A method for identifying Clematidis armandii Franch.

Abstract

The present invention relates to a method for identifying Clematidis Armandii, belonging to the technical field of pharmaceuticals, and comprising the following steps: S1, extracting the DNA of the sample to be detected; S2, performing PCR amplification using the DNA of the sample to be detected as a template; S3, performing electrophoresis detection on the amplification product. In the technical solution of the present invention, a modified magnetic bead is added during the process of extracting the DNA of the sample to be detected. The surface of the modified magnetic bead contains a high content of metal ions, and the metal ions can interact with the phosphate groups on the DNA so as to achieve the purpose of separating and extracting the DNA. The DNA extraction method provided by the present invention can effectively improve the purity and concentration of the DNA template, is beneficial to the subsequent PCR amplification reaction, can effectively identify the Clematidis Armandii medicinal materials, and improves the accuracy rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and specifically relates to a method for identifying Clematidis armandii Caulis. Background Art

[0002] The traditional Chinese medicine Clematidis armandii Caulis is the dried vine stem of the Ranunculaceae plant Clematis armandii Franch. or Clematis montana Buch.-Ham., and has the effects of promoting diuresis and relieving stranguria, clearing the heart and relieving restlessness, and promoting menstruation and lactation. Clematidis armandii Caulis is mainly produced in regions such as Sichuan, Guizhou, Yunnan, and Tibet, and has rich wild resources and is a commonly used traditional Chinese medicine. Clematidis armandii Caulis has high medicinal value and development value and low toxicity. However, the appearance of the vine stem of Clematidis armandii Caulis is highly similar to that of the vine stem of Akebia quinata (Thunb.) Decne. or Akebia trifoliata (Thunb.) Koidz. of the Lardizabalaceae family, and the names are easily confused, resulting in difficulties in conventional identification, causing chaos in the market circulation and bringing potential safety hazards.

[0003] Since DNA is a stable genetic material and the DNA of different species varies greatly, in recent years, the polymerase chain reaction (PCR) technology has often been used to amplify specific genes, and means such as sequence comparison are used to identify the authenticity of medicinal materials. Although the PCR amplification technology has been relatively mature, it is affected by various factors such as organic solvents and proteins, so this technology has high requirements for the purity and concentration of the DNA template. Therefore, there is an urgent need to provide a method for identifying Clematidis armandii Caulis that can solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for identifying Clematidis armandii Caulis. By adding modified magnetic beads during the process of extracting DNA from the sample to be detected, the surface of the modified magnetic beads contains a high content of metal ions, and the metal ions can interact with the phosphate groups on the DNA to achieve the purpose of separating and extracting DNA. The DNA extraction method provided by the present invention can effectively improve the purity and concentration of the DNA template, is beneficial to the subsequent PCR amplification reaction, can effectively identify Clematidis armandii Caulis medicinal materials, and improves the accuracy rate.

[0005] The technical problem to be solved by the present invention: Although the PCR amplification technology has been relatively mature, it is affected by various factors such as organic solvents and proteins, so this technology has high requirements for the purity and concentration of the DNA template.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for identifying Clematidis armandii Caulis, comprising the following steps:

[0008] S1. Extract the DNA of the sample to be detected;

[0009] S2. Perform PCR amplification using the DNA of the sample to be detected as a template;

[0010] Further, in step S2, the reaction conditions for PCR amplification are as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 35 s, annealing at 58°C for 30 s, extension at 72°C for 35 s, for 30 cycles, and then hold at 72°C for 6 min.

[0011] Further, in step S2, the reaction system for PCR amplification is as follows: 2.5 μL of 10× PCR reaction buffer, 1.5 μL of dNTP with a concentration of 2.5 mM, 2.0 μL of MgCl2 with a concentration of 25 mM, 1 μL of upstream primer with a concentration of 10 μg / mL, 1 μL of downstream primer with a concentration of 10 μg / mL, 0.2 μL of SpeedStar HS Taq DNA polymerase (Takara, 5 U / μL), 1 μL of DNA of the sample to be detected, and make up to 25 μL with sterile double-distilled water.

[0012] Further, the upstream primer is: 5’-TGCCCAGCCTGCACAAGA-3’; the downstream primer is: 5’-TGCCCAGCCTCAACAGTGT-3’.

[0013] S3. Electrophoretically detect the amplification product. Take 5 μL of the amplification product and 10× loading buffer, and use 2% agarose gel to detect the amplification result. If a target band with a size of approximately 478 bp is obtained, then the traditional Chinese medicine sample to be detected is Clematidis Armandii Caulis; if a target band with a size of approximately 478 bp is not obtained, then the traditional Chinese medicine sample to be detected is not Clematidis Armandii Caulis.

