the amplification system is denatured at 94-96 ℃ for 28-32 s, annealed at 60-62 ℃ for 26-32 s, and then extended at 70-73 ℃ for 28-35 s.
14. The identification method of claim 13, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 95 ℃ for 5min, then cycling 40 times under a preset program, and finally extending at 72 ℃ for 5 min;
wherein the preset program is: the amplification system was denatured at 95 ℃ for 30s, then annealed at 61 ℃ for 30s, and then extended at 72 ℃ for 30 s.
15. The method of claim 11, wherein the electrophoretic analysis method is: and diluting the amplification product, then spotting the diluted amplification product on 1.5-2% agarose gel, performing electrophoresis for 45-60 min under the condition of 145-160V voltage, and performing Gelred color development to obtain an electrophoresis pattern.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111235328.8A CN113981105A (en) | 2021-10-22 | 2021-10-22 | Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111235328.8A CN113981105A (en) | 2021-10-22 | 2021-10-22 | Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113981105A true CN113981105A (en) | 2022-01-28 |
Family
ID=79740566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111235328.8A Pending CN113981105A (en) | 2021-10-22 | 2021-10-22 | Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113981105A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101933960A (en) * | 2010-09-15 | 2011-01-05 | 四川农业大学 | Method for identifying medicinal cyathula root and easily-confused species thereof |
| CN102242114A (en) * | 2011-02-23 | 2011-11-16 | 中国检验检疫科学研究院 | Method for extracting total DNA from soy and application thereof |
| TW201425586A (en) * | 2012-12-28 | 2014-07-01 | Ind Tech Res Inst | Method for identifying a plant medicinal material, and database and primer pair thereof |
| CN105002160A (en) * | 2015-07-15 | 2015-10-28 | 澳门科技大学 | Method for extracting DNA of Chinese patent medicines or Chinese herbal medicine-containing health care products |
| CN107557486A (en) * | 2017-08-31 | 2018-01-09 | 河南工业大学 | EST SSR primer sets and its acquisition methods based on the exploitation of root of bidentate achyranthes transcript profile |
-
2021
- 2021-10-22 CN CN202111235328.8A patent/CN113981105A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101933960A (en) * | 2010-09-15 | 2011-01-05 | 四川农业大学 | Method for identifying medicinal cyathula root and easily-confused species thereof |
| CN102242114A (en) * | 2011-02-23 | 2011-11-16 | 中国检验检疫科学研究院 | Method for extracting total DNA from soy and application thereof |
| TW201425586A (en) * | 2012-12-28 | 2014-07-01 | Ind Tech Res Inst | Method for identifying a plant medicinal material, and database and primer pair thereof |
| CN105002160A (en) * | 2015-07-15 | 2015-10-28 | 澳门科技大学 | Method for extracting DNA of Chinese patent medicines or Chinese herbal medicine-containing health care products |
| CN107557486A (en) * | 2017-08-31 | 2018-01-09 | 河南工业大学 | EST SSR primer sets and its acquisition methods based on the exploitation of root of bidentate achyranthes transcript profile |
Non-Patent Citations (2)
| Title |
|---|
| 孟虎彪等: "牛膝及川牛膝配方颗粒位点特异性PCR鉴别研究", 中国中药杂志, no. 5, pages 946 * |
| 陈诗晴等: "基于ITS序列分析技术鉴定川牛膝与常见伪品麻牛膝", 中药与临床, no. 4, pages 4 - 6 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105543373B (en) | Glycyrrhiza Uralensis, glycyrrhiza glabra, swollen fruit Radix and its Hybrid rapid molecular identification method | |
| CN113981105A (en) | Primer for multiplex PCR identification of achyranthes and cyathula medicinal materials, standard decoction and traditional Chinese medicine formula granules and application thereof | |
| CN108866205A (en) | Identify the specific primer of hiruto based on molecular biology method | |
| CN114045331A (en) | Primers for multiple PCR identification of Bungarus parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof | |
| CN109609679A (en) | Identify characteristic nucleotide sequence, nucleic acid molecular probe, kit and the method for ganoderma strain GIMS1524 | |
| CN114317797B (en) | DNA barcode sequence combination for identifying rehmannia and identification method | |
| CN116004838A (en) | Multiple STR typing primer for Bungarus Parvus medicinal material, standard decoction and traditional Chinese medicine formula granule, application thereof and identification method | |
| CN114657256A (en) | Primer combination for PCR identification of Bungarus parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof | |
| CN111979352A (en) | System for detecting pinellia ternata infecting virus by mRT-PCR and application thereof | |
| CN106399475A (en) | Method of acquiring rDNA ITS2 sequence for identification of dendrobium candidum | |
| CN110923331B (en) | Primer pair and application thereof in identification of limnodrilus | |
| CN114657255A (en) | Primer combination for PCR identification of agkistrodon medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof | |
| CN116004839A (en) | Primer for multiple STR typing of Zaocys medicinal material, standard decoction and traditional Chinese medicine formula granule, application thereof and identification method | |
| CN114657257A (en) | Primer combination for PCR identification of zaocys dhumnade medicinal material, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof | |
| CN113652496A (en) | Kit and method for identifying pholidota chinensis | |
| CN116814797A (en) | Primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, application thereof and identification method | |
| CN111041117A (en) | Araliaceae traditional Chinese medicine product identification kit and method | |
| CN111434781A (en) | PCR identification method of novel rehabilitation liquid | |
| CN114032328B (en) | Huaihu PCR identification kit and application | |
| CN104328220B (en) | RT-LAMP primer sets, detection method and the kit of citrus yellow vein virus | |
| CN114231659B (en) | Primer pair for detecting thistle plants in alfalfa, application of primer pair and detection method | |
| CN114107543B (en) | Hangzhou white chrysanthemum PCR identification kit and application | |
| CN116287424B (en) | Primer, probe and detection method for detecting rhizoma zingiberis pseudo-ginseng | |
| CN115976247A (en) | Primer, kit and identification method for nondestructively identifying American ginseng and ginseng | |
| KR102643216B1 (en) | Primer set for determining species and origin of cnidium, and method for determining species and origin of cnidium using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |