Nucleic acid aptamer of theanine and application

Abstract

The invention belongs to the field of theanine detection, and particularly discloses a plurality of nucleic acid aptamers of theanine and application. The theanine aptamer screened based on the exponential enrichment ligand systematic evolution technology (SELEX) has high affinity and high specificity to theanine, theanine molecules can be recognized in a complex tea soup matrix environment, and a semi-quantitative rapid detection test can be carried out on a nano-gold test strip filled with the theanine aptamer. Compared with a traditional high performance liquid chromatography detection method, the rapid detection technology for theanine disclosed by the invention is low in cost, efficient and convenient, has a wide application prospect, can be used for rapid identification of theanine in a complex background, and provides a rapid detection means for theanine content of germplasm resources in a field environment for breeding workers.

Description

Technical Field

[0001] The invention belongs to the field of theanine detection, and particularly relates to a nucleic acid aptamer of theanine and applications thereof. Background Art

[0002] Theanine (L-Theanine) is a unique free amino acid in tea, and its content varies depending on the variety and part of the tea. It is the main component of tea that produces body fluid and sweetness, and has many functions such as improving learning ability and memory, calming, protecting nerve cells, lowering blood pressure, and enhancing the efficacy of anti-cancer drugs. Therefore, breeding tea varieties with high theanine content has become an important goal for tea breeders. At present, the detection method of theanine is mainly high-performance liquid chromatography. Although high-performance liquid chromatography can detect theanine well, it requires expensive equipment and complex sample pre-treatment steps. It is a labor- and resource-intensive detection method, which makes it impossible for tea breeders to quickly identify the level of theanine in planting resources in the field environment. Therefore, there is an urgent need for a new and convenient detection method to achieve rapid and quantitative detection of theanine.

[0003] The rapid development of nucleic acid aptamer technology in small molecule detection, sensor analysis and other applications has attracted the attention of researchers. Nucleic acid aptamers (Aptamer) are oligonucleotide fragments obtained by in vitro screening through exponential enrichment ligand system evolution technology. They can recognize a variety of target products with high specificity. Compared with other detection technologies, nucleic acid aptamers have the advantages of low preparation cost, good thermal stability, short cycle, low detection limit, simple and easy operation, etc. They can quickly bind to the corresponding targets and specifically recognize various targets. At present, nucleic acid aptamers have been successfully applied to many fields such as food safety analysis and biomedical research as chemical and biological sensor recognition molecules, but there are no reports on nucleic acid aptamer sequences and their applications for the detection of theanine in complex matrix backgrounds. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a number of nucleic acid aptamers with high affinity for theanine, and provide the application of these nucleic acid aptamers in the detection, separation, enrichment of theanine, and the preparation of theanine detection reagents, kits or sensors. The present invention uses the systematic evolution of ligands by exponential enrichment (SELEX technology) and combines specific positive screening and negative screening strategies to screen out nucleic acid aptamers (Apt.1) with high affinity and specific binding to theanine, and the obtained original theanine aptamer chain is truncated and optimized after the binding site is determined according to the molecular docking results of the Autodock software, so that the modified aptamers (Apt.2-3) have better affinity for the target, and discloses a set of sample pretreatment methods and provides a method for detecting theanine by nanogold test paper using theanine aptamers, so that the tea soup sample after pretreatment can be detected on a set of nanogold test paper strips developed based on this theanine nucleic acid aptamer.

[0005] The present invention is achieved through the following technical solutions:

[0006] The nucleic acid aptamers Apt.1-3 of theanine have nucleotide sequences as shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0007] SEQ ID NO: 1: 5'-ATAGGAGTCACGACGACCAGAAGAGGTCCCGCAT GAGAAAGCCCATCACCGC T ATGTGCGTCTACCTCTTGACTAAT-3';

[0008] SEQ ID NO: 2: 5'-AAGAGGTCCCGCATGAGAAAGCCCATCACCGCTAT GTGCGTCTACCTCTT-3';

[0009] SEQ ID NO: 3: 5'-AAGAGGTCCCGCATGAGAAATATGTGCGTCTACCT CTT-3';

[0010] SEQ ID NO: 2 and SEQ ID NO: 3 are sequences obtained by truncating and optimizing SEQ ID NO: 1. Among them, SEQ ID NO: 3 has the highest binding affinity with theanine, and the dissociation constant is close to the nM level.

[0011] A detection probe or detection kit for theanine comprises the above-mentioned nucleic acid aptamer.

[0012] Application of the nucleic acid aptamer in preparing a detection probe or a detection kit for theanine.

[0013] Application of the above-mentioned nucleic acid aptamer, detection probe or detection kit in detecting theanine.

[0014] Application of the above-mentioned nucleic acid aptamer, detection probe or detection kit in identifying tea varieties or tea tree breeding screening.

[0015] Preferably, the nano-gold test paper detection method is used for detection or screening. Preferably, the tea sample to be detected is extracted with alcohol (preferably 75% methanol solution), and the tea soup obtained by extraction is tested after polyphenols are removed (through SPE column).

[0016] Application of the above-mentioned nucleic acid aptamers in the separation and enrichment of theanine.

[0017] Application of the above nucleic acid aptamer in the preparation of theanine targeting reagent.

[0018] The advantage of the present invention is that it provides multiple nucleic acid aptamers with high affinity and high specificity for theanine, and the dissociation constant is close to the nM level. The aptamers for theanine provided by the present invention can identify theanine molecules in a complex tea soup matrix environment. After the tea soup is pre-treated to remove polyphenols, a semi-quantitative rapid test can be performed on a nanogold test strip equipped with theanine aptamers. Compared with the traditional high-performance liquid chromatography detection method, the theanine rapid detection method of the present invention is low in cost, efficient and convenient, has broad application prospects, can be used for the rapid identification of theanine in complex backgrounds, and provides breeders with a means of rapid detection of theanine content in germplasm resources in the field environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the secondary structure of theanine nucleic acid aptamer (Apt.1~3).

[0020] Figure 2 Figure 2 shows the ITC data of affinity detection of theanine nucleic acid aptamer (SEQ ID NO: 1) obtained by the present invention and K d value.

[0021] Figure 3 The figure shows the docking result and binding site of theanine nucleic acid aptamer (SEQ ID NO: 1) and theanine molecule.

[0022] Figure 4 ITC data of affinity detection of theanine nucleic acid aptamer (SEQ ID NO: 2) obtained by the present invention and K d value.

[0023] Figure 5Figure 2 shows the ITC data of affinity detection of theanine nucleic acid aptamer (SEQ ID NO: 3) obtained by the present invention and K d value.

[0024] Figure 6 This is a quantitative result diagram of the specific removal effect of theanine nucleic acid aptamer (SEQ ID NO: 3) in tea soup using high performance liquid chromatography-mass spectrometry.

[0025] Figure 7 This is a schematic diagram of the pre-treatment of tea soup.

[0026] Figure 8 It is a positive reference result of theanine aptamer (SEQ ID NO: 3) nanogold test paper detection. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is further illustrated and described below by specific embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0028] Example 1 Screening of theanine DNA aptamers based on magnetic beads-SELEX.

[0029] Theanine was used as the target and amino magnetic beads were used as the solid phase carrier of the target. An artificially synthesized single-stranded DNA with a fixed sequence of 22 bp at the 5' end, a fixed sequence of 25 bp at the 3' end and a random sequence of 40 bp in the middle was used as the initial ssDNA library to screen DNA aptamers. The sequence information of the random single-stranded oligonucleotide library was as follows SEQ ID NO: 4: SEQ ID NO: 4: 5'-ATAGGAGTCACGACGACCAGAA-N40-TATGTGCGTCTACCTCTTGACTAAT-3'.

[0030] 1. Amino magnetic beads coupled with theanine

[0031] 1.1 Activation of amino groups on the surface of magnetic beads

[0032] After mixing the magnetic beads, take 2 portions of 40 μL and 2 portions of 20 μL of amino magnetic beads into a 1.5 mL reaction tube, and take another empty EP tube, which are recorded as groups A, B, C, D, and E. Groups C, D, and E are used to verify the effect of magnetic bead coupling with theanine, and groups A and B are used for subsequent experiments. The supernatant was removed by magnetic liquid separation, and 500 μL of coupling buffer was added to each of the five groups to elute the magnetic beads (group E had no magnetic beads). After vortexing for 1 min, the supernatant was removed by magnetic liquid separation, and the elution was repeated once, and the supernatant was removed by magnetic liquid separation.