[0014] Further, the specific operation steps in step S1 are as follows:

[0015] A1. Crush the sample to be detected;

[0016] A2. Add the crushed sample to be detected into the lysis solution for lysis, centrifuge, and take the supernatant;

[0017] Further, in step A2, the lysis solution is: 0.5 - 1.5% CTAB, 0.1 mol / L Tris-HCl pH8.0, 0.01 - 0.02 mol / L EDTA pH8.0, 1 - 1.5 mol / L potassium chloride, 1 - 2% PVP40.

[0018] A3. Add an equal volume of magnetic bead binding solution to the supernatant, mix well, and let stand at room temperature for 1 - 5 min. Separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant;

[0019] Further, in step A3, the method for preparing the magnetic bead binding solution is as follows: Mix the modified magnetic beads with isopropanol and PEG8000, and make up the volume with ultrapure water to obtain the magnetic bead binding solution; the final concentration of the modified magnetic beads in the magnetic bead binding solution is 2 - 5 mg / mL, the final concentration of isopropanol is 70 - 80%, and the final concentration of PEG8000 is 10 - 15%;

[0020] Further, in step A3, the particle size of the modified magnetic beads is 250 - 700 nm.

[0021] A4. Add the washing solution to the magnetic beads in step A3 for washing, separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant;

[0022] Further, in step A4, the washing solution is 0.01 mol / L Tris-HCl pH8.0, 0.001 mol / L EDTA pH8.0, 75% ethanol, and make up the volume with ultrapure water;

[0023] A5. Add the elution solution to elute the DNA adsorbed on the modified magnetic beads, perform a water bath at 65°C for 5 - 10 min, mix intermittently, perform magnetic separation, carefully take the supernatant to a new centrifuge tube to obtain purified DNA.

[0024] Further, in step A5, the elution solution is: 0.01 - 0.015 mol / L Tris-HCl pH8.0, 0.001 mol / L EDTA pH8.0, and make up the volume with ultrapure water.

[0025] Further, the method for preparing the modified magnetic beads includes the following steps:

[0026] B1. First, perform degassing treatment on deionized water with nitrogen for 20 - 40 min to remove the dissolved oxygen in the solvent; then add FeSO4·7H2O and FeCl3·6H2O, heat and stir to dissolve at 70 - 90°C, then add the NaOH solution while stirring, adjust the pH to 12, the solution gradually turns black, and continue to maintain for 20 - 40 min; finally, perform solid-liquid separation with a magnet to obtain a black-brown magnetic solid, wash it several times with distilled water until neutral, and then perform separation with a magnet to obtain magnetic nanoparticles;

[0027] Further, in step B1, the mass ratio of FeSO4·7H2O to FeCl3·6H2O is (55.6 - 57.3):27.

[0028] Further, in step B1, the concentration of the NaOH solution is 1 - 2 mol / L.

[0029] B2. Disperse magnetic nanoparticles in Tris-HCl buffer solution (pH = 8.5) to obtain a magnetic nanoparticle dispersion; add dopamine to the magnetic nanoparticle dispersion, stir at room temperature for 6 - 10 h, perform solid-liquid separation with a magnet, and wash to obtain magnetic nanoparticles coated with polydopamine;

[0030] Furthermore, in step B2, the dosage ratio of magnetic nanoparticles, Tris-HCl buffer solution, and dopamine is (1 - 3) mg : (3 - 4) mL : (2 - 4) mg.

[0031] B3. Mix urea and sodium citrate and grind them evenly to obtain a mixture, then heat and react the mixture at 150 - 200 °C for 1 - 3 h. After the reaction, centrifuge, dialyze, and freeze-dry to obtain purified carbon nitride quantum dots;

[0032] Furthermore, in step B3, the mass ratio of urea to sodium citrate is 10 : (5 - 8).

[0033] B4. Add carbon nitride quantum dots and phytic acid to deionized water, stir evenly, heat to 60 - 90 °C and react for 1 - 2 h, centrifuge, wash, and dry to obtain a phytic acid-modified carbon nitride quantum dot composite;

[0034] Furthermore, in step B4, the dosage ratio of carbon nitride quantum dots, phytic acid, and deionized water is (1 - 1.3) g : (1 - 2) g : (100 - 120) mL.

[0035] B5. Disperse magnetic nanoparticles coated with polydopamine in deionized water to obtain a dispersion; add the phytic acid-modified carbon nitride quantum dot composite to deionized water, then add the dispersion, stir and react for 1 - 4 h, perform solid-liquid separation with a magnet, wash, and dry to obtain magnetic beads grafted with phosphate groups;

[0036] Furthermore, in step B5, the mass ratio of magnetic nanoparticles coated with polydopamine to the phytic acid-modified carbon nitride quantum dot composite is (20) : (18 - 25).

[0037] B6. Immerse the magnetic beads grafted with phosphate groups in an aqueous metal salt solution, react for 12 - 18 h, after the reaction, perform solid-liquid separation with a magnet, wash, and dry to obtain modified magnetic beads.

[0038] Furthermore, in step B6, the metal salt is an iron salt, and the iron salt includes ferric chloride or ferric sulfate.

[0039] Furthermore, in step B6, the concentration of the metal salt is 80 - 120 mmol / L.

[0040] In the above-mentioned preparation process, polydopamine is coated on the surface of magnetic nanoparticles, and then phytic acid modified carbon nitride quantum dots are coated, and finally the phosphoric acid groups on the phytic acid are used to complex with metal ions to obtain modified magnetic beads. Carbon nitride quantum dots have a high specific surface area and multiple active sites, which can increase the content of phytic acid attached to the surface of the modified magnetic beads, and then increase the content of metal ions complexed on the surface of the modified magnetic beads. The metal ions can interact with the phosphoric acid groups on the DNA to achieve the purpose of separating and extracting DNA. Therefore, the increase in the metal ion content can increase the loading rate of DNA, thereby increasing the extraction rate of the sample DNA to be tested. Moreover, the abundant active functional groups on the surface of polydopamine are also conducive to the adsorption of DNA, further increasing the loading rate of DNA.

[0041] Beneficial effects of the present invention:

[0042] (1) In the technical solution of the present invention, the DNA extraction method provided by the present invention can effectively improve the purity and concentration of the DNA template, which is beneficial to the subsequent PCR amplification reaction, can effectively identify the medicinal materials of Chuanmutong, and improves the accuracy rate.

[0043] (2) In the technical solution of the present invention, polydopamine is coated on the surface of magnetic nanoparticles, and then coated with phytic acid-modified carbon nitride quantum dots, and finally the phosphate groups on the phytic acid are used to complex with metal ions to obtain modified magnetic beads. Carbon nitride quantum dots have a high specific surface area and many active sites, which can increase the content of phytic acid attached to the surface of the modified magnetic beads, and then increase the content of metal ions complexed on the surface of the modified magnetic beads. The metal ions can interact with the phosphate groups on the DNA to achieve the purpose of separating and extracting DNA. Therefore, the increase in the metal ion content can increase the DNA loading rate, thereby increasing the extraction rate of DNA in the sample to be tested. In addition, the rich active functional groups on the surface of polydopamine are also conducive to the adsorption of DNA, further increasing the DNA loading rate. DETAILED DESCRIPTION

[0044] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1

[0046] This embodiment provides a method for preparing modified magnetic beads, comprising the following steps:

[0047] B1. First, degas 50 mL of deionized water with nitrogen for 20 - 40 min to remove the dissolved oxygen in the solvent; then add 5.56 g of FeSO4·7H2O and 2.7 g of FeCl3·6H2O, heat and stir to dissolve at 70 °C, then add 1 mol / L NaOH solution dropwise with stirring to adjust the pH to 12. The solution gradually turns black and is kept for another 20 min; finally, perform solid-liquid separation with a magnet to obtain a black-brown magnetic solid, wash it several times with distilled water until neutral, and then perform separation with a magnet again to obtain magnetic nanoparticles;

[0048] B2. Disperse 0.5 g of magnetic nanoparticles in 1.5 L of Tris-HCl buffer solution (pH = 8.5) to obtain a magnetic nanoparticle dispersion; add 1 g of dopamine to the magnetic nanoparticle dispersion, stir at room temperature for 6 h, perform solid-liquid separation with a magnet, and wash to obtain magnetic nanoparticles coated with polydopamine;

[0049] B3. Mix 10 g of urea and 5 g of sodium citrate and grind them evenly to obtain a mixture, then heat and react the mixture at 150 °C for 1 h. After the reaction, centrifuge, dialyze for 18 h, and freeze-dry to obtain purified carbon nitride quantum dots;

[0050] B4. Add 1 g of carbon nitride quantum dots and 1 g of phytic acid to 100 mL of deionized water, stir evenly, heat to 60 °C and react for 1 h, then centrifuge, wash, and dry to obtain a phytic acid-modified carbon nitride quantum dot composite;

[0051] B5. Disperse 0.5 g of magnetic nanoparticles coated with polydopamine in 500 mL of deionized water to obtain a dispersion; add 0.45 g of phytic acid-modified carbon nitride quantum dot composite to 400 mL of deionized water, then add the dispersion, stir and react for 1 h, perform solid-liquid separation with a magnet, wash, and dry to obtain magnetic beads grafted with phosphate groups;

[0052] B6. Immerse 300 mg of magnetic beads grafted with phosphate groups in an aqueous solution of ferric chloride with a concentration of 80 mmol / L and react for 12 h. After the reaction, perform solid-liquid separation with a magnet, wash, and dry to obtain modified magnetic beads.

[0053] Example 2

[0054] This example provides a method for preparing modified magnetic beads, which includes the following steps:

[0055] B1. First, degas 50 mL of deionized water with nitrogen for 20 - 40 min to remove the dissolved oxygen in the solvent; then add 5.64 g of FeSO4·7H2O and 2.7 g of FeCl3·6H2O, heat and stir to dissolve at 80 °C, then add 1 mol / L NaOH solution dropwise with stirring to adjust the pH to 12. The solution gradually turns black and is kept for another 30 min; finally, perform solid-liquid separation with a magnet to obtain a black-brown magnetic solid, wash it several times with distilled water until neutral, and then perform separation with a magnet again to obtain magnetic nanoparticles;

[0056] B2. Disperse 1 g of magnetic nanoparticles in 1.7 L of Tris-HCl buffer solution (pH = 8.5) to obtain a magnetic nanoparticle dispersion; add 1.6 g of dopamine to the magnetic nanoparticle dispersion, stir at room temperature for 8 h, perform solid-liquid separation with a magnet, and wash to obtain magnetic nanoparticles coated with polydopamine;

[0057] B3. Mix 10 g of urea and 6.9 g of sodium citrate and grind them evenly to obtain a mixture, then heat and react the mixture at 180 °C for 2 h. After the reaction, centrifuge, dialyze for 24 h, and freeze-dry to obtain purified carbon nitride quantum dots;

[0058] B4. Add 1.1 g of carbon nitride quantum dots and 1.6 g of phytic acid to 110 mL of deionized water, stir evenly, heat up to 75 °C and react for 1.5 h, centrifuge, wash, and dry to obtain a phytic acid-modified carbon nitride quantum dot composite;

[0059] B5. Disperse 0.5 g of magnetic nanoparticles coated with polydopamine in 500 mL of deionized water to obtain a dispersion; add 0.58 g of phytic acid-modified carbon nitride quantum dot composite to 400 mL of deionized water, then add the dispersion, stir and react for 3 h, perform solid-liquid separation with a magnet, wash, and dry to obtain magnetic beads grafted with phosphate groups;

[0060] B6. Immerse 300 mg of magnetic beads grafted with phosphate groups in an aqueous solution of ferric chloride with a concentration of 100 mmol / L and react for 15 h. After the reaction, perform solid-liquid separation with a magnet, wash, and dry to obtain modified magnetic beads.

[0061] Example 3

[0062] This example provides a method for preparing modified magnetic beads, including the following steps:

[0063] B1. First, degas 50 mL of deionized water with nitrogen for 20 - 40 min to remove the dissolved oxygen in the solvent. Then add 5.73 g of FeSO4·7H2O and 2.7 g of FeCl3·6H2O, heat and stir to dissolve at 70 - 90 °C. Next, add 2 mol / L NaOH solution dropwise with stirring to adjust the pH to 12. The solution gradually turns black and is maintained for another 40 min. Finally, perform solid-liquid separation with a magnet to obtain a black-brown magnetic solid, wash it several times with distilled water until neutral, and then perform separation with a magnet again to obtain magnetic nanoparticles.

[0064] B2. Disperse 1.5 g of magnetic nanoparticles in 2 L of Tris-HCl buffer solution (pH = 8.5) to obtain a magnetic nanoparticle dispersion. Add 2 g of dopamine to the magnetic nanoparticle dispersion, stir at room temperature for 10 h, perform solid-liquid separation with a magnet, and wash to obtain magnetic nanoparticles coated with polydopamine.

[0065] B3. Mix 10 g of urea and 8 g of sodium citrate and grind them evenly to obtain a mixture. Then heat and react the mixture at 200 °C for 3 h. After the reaction, centrifuge, dialyze for 30 h, and freeze-dry to obtain purified carbon nitride quantum dots.

[0066] B4. Add 1.3 g of carbon nitride quantum dots and 2 g of phytic acid to 120 mL of deionized water, stir evenly, heat to 90 °C and react for 2 h, centrifuge, wash, and dry to obtain a phytic acid-modified carbon nitride quantum dot composite.

[0067] B5. Disperse 0.5 g of magnetic nanoparticles coated with polydopamine in 500 mL of deionized water to obtain a dispersion. Add 0.625 g of the phytic acid-modified carbon nitride quantum dot composite to 400 mL of deionized water, then add the dispersion, stir and react for 4 h, perform solid-liquid separation with a magnet, wash, and dry to obtain magnetic beads grafted with phosphate groups.

[0068] B6. Immerse 300 mg of magnetic beads grafted with phosphate groups in an aqueous solution of ferric sulfate with a concentration of 120 mmol / L, react for 18 h. After the reaction, perform solid-liquid separation with a magnet, wash, and dry to obtain modified magnetic beads.

