CN114317799A - Specific primer pair for identifying angelica and common angelica mixed counterfeit and application thereof - Google Patents
Specific primer pair for identifying angelica and common angelica mixed counterfeit and application thereof Download PDFInfo
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Abstract
The invention discloses an angelica sinensis specific primer pair, a PCR-RFLP reagent, a PCR-RFLP kit and a PCR-RFLP detection method. The angelica sinensis specific primer pair consists of a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID No.1 and a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID No. 2. The enzyme used in PCR-RFLP is restriction endonuclease SmlI. The PCR-RFLP method can not only detect the Chinese angelica plants and Chinese angelica medicinal materials, but also can accurately detect whether Chinese angelica is contained in the Chinese medicinal formula particles. The identification method is accurate and quick, and has higher specificity in identifying angelica and common angelica mixed counterfeit products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a specific primer pair for identifying angelica and common angelica adulterants and application thereof.
Background
The traditional Chinese medicine formula particle completely loses the identification characteristics of the traditional Chinese medicine decoction pieces, and the identification and supervision become one of bottleneck problems restricting the formula particle industry. Due to different production and control processes, the traditional Chinese medicine formula particles lack uniform and controllable quality standards, and are not effectively supervised in production, circulation and use links, so that the phenomenon that the traditional Chinese medicine formula particles are false and inferior is caused, and the clinical efficacy of the formula particles is influenced. The general administration of food and drug supervision and management in 12 months in 2015 issued a notice on soliciting opinions of management methods of traditional Chinese medicine formula granules (solicited for an opinion) (283 in 2015), wherein the stipulation that species including subspecies, varieties or varieties and different traditional Chinese medicinal materials cannot be mixed with each other should be accurately identified provides a new challenge for the quality control problem of formula granules, and a method with high accuracy and good stability needs to be established for identifying the species of the raw materials of the formula granules.
In 29 months 4 in 2021, the national drug administration issued the first 160 national standards for traditional Chinese medicine formulation granules, including the angelica formulation granule. The national standard of the traditional Chinese medicine formula granule takes 'standard decoction' as a reference to measure 'consistency' of the formula granule and decoction pieces, establishes a quantity value transmission data table and a characteristic map control index, and realizes comprehensive control of the specificity and integrity of the quality of the formula granule. The DNA molecule identification technology is more effective for distinguishing closely related species, and the increase of the DNA molecule identification method is helpful for further improving the control level of the primordial specificity of the species of the formula particles.
Disclosure of Invention
The invention provides an angelica specific primer pair, which consists of a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.1 and a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 2.
Optionally, the molar ratio of the single-stranded DNA molecule with the nucleotide sequence of SEQ ID No.1 to the single-stranded DNA molecule with the nucleotide sequence of SEQ ID No.2 in the angelica-specific primer pair is 1: 1.
The angelica specific primer pair can be used for identifying or assisting in identifying angelica and common angelica pseudolites, wherein the common angelica pseudolites are levisticum, angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and/or other species of angelica sinensis.
The invention also provides a reagent for identifying angelica, which comprises the angelica specific primer pair and the restriction endonuclease SmlI.
The reagent can be used for identifying or assisting in identifying angelica and common angelica pseudolites, wherein the common angelica pseudolites are levisticum, angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and/or other species of the angelica. The reagent may be a PCR-RFLP reagent.
The invention also provides a kit for identifying angelica, which comprises the angelica specific primer pair and the restriction endonuclease SmlI or comprises the reagent.
The kit can be used for identifying or assisting in identifying the angelica and common angelica pseudoliterary compositions, wherein the common angelica pseudoliterary compositions are angelica, angelica dahurica, peucedanum root, ligusticum, pubescent angelica root and/or other species of the angelica. The kit can be a PCR-RFLP kit.
As described above, the Angelica sinensis specific primer pair and the restriction endonuclease SmlI can be packaged separately.
The application of the angelica-specific primer pair, the reagent or the kit is also within the protection scope of the invention.
The application is any one of A1) -A8) as follows:
A1) the application in identification or auxiliary identification of angelica and common angelica mixed counterfeit products, wherein the common angelica mixed counterfeit products are angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and/or other species of angelica sinensis;
A2) the application in preparing products for identifying or assisting in identifying angelica and common angelica mixed counterfeit products, wherein the common angelica mixed counterfeit products are angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and/or other species of angelica sinensis;
A3) use in authentication or to assist in authentication of a token;
A4) the application in preparing the product for identifying or assisting in identifying the angelica;
A5) the application in identification or auxiliary identification of whether the sample to be detected contains the angelica or not;
A6) the application in preparing and identifying or assisting in identifying whether the to-be-detected sample contains the product of the Chinese angelica or not;
A7) the application in preparing the product for identifying the authenticity of the angelica formula particles;
A8) application in identifying authenticity of radix Angelicae sinensis formula granule is provided.
The invention also provides a method for detecting or assisting in detecting whether a sample to be detected is angelica sinensis or contains angelica sinensis, which comprises the following steps:
a1) carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the angelica sinensis specific primer pair to obtain an amplification product;
a2) carrying out enzyme digestion on the amplification product by SmlI to obtain an enzyme digestion product;
a3) detecting the enzyme digestion product by gel electrophoresis, and determining whether the sample to be detected is angelica or contains angelica according to the detection result as follows: if the enzyme digestion product only has a single DNA band between 100-200 bp, the sample to be detected is or is candidate to be angelica, or contains or is candidate to contain angelica; if the enzyme digestion product does not have a single DNA band only between 100-200 bp, the sample to be detected is not or does not candidate to be angelica sinensis, or does not contain or does not candidate to contain angelica sinensis.
Specifically, in the above method, the single DNA band between 100-200 bp is divided into the following 3 cases:
b1) no band;
b2) there are a plurality of strips.
Specifically, in the method, the primer annealing condition adopted by the PCR amplification is 60 ℃ annealing for 2 min.
Specifically, in the above method, the PCR reaction procedure adopted in the PCR amplification is: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 2min, extension at 72 ℃ for 30s, and 40 cycles; final extension at 72 ℃ for 60 min.
Specifically, in the method, the concentration of the template DNA in the PCR reaction system is 4.5-13.3 ng/. mu.L, and the Taq DNA polymerase is 2 XM 5 SuperFast Taq PCR MasterMix.
Specifically, in the method, the amount of the SmlI restriction enzyme in the enzyme digestion system is 5000U/30 mu L, and the enzyme digestion time is 60 min.
The invention establishes a PCR-RFLP detection system which can simultaneously distinguish angelica and common angelica mixed counterfeit angelica, angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and/or other species of angelica.
The primer pair, the PCR-RFLP reagent and the PCR-RFLP kit are accurate and rapid, and have higher accuracy and adaptability and important application value in the aspect of identifying the angelica and common angelica mixed counterfeit products (European angelica, east angelica, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and/or other species of the angelica), especially whether the Chinese medicinal formula particles contain the angelica or not.
Drawings
FIG. 1 is a design diagram of the specific enzyme digestion identification primer for angelica sinensis of the present invention.
FIG. 2 shows the effect of annealing temperature of PCR reaction on the identification result of specific PCR-RFLP of Angelica sinensis in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 3 is a graph showing the influence of the number of PCR cycles on the result of the PCR-RFLP identification of Angelica sinensis in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 4 shows the effect of different polymerase species on the identification result of the PCR-RFLP of Angelica sinensis in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 5 is a graph showing the effect of the amount of the starting DNA template on the identification result of Angelica sinensis-specific PCR in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 6 shows the effect of different PCR amplification apparatus on the identification result of Angelica sinensis PCR-RFLP in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 7 shows the effect of the amount of restriction enzyme on the result of the PCR-RFLP identification of Angelica sinensis in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 8 shows the effect of restriction enzyme digestion time on the identification result of PCR-RFLP of Angelica sinensis in example 1 of the present invention. M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 9 shows the effect of restriction enzyme species on the result of PCR-RFLP identification of Angelica sinensis in example 1 of the present invention. In the figure, M: trans2K DNA Marker; as: radix Angelicae sinensis; AsE: angelica formula granule extract; AsG: angelica sinensis formula granules; lo: levisticum officinale; ad: radix Angelicae Dahuricae; pp: radix peucedani; ap: radix angelicae pubescentis; asp: wild angelica; ls: ligusticum; n: blank control.
FIG. 10 shows the PCR-RFLP identification results of different sources of Dang Gui and its mixture of counterfeit in example 2. In the figure, m.trans2K DNA Marker; 1-31: radix Angelicae sinensis of different sources; 32-33: angelica sinensis is disclosed; 34-35: the angelica dahurica is described in the specification; 36-37: radix peucedani; 38-39: radix angelicae pubescentis; 40: wild angelica; 41: ligusticum; n: blank control.
FIG. 11 shows the identification results of different batches of Dang Gui formula granules in example 2. In the figure, m.trans2k DNA Marker; 1-3: radix Angelicae sinensis granule extract (7190417C,7190207C-03,7190305C); 4-24: angelica sinensis formula granules, 4-13: huarun sanjiu (1903009W,1903003W,1903005W,1801009W,1801005W,1807001W,1903001W,1801007W,1901001W,1801011W), 14-15: conrentang (18007822, KRTDG02), 16-17: jiangyin Tianjiang (19070491,19070481), 18-22: guangdong (9050342,9059011,9025892,9049211,9050332), 23: sichuan new green (XLSDG03), 24: jiangxi Baishen (190925122512).
FIG. 12 shows the identification results of counterfeit mixture of angelica granules in different enterprises and batches in example 2 of the present invention. In the figure, m.trans2K DNA Marker; 1: chinese angelica medicinal material; 2: angelica sinensis formula (1903009W); 3-6: pubescent angelica root dispensing granule (1910004W,19112151,20021731,9056881); 7-9: peucedanum praeruptorum formula granules (1904003C,19121351,9075041); 10-12: radix Angelicae Dahuricae granule (1911001W,20020101,9081681); 13-15: ligusticum formulation granules (1909001W,19100171,9090451); n: blank control.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction and optimization of PCR-RFLP identification primer and identification method
Design of PCR-RFLP identification primer for angelica
The ITS sequences of genuine angelica and common angelica mixed counterfeit products (angelica archang lica, angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and other species of the angelica genus) are searched by sequencing and from a GenBank database. Performing sequence comparison by using BioEdit software, wherein the result is shown in figure 1, screening out a gene locus with interspecific specificity and proper length in the angelica genome, wherein the gene sequence is CTCAAG, accords with the characteristics of a recognition sequence (5'-CTYRAG-3') of the SmlI enzyme, and can be cut by the SmlI enzyme; the gene sequences of the common angelica mixed counterfeit products of the angelica sinensis, the angelica dahurica, the radix peucedani, the ligusticum, the radix angelicae pubescentis and the corresponding positions of other species of the angelica belong to do not accord with the characteristics of a SmlI recognition sequence (CTYRAG) and can not be cut by SmlI enzyme.
Aiming at the characteristics of the gene locus and the recognition sequence of SmlI, a primer pair consisting of Danggui-224F and Danggui-224R is designed, and the specific sequence is as follows:
Danggui-224F: 5'-CTCGTGGAGCTGTACTGGTA-3' (shown in SEQ ID No. 1);
Danggui-224R: 5'-GGTAGTCCCGCCTGACCT-3' (shown in SEQ ID No. 2).
After PCR amplification and enzyme digestion by SmlI endonuclease (polymerase chain reaction-restriction endonuclease digestion length polymorphism map, PCR-RFLP), the angelica can be digested into 162bp and 62bp short fragments due to the recognition sequence of the SmlI endonuclease, and common angelica counterfeit products (angelica sinensis, angelica dahurica, radix peucedani, ligusticum, radix angelicae pubescentis and other species in the same country) do not have the SmlI endonuclease recognition sequence and cannot be digested, and 224bp bands are still reserved or no bands are left due to the fact that the angelica sinensis cannot be amplified.
Construction of PCR-RFLP detection method
And (3) carrying out PCR amplification on the genome DNA of each sample to be detected by using angelica as a positive control and sterile double-distilled water as a blank control and adopting primers consisting of Danggui-224F and Danggui-224R.
The PCR reaction system is 2 XM 5 SuperFast Taq PCR Master Mix 12.5 muL, the upstream primer Danggui-224F (10 mumol/L) and the downstream primer Danggui-224R (10 mumol/L) are respectively 0.4 muL, the template DNA is 2 muL (20ng) (the concentration of the template DNA in the reaction system can be selected from 4.5-13.3 ng/muL), and the double-distilled sterile water is 9.7 muL.
The PCR amplification cycle program and parameters were as follows:
initial denaturation conditions: 95 ℃ for 5 min;
and (3) denaturation conditions: at 95 ℃ for 30 s;
annealing conditions: 30s at 60 ℃;
and (3) extension conditions: 72 ℃ for 30 s;
the number of cycles: 40, the number of the channels is 40;
final extension conditions: 72 ℃ for 5 min;
and (3) heat preservation: maintaining the temperature at 4 ℃.
The PCR amplification product was digested with restriction enzyme SmlI.
The enzyme digestion reaction system is as follows: PCR reaction product solution 25. mu.L, restriction enzyme buffer 10 X3. mu.L, SmlI restriction enzyme 0.5. mu.L, make up the reaction volume to 30. mu.L with sterile double distilled water.
The enzyme digestion time is 60 min.
5 mu L of the enzyme digestion reaction solution is subjected to 2% agarose gel electrophoresis, is stained by GelRed nucleic acid dye and is placed on a gel imager or an ultraviolet transmission instrument for imaging.
The result judgment standard is as follows: and under the condition that the positive control result and the blank control result meet the regulation, the positive result (namely the detected sample contains angelica) is obtained when only a single DNA band exists between 100 bp and 200bp (such as 162bp) on the corresponding lane of the detected sample, and the negative result (namely the detected sample does not contain angelica) is obtained otherwise.
The specific optimization process of the PCR amplification mode and the enzyme digestion mode is as follows:
1. optimization of PCR amplification parameters
Respectively selecting 1 of angelica medicinal material, angelica formula particle extract, angelica formula particle, and mixed counterfeit products of angelica sinensis, angelica dahurica, radix peucedani, radix angelicae pubescentis, wild angelica and ligusticum to be detected, extracting genome DNA as a template, and carrying out PCR parameter optimization.
1.1 annealing temperature investigation
The annealing temperatures are respectively set to be 58 ℃, 60 ℃, 62 and 64 ℃, other parameters are kept unchanged, the result shows that at 58-62 ℃, the angelica medicinal material, the extract and the formula particle positive product can be amplified to obtain a bright band of 224bp, the negative control is not amplified in the range, the band of the angelica formula particle is obviously weakened at 64 ℃, and the angelica extract has no band, as shown in figure 2. To ensure that the DNA product before the PCR-RFLP digestion is in sufficient quantity, the PCR annealing temperature is determined to be 60 ℃.
1.2 cycle number examination
36, 38, 40 and 42 cycles are respectively selected for investigation, and other parameters are kept unchanged, the result shows that PCR amplification bands of 224bp can be seen in gel electrophoresis pattern angelica medicinal materials, extractum and formula particles of 36 cycles, and DNA bands are obvious in 38-42 cycles (see figure 3). Because the content and the quality of the DNA of the angelica formula particles are lower than those of the angelica samples, 40 cycles of PCR reaction are subsequently selected to ensure the stability and the accuracy of the result.
1.3 Effect of polymerase on PCR identification
To further determine the applicability of different Taq DNA polymerases to the PCR identification of Angelica sinensis formula particles, Taq enzymes and Mix thereof from different companies were selected for testing, including 2 XT 5 SuperPCR Mix (Beijing Pongku New Biotechnology Co., Ltd.), 2 XM 5 SuperFast Taq PCR Mastermix (Med., Ltd.), MightyAmp DNA Polymerase Ver.2(Takara Biotechnology Co., Ltd.), 2 for the purpose of
FastPfu Fly PCR SuperMix (all-gold Biotechnology Co., Ltd.) was tested. Configuring cycle parameters of a PCR amplification system: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 2min, extension at 72 ℃ for 30s, and 40 cycles; final extension at 72 deg.C for 60 min; keeping the temperature at 4 ℃.
The result is shown in figure 4, 2 XM 5 SuperFast Taq PCR MasterMix and MightyAmp DNA Polymerase Ver.2 can amplify a single strip of 224bp in angelica medicinal material, angelica formula granule extract and angelica formula granule, 2F
The FastPfu Fly PCR SuperMix can not amplify a band from the formula particle DNA, the 2 XT 5 SuperPCR Mix amplification band is weak, downstream enzyme digestion identification operation is difficult to carry out, and 2 XM 5 SuperFast Taq PCR MasterMix is selected for subsequent research.
1.4 template DNA concentration investigation
Adjusting the concentration of the DNA template to 40 ng/. mu.L, performing gradient dilution to form concentration series of 40 ng/. mu.L, 13.3 ng/. mu.L, 4.5 ng/. mu.L and 1.5 ng/. mu.L, performing PCR amplification, taking 2 XM 5 SuperFast Taq PCR MasterMix as Taq DNA polymerase, and performing PCR amplification system parameters: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 2min, extension at 72 ℃ for 30s, and 40 cycles; final extension at 72 deg.C for 60 min; keeping the temperature at 4 ℃.
The result is shown in figure 5, which shows that the angelica sinensis medicinal material, the extract and the formula particle can be amplified to obtain a 224bp band when the concentration of the template DNA is 4.5-13.3 ng/mu L, however, the PCR inhibition phenomenon exists when the amount of the PCR initial DNA template is too high (40ng), the extract and the formula particle cannot be amplified, and the initial DNA template amount is determined to be 4.5-13.3 ng/mu L.
1.5 influence of PCR Instrument on PCR identification of Angelica sinensis formula granules
The concentration of template DNA in the PCR reaction system is 10 ng/. mu.L, 2 XM 5 SuperFast Taq PCR MasterMix is used as Taq DNA polymerase, and the PCR amplification system parameters are as follows: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 2min, extension at 72 ℃ for 30s, and 40 cycles; final extension at 72 deg.C for 60 min; keeping the temperature at 4 ℃. Different manufacturers are respectively selected for PCR amplification, except that 7500 type PCR instruments (Applied Biosystems company) are weak in amplification bands, Veriti type PCR instruments (Applied Biosystems company), PCT-100 type PCR instruments (Gene company) and TC-512 type PCR instruments (Techne company) are used, and the Angelica sinensis medicinal material, the Angelica sinensis formula particle extract and the Angelica sinensis formula particles can be amplified to obtain 224bp specific bands (shown in figure 6), which indicates that different PCR instruments have no influence on the Angelica sinensis PCR-RFLP identification result.
After the examination of the above conditions, the optimal PCR reaction parameters are obtained: the concentration of template DNA in a PCR reaction system is 4.5-13.3 ng/. mu.L, 2 XM 5 SuperFast Taq PCR MasterMix is used as Taq DNA polymerase, and an amplification system: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 2min, extension at 72 ℃ for 30s, and 40 cycles; final extension at 72 deg.C for 60 min; keeping the temperature at 4 ℃.
2. Optimization of SmlI enzyme digestion conditions
Respectively selecting 1 of angelica medicinal material, angelica formula particle extract, angelica formula particle, and mixed counterfeit products of angelica sinensis, angelica dahurica, radix peucedani, radix angelicae pubescentis, wild angelica and ligusticum to be detected, extracting genome DNA, and taking the amplification product to perform enzyme digestion condition investigation by using the PCR reaction parameters optimized in the step 1.
2.1 examination of the amount of restriction enzyme
Firstly, carrying out restriction enzyme dosage investigation, and configuring a series of restriction enzyme digestion reaction systems: the PCR reaction product solution (25. mu.L), 10 Xrestriction enzyme buffer (3. mu.L, NEB Co.), containing SmlI restriction enzyme (10000U/. mu.L, NEB Co.), was 0.125. mu.L, 0.25. mu.L, 0.5. mu.L or 1. mu.L, respectively, and the reaction volume was made up to 30. mu.L with sterile double distilled water, respectively. The reaction solution is shaken and mixed evenly and then is centrifuged instantly. The reaction solution was placed in a PCR apparatus and incubated at 55 ℃ for 60 min.
After gel electrophoresis of the cleavage products, the results are shown in FIG. 7: when the restriction enzyme is more than or equal to 0.25 mu L, the angelica medicinal material, the formula particle extract and the formula particle PCR product can be completely cut by enzyme to obtain a specific enzyme cutting strip of about 160bp, the mixed counterfeit product can not be cut by enzyme, the amplification strip of 224bp or no strip can be still kept, the restriction enzyme can not be completely cut when the dosage of the restriction enzyme is 0.125 mu L to form a strip of 224bp +160bp (see figure 7), and the SmlI restriction enzyme in the enzyme cutting system is 5000U/30 mu L to ensure the enzyme cutting effect.
2.2 restriction enzyme digestion time study
Preparing a series of restriction enzyme digestion reaction systems: the PCR reaction product solution (25. mu.L), 10 Xrestriction enzyme buffer (3. mu.L, NEB Co.), containing SmlI restriction enzyme (10000U/. mu.L, NEB Co.), 0.5. mu.L, was made up to a reaction volume of 30. mu.L with sterile double distilled water. The reaction solution is shaken and mixed evenly and then is centrifuged instantly. The reaction solution was placed in a PCR apparatus and heat-preserved at 55 ℃. And respectively setting the heat preservation time to be 30min, 60min, 120min or 240min for enzyme digestion, wherein the PCR product can be completely digested after 30min, star activity does not occur even if the incubation time is 240min (see figure 8), and the restriction enzyme digestion time is determined to be 60 min.
2.3 restriction enzyme species investigation
In order to further determine the applicability of different endonucleases to the PCR identification of the angelica formula particles, SmlI endonucleases of 2 different production enterprises such as NEB, Thermo and the like are selected to carry out enzyme digestion tests, the endonucleases of the different enterprises obtain correct enzyme digestion results, the angelica medicinal material, the angelica formula particle extract and the angelica formula particle PCR product are all subjected to enzyme digestion to obtain a specific strip of about 160bp, a mixed counterfeit product cannot be subjected to enzyme digestion, and an amplification strip of 224bp or no strip is still kept (see figure 9).
Through the investigation of the conditions, the SmlI restriction enzyme in the optimized enzyme digestion system is 5000U/30 mu L, and the enzyme digestion time is 60 min.
Example 2 accuracy and suitability examination of PCR-RFLP method
According to the national traditional Chinese medicine resource general survey specimen bank specimen, 31 batches of angelica are collected from Tibet, Sichuan, Gansu, Hubei and the like, and 34 batches of medicinal material samples are counted in total from the samples of the European angelica, wild angelica, angelica dahurica, radix peucedani and ligusticum, which are detailed in table 1.
Formula particle samples such as angelica sinensis (24 batches), angelica dahurica (3 batches), peucedanum root (3 batches), radix angelicae pubescentis (3 batches), ligusticum (3 batches) and the like are collected from mainstream formula particle manufacturers such as sanjiu huan group, tianjiang-yin tianjiang pharmaceutical industry, beijing kannren pharmaceutical industry, guangdong party pharmaceutical industry, sichuan new lotus pharmaceutical industry, Hunan Chunshou Jiuhui pharmaceutical industry and the like, and the detailed table is shown in table 2. After the medicinal materials are subjected to character identification, DNA sequencing is used for auxiliary identification, and the result of consistency of the two is taken as the final identification result.
TABLE 1 original plant and medicinal material sample Table
| Serial number | Sample name | Type of sample | Name of scholars | Source ground | Number of batches (batch) | |
| 1 | Radix Angelicae sinensis | Original plant | Angelica sinensis | All-grass of Tibetan |
10 | |
| 2 | Radix Angelicae sinensis | Original plant | Angelica sinensis | Wenchun, |
5 | |
| 3 | Radix Angelicae sinensis | Original plant | Angelica sinensis | Enshi in |
10 | |
| 4 | Radix Angelicae sinensis | Medicinal materials | Angelica sinensis | (Anguo) | 3 | |
| 5 | Radix Angelicae sinensis | Medicinal materials | Angelica sinensis | |
3 | |
| 6 | Radix Angelicae sinensis | Original plant | Levisticum officinale | Qinghai Xining (a medicine for treating psoriasis) | 8 | |
| 7 | Radix Angelicae sinensis | Medicinal materials | Levisticum officinale | (Anguo) | 2 | |
| 8 | Radix Angelicae sinensis | Medicinal materials | Angelica sp. | Sichuan | 4 | |
| 9 | Radix Angelicae sinensis | Medicinal materials | Angelica sp. | Sichuan Ganzui (rhizoma Et radix Valerianae) | 3 | |
| 10 | Radix Angelicae Pubescentis | Medicinal materials | Angelica pubescens | (Anguo) | 3 | |
| 11 | Root of Dahurian Angelica | Medicinal materials | Angelica dahurica | (Anguo) | 7 | |
| 12 | Radix peucedani | Medicinal materials | Peucedanum | Bozhou nationality | 7 | |
| 13 | Ligusticum sinense (oliv.) Diels | Medicinal materials | Ligusticum sinense | |
3 |
Table 2 sample tables of formula granules
Note: in Table 2, CR is Huarun Sanjiu group, BTC is Beijing Kanrandtang pharmaceutical industry, JTP is Jiangyin Tianjiang pharmaceutical industry, Yifang is Guangdong one-party pharmacy, Neo is Sichuan new green pharmaceutical industry, and Bai is Jiangxi Baishen pharmaceutical industry.
1 accuracy examination of identification method
The PCR-RFLP primer pairs prepared in example 1 are used for detecting the angelica original plant and the angelica medicinal material with different sources in the table 1 respectively by adopting the PCR-RFLP detection method in example 1.
The result is shown in figure 10, all angelica genuine products are subjected to PCR-RFLP reaction to obtain a specific identification strip of 160bp, and common angelica mixed counterfeit products from different sources, including angelica archang lica, angelica dahurica, peucedanum root, radix angelicae pubescentis, wild angelica and ligusticum sinensis, are not amplified or are 224bp strips.
2 method suitability examination
The PCR-RFLP detection method in example 1 was used to detect different sources of Angelica sinensis formula particles in Table 2, respectively, using the PCR-RFLP primer pair prepared in example 1.
The results are shown in fig. 11 and fig. 12, which show that the system can stably and accurately identify the angelica formula particles, all the angelica formula particles can be subjected to enzyme digestion to obtain a specific identification band of about 160bp, and the mixed counterfeit formula particles have no amplification band at corresponding positions.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine
<120> specific primer pair for identifying angelica and common angelica mixed counterfeit and application thereof
<130> GNCSY213299
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (10)
1. The angelica specific primer pair is characterized in that: the angelica sinensis specific primer pair consists of a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.1 and a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 2.
2. The angelica-specific primer pair according to claim 1, wherein: the molar ratio of the single-stranded DNA molecule with the nucleotide sequence of SEQ ID No.1 to the single-stranded DNA molecule with the nucleotide sequence of SEQ ID No.2 in the angelica specific primer pair is 1: 1.
3. A reagent for identifying angelica, characterized in that: comprises the angelica-specific primer pair of claim 1 or 2 and a restriction endonuclease SmlI.
4. The kit for identifying the angelica sinensis is characterized by comprising the following components in parts by weight: comprising the angelica-specific primer pair of claim 1 or 2 and the restriction endonuclease SmlI, or comprising the reagent of claim 3.
5. Use of the angelica-specific primer pair of claim 1 or 2, the reagent of claim 3, or the kit of claim 4) in any one of the following a1) -A8);
A1) the application in identification or auxiliary identification of angelica and common angelica mixed counterfeit products, wherein the common angelica mixed counterfeit products are angelica sinensis, angelica dahurica, radix peucedani, ligusticum and/or radix angelicae pubescentis;
A2) the application in preparing products for identifying or assisting in identifying angelica and common angelica pseudolites is that the common angelica pseudolites are levisticum, angelica sinensis, angelica dahurica, radix peucedani, ligusticum and/or radix angelicae pubescentis;
A3) use in authentication or to assist in authentication of a token;
A4) the application in preparing the product for identifying or assisting in identifying the angelica;
A5) the application in identification or auxiliary identification of whether the sample to be detected contains the angelica or not;
A6) the application in preparing and identifying or assisting in identifying whether the to-be-detected sample contains the product of the Chinese angelica or not;
A7) the application in preparing the product for identifying the authenticity of the angelica formula particles;
A8) application in identifying authenticity of radix Angelicae sinensis formula granule is provided.
6. A method for detecting or assisting in detecting whether a sample to be detected is angelica sinensis or contains angelica sinensis is characterized in that: the method comprises the following steps:
a1) carrying out PCR amplification on the genome DNA of the sample to be detected by adopting the angelica-specific primer pair of claim 1 or 2 to obtain an amplification product;
a2) carrying out enzyme digestion on the amplification product by SmlI to obtain an enzyme digestion product;
a3) detecting the enzyme digestion product by gel electrophoresis, and determining whether the sample to be detected is angelica or contains angelica according to the detection result as follows: if the enzyme digestion product only has a single DNA band between 100-200 bp, the sample to be detected is or is candidate to be angelica, or contains or is candidate to contain angelica; if the enzyme digestion product does not have a single DNA band only between 100-200 bp, the sample to be detected is not or does not candidate to be angelica sinensis, or does not contain or does not candidate to contain angelica sinensis.
7. The method of claim 6, wherein: the primer annealing condition adopted by the PCR amplification is 60 ℃ annealing for 2 min.
8. The method of claim 7, wherein: the PCR reaction procedure adopted in the PCR amplification is as follows: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 2min, extension at 72 ℃ for 30s, and 40 cycles; final extension at 72 ℃ for 60 min.
9. The method according to claim 6 or 7, characterized in that: in the method, the concentration of template DNA in a PCR reaction system is 4.5-13.3 ng/mu L, and Taq DNA polymerase is 2 XM 5 SuperFast Taq PCR MasterMix.