[0033] 1.2 Magnetic beads coupled to theanine

[0034] Dissolve 600ug of theanine in 1500μL of amino magnetic bead coupling buffer (0.05M MES, 0.5M NaCl), and add 500μL of this mixed solution to groups A, C, and E, and only add 500μL of amino magnetic bead coupling buffer to groups B and D, and vortex for 1min. Weigh 0.01g of EDC powder and mix it with 1mL of coupling buffer, and add 10μL to each group, and vortex for 1min.

[0035] Incubate at 25℃ for 2 hours in a constant temperature shaker to ensure that the magnetic beads are always in suspension. Take the supernatant from both groups C and D, record it as F and G, and add 500μL of 1×PBS solution to both groups to rinse the magnetic beads twice, add 100μL of 1×PBS solution, and place in a 95℃ water bath for 8min. Take the clear solution and discard the magnetic beads, take out the supernatant from groups C and D again, record it as H and I, and use Nanodrop micro-spectrophotometer to measure the absorbance of the five groups of solutions E, F, G, H, and I to test the coupling effect of the magnetic beads. The results showed that the absorbance of group E was greater than that of group F, and the absorbance of group H was greater than that of group I, indicating that the coupling of theanine to amino magnetic beads was successful, and A and B can continue to be used in subsequent experiments.

[0036] 1.3 Block the remaining sites on the magnetic beads

[0037] Place the magnetic bead mixture of groups A and B on a magnetic rack, aspirate the supernatant, keep the magnetic beads, add 500 μL of 0.1 M Tris blocking solution, and place in a shaker at 220 r / min and 25°C for 3 hours.

[0038] 2. Screening of aptamers from the library

[0039] Denature the initial ssDNA library in a 95°C water bath for 8 minutes, and then immediately renature it in an ice bath for 8 minutes to form a specific structure. Add 400uLBB buffer (50mM Tris-HCl, 5mM KCl, 100mMNaCl, 1mM MgCl2, pH 7.4) to the EP tube of group B of amino magnetic beads. Add 200uL of the initial ssDNA library to the above EP tube and incubate at room temperature for 30min. After incubation, aspirate all the supernatant and add it to the tube A of amino magnetic beads coupled with theanine, and incubate for 1h. Aspirate and discard the supernatant, add 100uLddH2O, and bathe at 95°C for 8 minutes. After cooling, collect the supernatant, which is the first round of aptamer library obtained by amino magnetic bead screening, and use it as a template for PCR amplification.

[0040] 3. Post-screening library enrichment monitoring

[0041] The aptamer library obtained by amino magnetic beads screening was used as a PCR template, and the LAtaq enzyme system was selected to perform PCR amplification. The specific dosage is shown in Table 1 below;

[0042] Table 1

[0043] name Dosage LATaq enzyme 0.5μL <![CDATA[10×LATaqBufferII(Mg 2+ Plus)]]> 5μL dNTP Mixture (2.5 mM) 8μL Template 3μL Primer 1 (Fam) 2.5μL Primer 2 (intermediate arm) 2.5μL dd sterile water 28.5μL

[0044] Primer 1 (FAM) sequence SEQ ID NO: 5: 5'-ATAGGAGTCACGACGACCAGAA-3'

[0045] Primer 2 (spacer) sequence SEQ ID NO: 6:

[0046] 5'-dA20-Spacer18-ATTAGTCAAGAGGTAGACGCACATA-3'

[0047] Take 20 200μL EP tubes and divide them into two groups. Add the above ingredients in turn. Vortex and centrifuge at low speed before placing the EP tube into the PCR instrument. Edit the PCR program: 94℃ pre-denaturation for 5 minutes; 30 cycles of the following: 95℃ denaturation for 45 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 15 seconds; 72℃ final extension for 1 minute; 20℃ incubation for 15 minutes.

[0048] Make 2% agarose gel: weigh 0.5g agarose and add it to a conical flask, then add 25mL of 0.5% TBE solution to the conical flask, heat and boil in a microwave until the agarose dissolves, add 2.5μL nucleic acid dye, shake well and pour it onto the gel plate, insert a comb, and let it stand for 30 minutes. After the gel solidifies, put the gel plate into the electrophoresis tank, add 0.5×TBE solution, unplug the comb, and load 5μL. Use a Bio-Rad electrophoresis instrument for 120V electrophoresis for 30 minutes. After taking out the gel, use a Bio-Rad gel imaging analyzer to image and judge the PCR effect. From the gel image, it can be seen that the bands are single and neat, and the fragment length is about 87nt, which is consistent with the size of the initial library, indicating that the screening process is reasonable and can be purified and recovered to continue the next round of screening.

