CN117187251A - Nucleic acid aptamer of pentamethyltetrahydrofolate and application of nucleic acid aptamer in animal product detection - Google Patents

Abstract

The invention discloses a nucleic acid aptamer for identifying pentamethyltetrahydrofolate and application thereof in animal product detection. The invention uses the systematic evolution technology of exponential enrichment ligand, takes magnetic beads as separation medium, takes pentamethyltetrahydrofolate as target, obtains the nucleic acid aptamer with the polynucleotide sequence shown as SEQ ID No.1 specifically combined with the target through 9 rounds of screening, has extremely high affinity with pentamethyltetrahydrofolate, has dissociation constant reaching mu M, can not combine with main interfering substances folic acid and tetrahydrofolate of pentamethyltetrahydrofolate, only specifically combines with pentamethyltetrahydrofolate, has good affinity and specificity, can be artificially synthesized, has low cost and short production period, is easy to chemically modify, and can be prepared into sensors, molecular probes, detection reagents and the like for detecting the pentamethyltetrahydrofolate in foods.

Description

Nucleic acid aptamer of pentamethyltetrahydrofolate and application of nucleic acid aptamer in animal product detection

Technical Field

The invention relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer for recognizing pentamethyltetrahydrofolate, a sensor, a detection probe or a detection kit containing the nucleic acid aptamer and application of the nucleic acid aptamer in detecting pentamethyltetrahydrofolate of animal products, and belongs to the field of nucleic acid aptamer and animal product detection.

Background

Pentamethyltetrahydrofolate (5-mTHF) is a biologically active form of folic acid. Folic acid, also known as vitamin B9, a micronutrient that is closely related to human health, deficiency of folic acid can cause various diseases and is particularly important for pregnant women. However, folic acid cannot be directly synthesized in the human body and needs to be obtained from diet. The folic acid enters organisms and is subjected to enzymatic reaction to generate the pentamethyltetrahydrofolate, and then the pentamethyltetrahydrofolate further participates in the reactions of the organisms, and almost 25% of people worldwide have methylene tetrahydrofolate reductase gene variability, so that only the folic acid is supplemented by the human body, and the people can not be utilized by the human body, and the pentamethyltetrahydrofolate needs to be directly supplemented.

At present, the main detection method for detecting folic acid and the active form thereof adopts a high performance liquid chromatography-mass spectrometry (HPLC-MS) method, and the combined technology is based on the high performance liquid chromatography-mass spectrometry method, but has high cost and complex operation, and greatly limits the wide application of the folic acid and the active form thereof in the food industry. The rapid detection kit sold on the market is mostly aimed at a folic acid receptor, and almost no folic acid or pentamethyltetrahydrofolate direct detection kit is found. In view of the problems of high difficulty, high cost and the like in the conventional detection of the pentamethyltetrahydrofolate, the development of a novel recognition molecule has great significance for the content determination of the pentamethyltetrahydrofolate in food, and the rapid detection method of the pentamethyltetrahydrofolate, such as a kit and the like, can be further developed.

Nucleic acid Aptamer (Aptamer) is a novel recognition molecule and has been attracting attention in recent years. The aptamer can be efficiently combined with target molecules through folding into a secondary or tertiary structure in a mode of action such as hydrogen bonding action and shape matching, and targets comprise small molecules, antibiotics, proteins, metal ions and the like. The molecular recognition molecule has the advantages of high affinity, good specificity, easy synthesis and the like, is an ideal recognition molecule, and can be widely applied to the field of analysis and detection. So far, no research report for specifically recognizing the pentamethyltetrahydrofolate aptamer exists.

Disclosure of Invention

It is an object of the present invention to provide nucleic acid aptamers recognizing pentamethyltetrahydrofolate;

it is another object of the present invention to provide a sensor, a detection probe or a kit comprising the nucleic acid aptamer recognizing pentamethyltetrahydrofolate;

the invention also provides a nucleic acid aptamer for recognizing pentamethyltetrahydrofolate, which is used for detecting pentamethyltetrahydrofolate in a sample.

The above object of the present invention is achieved by the following technical solutions:

in one aspect, the present invention provides a nucleic acid aptamer recognizing pentamethyltetrahydrofolate, said nucleic acid aptamer being selected from any one of the nucleotide sequences set forth in the following (a) - (d):

(a) A polynucleotide sequence shown in SEQ ID No. 1;

or (b) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID No.1, which polynucleotide sequence is still capable of specifically binding pentamethyltetrahydrofolate;

or (c) an RNA sequence transcribed from the polynucleotide sequence shown in SEQ ID No. 1;

or (d) a polynucleotide sequence having at least 60% homology to the polynucleotide sequence of SEQ ID No.1, and which is still capable of specifically binding pentamethyltetrahydrofolate; preferably, the polynucleotide sequence has at least 80% homology with the polynucleotide sequence of SEQ ID No.1, and the polynucleotide sequence can still specifically bind to pentamethyltetrahydrofolate; more preferably, the polynucleotide sequence has at least more than 90% homology with the polynucleotide sequence of SEQ ID No.1, and still is capable of specifically binding pentamethyltetrahydrofolate.

In a preferred embodiment of the invention, the nucleic acid aptamer sequence may be modified, including phosphorylation, methylation, amination, carboxylation, sulfhydrylation or isotopic modification; alternatively, the 5 'or 3' end of the sequence of the nucleic acid aptamer is linked to a fluorescent label, a radioactive substance, biotin, streptavidin, digoxin, a nano-luminescent material or an enzyme.

In another aspect, the invention provides a nucleic acid aptamer sensor for detecting pentamethyltetrahydrofolate, the nucleic acid aptamer sensor comprising a PCR primer and a nucleic acid aptamer specifically binding to pentamethyltetrahydrofolate; wherein, the nucleic acid aptamer specifically combined with the pentamethyltetrahydrofolate is the nucleic acid aptamer screened by the invention, and a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxin, a nano luminescent material or enzyme is modified at the 5 'end or the 3' end of the nucleic acid aptamer.

Yet another aspect of the invention is a kit for detecting pentamethyltetrahydrofolate, comprising: the invention provides a PCR tube modification solution and a PCR system solution, and provides a nucleic acid aptamer specifically combined with pentamethyltetrahydrofolate.

The PCR system solution can be reaction liquid carried by commercial DNA polymerase or prepared according to the reaction principle, and comprises the following components: SYBR Green dye, taq DNA polymerase, PCR buffer, dNTP and water.

The invention utilizes systematic evolution technology (SELEX) of exponential enrichment ligand, takes magnetic beads as separation medium, takes pentamethyltetrahydrofolate (5-mTHF) as target, obtains the aptamer specifically combined with the target through 9 rounds of screening, has extremely high affinity and dissociation constant reaching mu M, and specifically combines with 5-mTHF small molecules; the aptamer obtained by screening has good affinity and specificity, can be synthesized artificially, has low cost and short production period, is easy to chemically modify, and can be prepared into a sensor, a molecular probe, a detection reagent and the like for detecting 5-mTHF small molecules in foods.

Definition of terms in connection with the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The term "homology" refers to sequence similarity to a native nucleic acid sequence. "homology" includes nucleotide sequences having preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity to the nucleotide sequence of the regulatory fragment of the present invention. Homology can be assessed visually or by computer software. Using computer software, homology between two or more sequences can be expressed in percent (%), which can be used to evaluate homology between related sequences.

The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other after hydrogen bonding between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are complementary to each other in reverse when the sequences are all seen in the 5 'to 3' direction. It is also known in the art that two sequences which hybridize to each other under a given set of conditions do not necessarily have to be 100% completely complementary.

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature as known in the art. Typically, the probe hybridizes to its target sequence to a greater degree of detectability (e.g., at least 2-fold over background) under stringent conditions than to other sequences. Stringent hybridization conditions are sequence dependent and will be different under different environmental conditions, longer sequences hybridizing specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. For a detailed guidance on nucleic acid hybridization, reference is made to the literature (Tijssen, techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays 1993). More specifically, the stringent conditions are typically selected to be about 5-10℃below the thermal melting point (Tm) for the specific sequence at the defined ionic strength pH. Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (50% of the probes at equilibrium are occupied at Tm because the target sequence is present in excess). Stringent conditions may be the following conditions: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at a pH of 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes, including but not limited to 10 to 50 nucleotides, and at least about 60 ℃ for long probes, including but not limited to greater than 50 nucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least twice background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, at 42 ℃; or 5 XSSC, 1% SDS, at 65℃in 0.2 XSSC and at 65℃in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.

Drawings

FIG. 1 shows the ITC data graph and K values of the aptamer (SEQ ID No. 1) obtained by screening according to the present invention and 5-mTHF small molecule affinity detection.