10. The method according to claim 6 or 7, characterized in that: in the method, the using amount of the SmlI restriction enzyme in the enzyme digestion system is 5000U/30 mu L, and the enzyme digestion time is 60 min.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007175044A (en) * | 2005-12-28 | 2007-07-12 | Univ Of Miyazaki | Quick discrimination kit of angelica furcijuga by method for analyzing dna polymorphism, and quick method for discriminating the same |
| US20080311627A1 (en) * | 2004-06-17 | 2008-12-18 | Epigenomics Ag | Compositions and Methods for Preventing Carry-Over Contamination in Nucleic Acid Amplification Reactions |
| CN104004818A (en) * | 2013-02-26 | 2014-08-27 | 中国医学科学院药用植物研究所 | Method for identifying Chinese angelica and its adulterants by utilizing ITS sequence |
| US20140322717A1 (en) * | 2011-11-18 | 2014-10-30 | The Trustees Of Columbia University In The City Of New York | Selective amplification and detection of mutant gene alleles |
| CN104911256A (en) * | 2015-03-10 | 2015-09-16 | 泸州医学院 | Chinese angelica SCAR molecular marker, its identification method and specific primer pair |
| CN105274213A (en) * | 2015-09-24 | 2016-01-27 | 郑州大学 | Method and kit for determination of human PLIN1 gene rs894160 site polymorphism |
| CN106367502A (en) * | 2016-08-31 | 2017-02-01 | 甘肃农业大学 | Gene SPP1 as molecular marker for ovine growth traits and application of molecular marker |
| US20170335369A1 (en) * | 2014-08-01 | 2017-11-23 | Dovetail Genomics, Llc | Tagging nucleic acids for sequence assembly |
| CN109266773A (en) * | 2017-07-14 | 2019-01-25 | 贵州汉方药业有限公司 | A kind of DNA bar code identification method of Qijiaoshenbai capsule raw medicinal material |
| US20200370096A1 (en) * | 2018-01-31 | 2020-11-26 | Dovetail Genomics, Llc | Sample prep for dna linkage recovery |
-
2021
- 2021-12-21 CN CN202111572614.3A patent/CN114317799B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080311627A1 (en) * | 2004-06-17 | 2008-12-18 | Epigenomics Ag | Compositions and Methods for Preventing Carry-Over Contamination in Nucleic Acid Amplification Reactions |
| JP2007175044A (en) * | 2005-12-28 | 2007-07-12 | Univ Of Miyazaki | Quick discrimination kit of angelica furcijuga by method for analyzing dna polymorphism, and quick method for discriminating the same |
| US20140322717A1 (en) * | 2011-11-18 | 2014-10-30 | The Trustees Of Columbia University In The City Of New York | Selective amplification and detection of mutant gene alleles |
| CN104004818A (en) * | 2013-02-26 | 2014-08-27 | 中国医学科学院药用植物研究所 | Method for identifying Chinese angelica and its adulterants by utilizing ITS sequence |
| US20170335369A1 (en) * | 2014-08-01 | 2017-11-23 | Dovetail Genomics, Llc | Tagging nucleic acids for sequence assembly |
| CN104911256A (en) * | 2015-03-10 | 2015-09-16 | 泸州医学院 | Chinese angelica SCAR molecular marker, its identification method and specific primer pair |
| CN105274213A (en) * | 2015-09-24 | 2016-01-27 | 郑州大学 | Method and kit for determination of human PLIN1 gene rs894160 site polymorphism |
| CN106367502A (en) * | 2016-08-31 | 2017-02-01 | 甘肃农业大学 | Gene SPP1 as molecular marker for ovine growth traits and application of molecular marker |
| CN109266773A (en) * | 2017-07-14 | 2019-01-25 | 贵州汉方药业有限公司 | A kind of DNA bar code identification method of Qijiaoshenbai capsule raw medicinal material |
| US20200370096A1 (en) * | 2018-01-31 | 2020-11-26 | Dovetail Genomics, Llc | Sample prep for dna linkage recovery |
Non-Patent Citations (3)
| Title |
|---|
| 史中飞 等: "PCR-RFLP鉴别当归药材及饮片中掺混伪品——欧当归的方法", 中国实验方剂学杂志, vol. 27, no. 9, pages 168 - 175 * |
| 时圣明 等: "分子鉴定技术在中药中的应用", 中草药, vol. 47, no. 17, pages 3121 - 3126 * |
| 赵国平 等: "伞形科常用中药品种rDNA序列特征及其标记方法研究", 中药材, vol. 29, no. 11, pages 1148 - 1153 * |
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