[0049] 4. Separation of required aptamer library by DNA-PAGE electrophoresis

[0050] Prepare 30mL of 8% PAGE gel and shake it evenly. Pour the prepared gel into the gel plate, stop pouring the gel when the gel surface reaches the top, and insert the comb. Polymerize at room temperature for 60 minutes. After the gel solidifies, pull out the comb and rinse the gel wells with 1×TBE solution. Mix the PCR amplified product sample with 2×DNA urea loading buffer in a volume ratio of 1:1, denature at 95℃ for three minutes, and put on ice before use.

[0051] Fix the gel plate on the 6-1 electrophoresis instrument and add enough 1×TBE to the upper and lower tanks. After 30 minutes of pre-electrophoresis, load the samples placed on ice, spot a marker in one well of each gel, and spot PCR samples in the remaining wells. Connect the power cord, turn on the switch, and select a power of 5-6w. Stop the electrophoresis, take out the gel, cut a strip of gel along the spotting hole where the marker of each gel is located, place it in the staining solution for staining for 30 minutes (staining solution formula: 15μL Gelred, 5mL 1M NaCl, 45mLH2O) After the two parts of the gel are assembled, use the Bio-Rad gel imaging analyzer to image, find the corresponding position of the library and cut the entire row horizontally.

[0052] 5. PAGE gel DNA recovery:

[0053] The desired bands were recovered using a UNIQ-10 column PAGE gel DNA recovery kit (Sangon Biotech (Shanghai) Co., Ltd., catalog number: B511135-0100). The resulting aptamer library will be used for the next round of screening.

[0054] 6. Repeat the second to fifteenth rounds of screening

[0055] Among them, in the thirteenth round of screening, five different amino acids were coupled to amino magnetic beads and tea content simulation solution for negative screening. The specific operation was to denature the ssDNA recovery product screened in the 12th round in a 95°C water bath for 8 minutes, and then immediately renature it in an ice bath for 8 minutes to form a specific structure. Aspartic acid, serine, glutamic acid, histidine, and arginine were coupled to amino magnetic beads according to the method described in step 1.2 (that is, 146ug aspartic acid, 88ug serine, 123ug glutamic acid, 130ug histidine, and 146ug arginine were dissolved in 500μL of amino magnetic bead coupling buffer (0.05MMES, 0.5M NaCl), and the other steps were consistent with 1.2) to obtain negative screening amino acid magnetic beads. The three major functional components of tea, catechins, alkaloids, and amino acids, were prepared into 1mL tea content simulation solution according to the content in fresh tea leaves, and the specific dosage is shown in Table 2. After the denatured and renatured 12th round aptamer library was incubated with the negative screening amino acid magnetic beads, all the supernatants were aspirated and added to the amino magnetic beads coupled with theanine, and then 10 μL of the simulation solution was added and incubated for 1 hour. The supernatant was discarded, 100 μL of ddH2O was added, and the mixture was incubated at 95°C for 8 minutes. After cooling, the supernatant was collected, which was the 13th round aptamer library obtained by amino magnetic bead screening, and used as a template for PCR amplification. After gel recovery, the next round of positive screening was continued.

[0056] Table 2 Composition of simulated solution of tea contents

[0057]

[0058] Example 2: Selection of DNA aptamer library for sequencing and prediction of candidate aptamer secondary structures.

[0059] 1. Aptamer library affinity verification

[0060] The Fam-modified initial library and the ssDNA recovery products screened in fifteen rounds of experiments were diluted to 60nM respectively, first denatured in a 95°C water bath for 8 minutes, and then immediately renatured in an ice bath for 8 minutes to form a specific structure. Prepare 16 copies of amino magnetic beads that have completed the theanine coupling step, recorded as A1-A16EP tubes. In addition, prepare 16 copies of unloaded amino magnetic beads, add 133.4uLBB buffer to each of them, recorded as B1-B16EP tubes. Add 66.6uL60nM of the Fam-modified initial library and the ssDNA library screened in fifteen rounds of experiments to the B1-B16EP tubes, and incubate at room temperature for 30min. After incubation, aspirate all the supernatants, add them to the A1-A16EP tubes, and incubate at room temperature for 30min. After incubation, the supernatant was discarded, the ssDNA bound to the magnetic beads was eluted, and the fluorescence value was measured using a Nanodrop 3300 ultra-micro fluorescence spectrophotometer (Thermo Scientific, ). The aptamer libraries with the highest affinity in the 5th, 7th, and 13th screening rounds were selected for PCR amplification using unlabeled primers. The PCR products were purified and sent to Novogene for library construction and High-throughput sequencing (HTS) sequencing was completed using the Illumina sequencing platform. The obtained sequence library was analyzed using self-compiled software, and sequences with high enrichment and low binding energy were selected to obtain the candidate theanine nucleic acid aptamer Apt-1 to be tested. The sequence is shown in SEQ ID NO: 1.