[0069] Comparative Example 1

[0070] Compared with Example 2, in Comparative Example 1, the surface of the modified magnetic beads does not contain carbon nitride quantum dots, and phytic acid is directly reacted with magnetic nanoparticles coated with polydopamine. Other steps and raw materials are the same as in Example 2.

[0071] Comparative Example 2

[0072] Compared with Example 2, in Comparative Example 2, the modified magnetic beads are magnetic nanoparticles coated with polydopamine, and other steps and raw materials are the same as those in Example 2.

[0073] Example 4

[0074] A method for identifying Clematis armandii Franch. includes the following steps:

[0075] S1. Extract the DNA of the sample to be detected; the specific operation steps are as follows:

[0076] A1. Grind the sample to be detected into powder using liquid nitrogen;

[0077] A2. Add the ground sample to be detected into the lysis solution and lyse at 65 °C for 10 min, centrifuge for 5 min, and take the supernatant; the lysis solution is: 0.5% CTAB, 0.1 mol / L Tris-HCl pH 8.0, 0.01 mol / L EDTA pH 8.0, 1 mol / L potassium chloride, 1.5% PVP40;

[0078] A3. Add an equal volume of magnetic bead binding solution to the supernatant, mix well and let stand at room temperature for 1 min, separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant; the preparation method of the magnetic bead binding solution is: mix the modified magnetic beads with an average particle size of 430 nm prepared in Example 2 with isopropanol and PEG8000, and make up the volume with ultrapure water to obtain the magnetic bead binding solution; the final concentration of the modified magnetic beads in the magnetic bead binding solution is 2 mg / mL, the final concentration of isopropanol is 80%, and the final concentration of PEG8000 is 10%;

[0079] A4. Add the washing solution to the magnetic beads in step A3 and wash twice, separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant; the washing solution is 0.01 mol / L Tris-HCl pH 8.0, 0.001 mol / L EDTA pH 8.0, 75% ethanol, and make up the volume with ultrapure water;

[0080] A5. Add the elution solution to elute the DNA adsorbed on the modified magnetic beads, perform a water bath at 65 °C for 5 min, mix intermittently, perform magnetic separation, and carefully take the supernatant to a new centrifuge tube to obtain purified DNA; the elution solution is: 0.01 mol / L Tris-HCl pH 8.0, 0.001 mol / L EDTA pH 8.0, and make up the volume with ultrapure water.

[0081] S2. Perform PCR amplification using the DNA of the sample to be detected as a template;

[0082] The PCR amplification reaction is carried out in a PCR reaction tube with a total reaction volume of 25 μL, including the following reagents: 2.5 μL of 10×PCR reaction buffer, 1.5 μL of dNTP with a concentration of 2.5 mM, 2.0 μL of MgCl2 with a concentration of 25 mM, 1 μL of upstream primer with a concentration of 10 μg / mL, 1 μL of downstream primer with a concentration of 10 μg / mL, 0.2 μL of SpeedStar HS Taq DNA polymerase (Takara, 5 U / μL), 1 μL of the DNA sample to be detected, and sterile double-distilled water is added to make up to 25 μL; the upstream primer is: 5’-TGCCCAGCCTGCACAAGA-3’; the downstream primer is: 5’-TGCCCAGCCTCAACAGTGT-3’;

[0083] After the PCR reaction solution is prepared, gently shake and mix well. Then place the PCR tube into a PCR instrument for PCR amplification. The specific reactions are as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 35 s, annealing at 58°C for 30 s, extension at 72°C for 35 s, for 30 cycles, and then hold at 72°C for 6 min to obtain the amplification product;

[0084] S3. Carry out electrophoresis detection on the amplification product. Take 5 μL of the amplification product and 10×loading buffer, and use 2% agarose gel (containing 0.005% ethidium bromide) to detect the amplification result. If a target band with a size of approximately 478 bp is obtained, then the Chinese medicine sample to be detected is Clematidis Armandii Caulis; if a target band with a size of approximately 478 bp is not obtained, then the Chinese medicine sample to be detected is not Clematidis Armandii Caulis.

[0085] Example 5

[0086] A method for identifying Clematidis Armandii Caulis, comprising the following steps:

[0087] S1. Extract the DNA of the sample to be detected; the specific operation steps are as follows:

[0088] A1. Use liquid nitrogen to grind the sample to be detected into powder;

[0089] A2. Add the ground sample to be detected into the lysis solution and lyse at 65°C for 20 min, centrifuge for 7 min, and take the supernatant; the lysis solution is: 1% CTAB, 0.1 mol / L Tris-HCl pH8.0, 0.016 mol / L EDTA pH8.0, 1.3 mol / L potassium chloride, 1.5% PVP40;

[0090] A3. Add an equal volume of magnetic bead binding solution to the supernatant. After mixing evenly, let it stand at room temperature for 1 - 5 min. Separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant. The preparation method of the magnetic bead binding solution is as follows: Mix the modified magnetic beads with an average particle size of 430 nm prepared in Example 2 with isopropanol and PEG8000, and make up the volume with ultrapure water to obtain the magnetic bead binding solution. The final concentration of the modified magnetic beads in the magnetic bead binding solution is 3.7 mg / mL, the final concentration of isopropanol is 70%, and the final concentration of PEG8000 is 15%.

[0091] A4. Add the washing solution to the magnetic beads in step A3 and wash twice. Separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant. The washing solution is 0.01 mol / L Tris - HCl pH8.0, 0.001 mol / L EDTA pH8.0, 75% ethanol, and make up the volume with ultrapure water.

[0092] A5. Add the elution solution to elute the DNA adsorbed on the modified magnetic beads. Incubate in a water bath at 65°C for 10 min, mix intermittently, perform magnetic separation, and carefully transfer the supernatant to a new centrifuge tube to obtain purified DNA. The elution solution is: 0.015 mol / L Tris - HCl pH8.0, 0.001 mol / L EDTA pH8.0, and make up the volume with ultrapure water.

[0093] S2. Use the DNA of the sample to be detected as a template for PCR amplification.

[0094] The PCR amplification reaction is carried out in a PCR reaction tube. The total reaction volume is 25 μL, including the following reagents: 2.5 μL of 10×PCR reaction buffer, 1.5 μL of dNTP with a concentration of 2.5 mM, 2.0 μL of MgCl2 with a concentration of 25 mM, 1 μL of the upstream primer with a concentration of 10 μg / mL, 1 μL of the downstream primer with a concentration of 10 μg / mL, 0.2 μL of SpeedStar HS Taq DNA polymerase (Takara, 5 U / μL), 1 μL of the DNA of the sample to be detected, and make up to 25 μL with sterile double - distilled water. The upstream primer is: 5’ - TGCCCAGCCTGCACAAGA - 3’; the downstream primer is: 5’ - TGCCCAGCCTCAACAGTGT - 3’.

[0095] After preparing the PCR reaction solution, gently mix it by shaking. Place the PCR tube in a PCR instrument for PCR amplification. The specific reaction is as follows: Pre - denature at 95°C for 5 min, denature at 95°C for 35 s, anneal at 58°C for 30 s, extend at 72°C for 35 s, for 30 cycles, and then hold at 72°C for 6 min to obtain the amplification product.

[0096] S3. Perform electrophoresis detection on the amplification product. Take 5 μL of the amplification product and 10× loading buffer, and use a 2% agarose gel (containing 0.005% ethidium bromide) to detect the amplification result. If a target band with a size of approximately 478 bp is obtained, the tested traditional Chinese medicine sample is Clematidis Armandii Caulis; if a target band with a size of approximately 478 bp is not obtained, the tested traditional Chinese medicine sample is not Clematidis Armandii Caulis.

[0097] Example 6

[0098] A method for identifying Clematidis Armandii Caulis, comprising the following steps:

[0099] S1. Extract the DNA of the sample to be detected; the specific operation steps are as follows:

[0100] A1. Use liquid nitrogen to grind and crush the sample to be detected;

[0101] A2. Add the crushed sample to the lysis solution and lyse at 70 °C for 30 min, centrifuge for 10 min, and take the supernatant; the lysis solution is: 1.5% CTAB, 0.1 mol / L Tris-HCl pH 8.0, 0.02 mol / L EDTA pH 8.0, 1.4 mol / L potassium chloride, 1.2% PVP40;

[0102] A3. Add an equal volume of magnetic bead binding solution to the supernatant, mix well, and let stand at room temperature for 5 min. Separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant; the preparation method of the magnetic bead binding solution is: mix the modified magnetic beads with an average particle size of 430 nm prepared in Example 2 with isopropanol and PEG8000, and make up the volume with ultrapure water to obtain the magnetic bead binding solution; the final concentration of the modified magnetic beads in the magnetic bead binding solution is 5 mg / mL, the final concentration of isopropanol is 75%, and the final concentration of PEG8000 is 15%;

[0103] A4. Add the washing solution to the magnetic beads in step A3 and wash twice. Separate the modified magnetic beads and the liquid by applying an external magnetic force, and discard the supernatant; the washing solution is 0.01 mol / L Tris-HCl pH 8.0, 0.001 mol / L EDTA pH 8.0, 75% ethanol, and make up the volume with ultrapure water;

[0104] A5. Add the elution solution to elute the DNA adsorbed on the modified magnetic beads, perform a water bath at 65 °C for 10 min, mix intermittently, perform magnetic separation, and carefully take the supernatant to a new centrifuge tube to obtain purified DNA; the elution solution is: 0.015 mol / L Tris-HCl pH 8.0, 0.001 mol / L EDTA pH 8.0, and make up the volume with ultrapure water.