[0061] 2. Apt-1 secondary structure prediction

[0062] The secondary structure of aptamer Apt-1 was predicted using Mfold software. Figure 1 As shown in the middle left figure, the results show that the secondary structure of Apt-1 is a stem-loop structure with high stability.

[0063] 3. Affinity verification of Apt-1

[0064] Determination of the K of theanine aptamer Apt-1 by ITC dvalue. Use binding buffer to prepare 50mM theanine solution, and use binding buffer to prepare 25uM aptamer solution (binding buffer: 50mMTris, 5mMKCl, 100mMNaCl, 1mMMgCl2, pH=7.4). Apt-1 was first denatured in a 95℃ water bath for 8 minutes, and then immediately renatured in an ice bath for 8 minutes to form a specific structure. Apt-1 was then isothermally titrated using an isothermal calorimetric titrator (TAInstruments). When the instrument vacuum was higher than 400mmHg, the sample was degassed for 10min. 350uL of 25uM aptamer was added to the titration needle. 50uL of 50mM theanine was added to the sample pool. The total number of titrations was set to 20, the volume of each titration was set to 2.5μL, the time was set to 200s, the reaction temperature was set to 25℃, and the stirring speed was set to 250rpm. Figure 2 Figure 2 is the ITC data of theanine nucleic acid aptamer screened by the present invention and the affinity detection of theanine and the dissociation constant (K d The isothermal calorimetric titration results showed that Apt-1 had an affinity for theanine, and its dissociation constant (K d ) value is 1.151×10 -2 mol / L.

[0065] Example 3: Truncation optimization and affinity and specificity verification of theanine aptamer.

[0066] 1. Molecular docking

[0067] The secondary structure of the aptamer Apt-1 was predicted using the NUPACK website (http: / / www.nupack.org / ), and the tertiary structure of the aptamer was generated using XiaoLab. The 3D structure of theanine was obtained from the PubChem website. The folder containing the Autodock run file, theanine molecule 3D structure file, and the aptamer molecule tertiary structure file was set as the default working directory. Import the theanine molecular structure and the aptamer molecular structure, select the theanine molecule and the aptamer molecule after editing, set the Autodock docking parameters and calculation method, and save them as a dpf file. Import the dpf file, run Autodock to obtain the results, and use PyMol to draw the graph. Figure 3 This is the docking result diagram and binding site of theanine nucleic acid aptamer Apt-1 and theanine molecule.

[0068] 2. Truncation optimization

[0069] Based on the molecular docking results, the binding sites of theanine and the aptamer were determined. According to the secondary structure of the aptamer, the aptamer sequence was truncated twice in the stem-loop unit to cut off the non-binding sites and obtain the truncated aptamers Apt-2 and Apt-3. The secondary structure of the aptamer Apt-2 is shown in Figure 1 The sequence is shown in SEQ ID NO: 2, and the secondary structure of the aptamer Apt-3 is shown in Figure 1 As shown in the middle right figure, the sequence is shown as SEQ ID NO: 3.

[0070] 3. Affinity verification

[0071] The affinity of the first truncated aptamer Apt-2 sequence and the second truncated aptamer Apt-3 sequence was verified by isothermal calorimetric titration, and the affinity verification method was the same as that of Apt-1. Figure 4 ITC data of affinity detection between theanine nucleic acid aptamer Apt-2 and theanine and K d value. Figure 5 ITC data of affinity detection between theanine nucleic acid aptamer Apt-3 and theanine and K d The isothermal calorimetric titration results showed that after truncation optimization, the Kd value of Apt-2 was 1.781×10 -5 mol / L, K of Apt-3 d The value is 5.942×10 -8 mol / L.