[0105] S2. Perform PCR amplification using the DNA of the sample to be detected as a template;

[0106] The PCR amplification reaction is carried out in a PCR reaction tube. The total reaction volume is 25 μL and includes the following reagents: 2.5 μL of 10×PCR reaction buffer, 1.5 μL of dNTP with a concentration of 2.5 mM, 2.0 μL of MgCl2 with a concentration of 25 mM, 1 μL of the upstream primer with a concentration of 10 μg / mL, 1 μL of the downstream primer with a concentration of 10 μg / mL, 0.2 μL of SpeedStar HS Taq DNA polymerase (Takara, 5 U / μL), 1 μL of the DNA sample to be detected, and sterile double-distilled water is added to make up to 25 μL;

[0107] The upstream primer is: 5’-TGCCCAGCCTGCACAAGA-3’;

[0108] The downstream primer is: 5’-TGCCCAGCCTCAACAGTGT-3’;

[0109] After the PCR reaction solution is prepared, gently shake and mix well. Then place the PCR tube into a PCR instrument for PCR amplification. The specific reactions are as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 35 s, annealing at 58 °C for 30 s, extension at 72 °C for 35 s, for 30 cycles, and then hold at 72 °C for 6 min to obtain the amplification product;

[0110] S3. Electrophoresis detection is carried out on the amplification product. Take 5 μL of the amplification product and 10× loading buffer, and use 2% agarose gel (containing 0.005% ethidium bromide) to detect the amplification result. If a target band with a size of approximately 478 bp is obtained, then the Chinese medicine sample to be detected is Clematidis Armandii Caulis; if a target band with a size of approximately 478 bp is not obtained, then the Chinese medicine sample to be detected is not Clematidis Armandii Caulis.

[0111] Comparative Example 3

[0112] Compared with Example 4, in Comparative Example 3, the modified magnetic beads are replaced with the substance prepared in Comparative Example 1, and phytic acid is directly added. Other steps and raw materials are the same as those in Example 4.

[0113] Comparative Example 4

[0114] Compared with Example 4, in Comparative Example 4, the modified magnetic beads are replaced with the substance prepared in Comparative Example 2, and other steps and raw materials are the same as those in Example 4.

[0115] Performance detection

[0116] The DNA of the samples to be detected extracted in Step S1 of Examples 4 - 6 and Comparative Examples 3 - 4 is subjected to purity and concentration detection: Take 1 μL of the extracted DNA and use a NanoDrop 2000 ultra-micro spectrophotometer to detect its concentration, A 260 / A 280 and A260 / A 230 The ratio is used to evaluate the quality of the extracted DNA and the removal of impurities, and the results are shown in Table 1.

[0117] It can be seen from the data in Table 1 that the DNA extracted by the DNA extraction method provided by the present invention has high concentration and purity. By comparing the data of Example 4 and Comparative Example 3, it can be seen that the addition of carbon nitride quantum dots can increase the DNA concentration and purity. By comparing the data of Example 4 and Comparative Example 4, it can be seen that coating carbon nitride quantum dots complexed with metal ions on the surface of polydopamine-coated magnetic nanoparticles can significantly increase the concentration and purity of the extracted DNA.