[0072] 4. Specificity Verification

[0073] Take 3g of Taiping Houkui dry tea and extract it with 50mL 75% methanol solution for 30min to obtain tea soup solution. Take the tea soup solution for pretreatment to remove polyphenols. The pretreatment diagram is shown in Figure 7 The specific steps are as follows: 2 mL of tea soup solution extracted with 75% methanol solution was added to a 15 mL centrifuge tube containing 200 mg of SPE filler (NH2), vortexed for 3 minutes, centrifuged at 4000 r / min for 5 minutes, and 1 mL of supernatant was filtered through a microporous filter membrane to obtain a pre-treated tea soup solution as the tea soup sample to be tested. 90 ul of 2.4 mM aptamer Apt-3 was added to the tea soup sample to be tested and immediately placed in a high performance liquid chromatography-mass spectrometer for detection. Figure 6This is a quantitative result diagram of the specific removal effect of theanine nucleic acid aptamer Apt-3 in tea soup by high performance liquid chromatography-mass spectrometry. The quantitative analysis results show that theanine aptamer Apt-3 has a high removal effect on theanine, with a removal rate of more than 80%, while it has no significant removal effect on other substances in tea soup such as histidine, aspartic acid, caffeine, EGC, GC, etc.

[0074] Example 4 Detection of theanine aptamer nanogold test paper.

[0075] 1. Preparation of Gold Nanoparticles

[0076] Heat 100 mL of 0.01% HAuCl4 solution to boil under magnetic stirring conditions. Immediately after boiling, add 2 mL of 1% trisodium citrate solution and continue heating with magnetic stirring. The solution changes from light yellow to dark purple and finally to wine red. Continue heating for 10 min, then stop heating, cool to room temperature, and refrigerate at 4°C for later use.

[0077] 2. Test strip T and C line processing

[0078] 30 μL of 1 mg / mL streptavidin solution was mixed with 240 μL of 10 μM Apt-3 aptamer solution, incubated at 4°C for 2 h, centrifuged and filtered in an ultrafiltration tube for three times, streaked, and dried in a 37°C incubator for 12 h.

[0079] 3. Coupling of gold nanoparticles with theanine

[0080] After mixing 2 mL of the pre-treated tea solution (treated according to the specificity verification experimental steps in Example 3) with 900 μL of pH 13 NaOH solution, 1 mL of the nano-gold solution was added, mixed and allowed to stand for 2 hours, and washed three times with ultrapure water (1000 r / min, 20 min) for later use.

[0081] 4. Test strip detection

[0082] Add 20μL of theanine-nanogold solution to the assembled test strip, the T line turns red, and add 5μL of sample solution for detection. In the legend, the negative sample is ddH2O, and the strong positive sample is 3g of Taiping Houkui dry tea extracted with 50mL75% methanol solution for 30min to obtain a tea soup solution diluted 5 times after removing polyphenols. The medium positive sample is a solution diluted 5 times of the strong positive sample, and the weak positive sample is a solution diluted 25 times of the strong positive sample. (The concentration of theanine in the sample competes with the amino acids on the aptamer to affect the binding of the T line. The more theanine, the more the red T line fades, until there is no color, that is, if the position of the T line does not show a red line, it is a strong positive, light red is a weak positive, and red is a negative. Negative means there is no theanine in the sample, and positive means there is theanine in the sample.)

[0083] Figure 8 It is the positive reference result of theanine aptamer nanogold test paper detection.

[0084] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A theanine nucleic acid aptamer, characterized in that: The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

2. A detection probe or detection kit for theanine, characterized in that: Comprising the nucleic acid aptamer according to claim 1.

3. Use of the nucleic acid aptamer according to claim 1 or the detection probe or detection kit according to claim 2 in detecting theanine.

4. Use of the nucleic acid aptamer according to claim 1 or the detection probe or detection kit according to claim 2 in identifying tea varieties or tea tree breeding screening.

5. The use according to claim 3 or 4, characterized in that: The nanogold test paper method is used for detection or screening.

6. The use according to claim 5, characterized in that: The tea samples to be tested are extracted with alcohol, and the tea soup obtained by extraction is tested after removing polyphenols.

7. The use according to claim 6, characterized in that: The tea sample to be tested is extracted with 75% methanol solution, and the tea soup obtained by extraction is tested after removing polyphenols through an SPE column.

8. Use of the nucleic acid aptamer according to claim 1 in the separation and enrichment of theanine.

9. Use of the nucleic acid aptamer according to claim 1 in preparing a theanine targeting agent.

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