[0118] In the description of the specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0119] The above content is only an example and illustration of the present invention. Those skilled in the art of this technology can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, as long as they do not deviate from the invention or exceed the scope defined by the claims of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for identifying Caulis Akebiae, characterized in that: S1, extracting DNA from samples to be tested; S2, using the DNA of the sample to be tested as a template for PCR amplification; In step S2, an upstream primer and a downstream primer are added to the reaction system during PCR amplification; the upstream primer is: 5'-TGCCCAGCCTGCACAAGA-3'; the downstream primer is: 5'-TGCCCAGCCTCAACAGTGT-3'; S3, electrophoresis detection of the amplified product, taking 5 μL of the amplified product and 10× loading buffer, using 2% agarose gel to detect the amplification result, if a target band of about 478 bp is obtained, the Chinese medicine sample to be tested is Chuanmutong; if no target band of about 478 bp is obtained, the Chinese medicine sample to be tested is not Chuanmutong; The specific operation steps in step S1 are: A1. Crush the sample to be tested; A2. Add the crushed sample to be tested into a lysis solution for lysis, centrifuge, and take the supernatant; A3. Add an equal volume of magnetic bead binding solution to the supernatant, mix well and let stand at room temperature for 1-5 minutes. Separate the modified magnetic beads and liquid by external magnetic force and discard the supernatant. A4, adding washing solution to the magnetic beads in step A3 for washing, separating the modified magnetic beads and the liquid by external magnetic force, and discarding the supernatant; A5. Add elution buffer to elute the DNA adsorbed on the modified magnetic beads. Incubate in a 65°C water bath for 5-10 minutes, mix occasionally, perform magnetic separation, and carefully transfer the supernatant to a new centrifuge tube to obtain purified DNA. The specific preparation method of the modified magnetic beads comprises the following steps: B1. Deionized water was first degassed with nitrogen for 20-40 minutes to remove dissolved oxygen in the solvent; FeSO4·7H2O and FeCl3·6H2O were then added, heated and stirred at 70-90°C to dissolve, and then NaOH solution was added while stirring to adjust the pH to 12. The solution gradually turned black and was maintained for 20-40 minutes; finally, a magnet was used for solid-liquid separation to obtain a dark brown magnetic solid, which was then washed with distilled water several times until neutral, and then separated with a magnet to obtain magnetic nanoparticles; B2, dispersing the magnetic nanoparticles in a Tris-HCl buffer solution at pH=8.5 to obtain a magnetic nanoparticle dispersion; adding dopamine to the magnetic nanoparticle dispersion, stirring at room temperature for 6-10 hours, performing solid-liquid separation with a magnet, and washing to obtain polydopamine-coated magnetic nanoparticles; B3, mixing urea and sodium citrate and grinding them evenly to obtain a mixture, and then heating the mixture at 150-200° C. for reaction for 1-3 hours, centrifuging after the reaction, dialyzing, and freeze-drying to obtain purified carbon nitride quantum dots; B4, adding carbon nitride quantum dots and phytic acid into deionized water, stirring evenly, heating to 60-90°C for reaction for 1-2h, centrifuging, washing, and drying to obtain a phytic acid-modified carbon nitride quantum dot complex; B5, dispersing polydopamine-coated magnetic nanoparticles in deionized water to obtain a dispersion; adding the phytic acid-modified carbon nitride quantum dot complex to deionized water, and then adding the dispersion, stirring the reaction for 1-4 hours, performing solid-liquid separation with a magnet, washing, and drying to obtain magnetic beads grafted with phosphate groups; B6. Immerse the magnetic beads grafted with phosphate groups in a metal salt aqueous solution and react for 12-18 hours. After the reaction is completed, use a magnet to separate the solid and liquid, wash and dry to obtain modified magnetic beads.

2. The method for identifying Caulis Atractylodes lancea according to claim 1, wherein: In step S2, the reaction conditions for PCR amplification are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 35 s, annealing at 58°C for 30 s, extension at 72°C for 35 s, 30 cycles, and then maintaining at 72°C for 6 min.

3. The identification method of Caulis Atractylodes lancea according to claim 1, characterized in that: In the step S2, the reaction system during PCR amplification is: 2.5 μL 10×PCR reaction buffer, 1.5 μL 2.5 mM dNTP, 2.0 μL 25 mM MgCl2, 1 μL 10 μg / mL upstream primer, 1 μL 10 μg / mL downstream primer, 0.2 μL SpeedStar HS Taq DNA polymerase, SpeedStar HS Taq DNA polymerase purchased from Takara, with an enzyme activity of 5 U / μL, 1 μL sample DNA to be tested, and sterile double distilled water is added to 25 μL.

4. The identification method of Caulis Atractylodes lancea according to claim 1, characterized in that: In step A2, the lysis solution is: 0.5-1.5% CTAB, 0.1 mol / L Tris-HCl pH 8.0, 0.01-0.02 mol / L EDTA pH 8.0, 1-1.5 mol / L potassium chloride, and 1-2% PVP40.

5. The identification method of Caulis Atractylodes lancea according to claim 1, characterized in that: In step A3, the preparation method of the magnetic bead binding solution is: mixing the modified magnetic beads with isopropanol and PEG8000, and making up the volume with ultrapure water to obtain the magnetic bead binding solution; the final concentration of the modified magnetic beads in the magnetic bead binding solution is 2-5 mg / mL, the final concentration of isopropanol is 70-80%, and the final concentration of PEG8000 is 10-15%; the particle size of the modified magnetic beads is 250-700 nm.

6. The method for identifying Caulis Akebiae according to claim 1, characterized in that: In the step B2, the ratio of the amount of magnetic nanoparticles, Tris-HCl buffer solution and dopamine is (1-3) mg: (3-4) mL: (2-4) mg.

7. The identification method of Caulis Akebiae according to claim 1, characterized in that: In step B4, the usage ratio of carbon nitride quantum dots, phytic acid and deionized water is (1-1.3) g: (1-2) g: (100-120) mL.

8. The identification method of Caulis Atractylodes lancea according to claim 1, characterized in that: In the step B5, the mass ratio of the polydopamine-coated magnetic nanoparticles to the phytic acid-modified carbon nitride quantum dot composite is (20): (18-25).

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