FIG. 2 shows a graph of detection data of affinity of aptamer (SEQ ID No. 1) screened by the invention with small molecules of interfering Folic Acid (FA) and Tetrahydrofolate (THF); a: adopting the affinity detection result of ITC to SEQ ID No.1 and the small interfering substance FA molecule; b: the affinity detection result of ITC on the small molecule of SEQ ID No.1 and the interfering THF is adopted.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.

Experimental materials and instruments:

1. SA magnetic bead

2. DPBS

3. Q-PCRmix：

TABLE 1Q-PCRmix reaction System

4. PCRmix (for ssDNA preparation)

TABLE 2 PCRmix reaction System

5.8% denaturing PAGE gel

6. The tag primer PCRmix;

library tag primer of lib3 PCRmix

TABLE 3 PCRmix reaction System

7. Screening the obtained pool in each round;

8. UNIQ-10 oligonucleotide purification kit (Shanghai);

9. absolute ethyl alcohol;

10. DNA marker；

denaturation DNA loading buffer by 11.10;

12.4S Red Plus nucleic acid stain (10,000 Xaqueous solution);

13. a TAE solution;

14.5-methyltetrahydrofolate (5-mTHF);

15. 20*PBS；

16. ultrapure water;

17. a Bio-rad fluorescent quantitative PCR instrument;

18. a Bio-rad T100 common PCR instrument;

19. Bio-radPowerPac Basic electrophoresis apparatus;

20. ITC 200。

test example 1 screening of pentamethyltetrahydrofolate nucleic acid aptamer

Test method

1.1 Synthesis of random Single-stranded DNA (ssDNA) library and primers

The random single-stranded DNA (ssDNA) Lib2-76nt library was: primer region sequence +58nt random sequence +primer region sequence, 58 variable region base sequences, 20 base numbers of primer region sequences at two ends:

5’-GGGACCAGCACACGCATAAC-N36-GAGTTATGCGTGCTACCGTG -3’；

an upstream primer: 5 '-FAM-GGGACCAGCACACGCATAAC-3',

a downstream primer: 5 '-15A-spacer 18-CACGGTAGCACGCATAACGC-3',

in the downstream primer, 15A represents a polyA tail composed of 15 adenylates (A), and Spacer 18 represents a hexaethylene glycol Spacer of 18 atoms, which were all synthesized by the company Shanghai, inc.

1.2 immobilization and screening of libraries

(1) 1OD library (pool 0, lib3-76 nt) was taken as a dry powder and centrifuged at 12000g for 10min. After adding 137. Mu.L of DPBS and vortexing, 12000g was centrifuged again for 10min.

(2) Adding the lib1S1CS-biotin primer 205.5 mu L of 10 mu M into the dissolved pool, centrifugally mixing, packaging into PCR tubes, packaging into a PCR 8 row, renaturating the PCR instrument for 10min at 95 ℃ and 1min at 57 ℃, and finally cooling to 25 ℃ (the speed is 0.1 ℃). The concentration of the mixture of the UV detection library and the biotin modification primer is C1.

(3) The 1500. Mu.L SA beads were aspirated, and 200. Mu.L DPBS was washed 5 times, with the final pass being to temporarily store the beads in buffer.

(4) Adding the library with good renaturation into magnetic beads, and shaking for 30min at room temperature. The magnet is used for fishing the magnetic beads, the supernatant is removed, and the concentration of the supernatant is measured by UV to be C2.

(5) 200 μLDPBS was added to the beads and rinsed for a total of 6 washes.

(6) Adding 100 mu LDPBS into the SA magnetic beads of the previous step, incubating for 30min by a shaking table, fishing the magnetic beads by a magnet, sucking the supernatant into an EP tube, washing the magnetic beads once by 100 mu L DPBS, adding the magnetic beads into the EP tube, and marking as the term "Elutation".

(7) Small molecular targets (5-methyltetrahydrofolate, dissolved in DPBS to 1mm x 100 μl) were added to the SA beads from the previous step and incubated for 30min on a shaker. The magnet was used to fish the beads, the supernatant was pipetted into an EP tube, the beads were washed once with 100. Mu.L DPBS and added to the Elutation EP tube, designated as Elutation+.

(8) Eight-joint tubes were taken, 30. Mu.L of mix was added to each well, 1. Mu.L of each of the solution-and solution+ was diluted 10-fold with DPBS, 1. Mu.L of each was added to mix, and the mixture was centrifuged and homogenized. And (5) fluorescence quantitative PCR detection.

1.3 preparation of Single Strand

(1) 2ml of ePCR mix was removed from-20℃and added to the remaining Elutation+ and transferred to a 50ml centrifuge tube for mixing.

(2) 8ml of EM90 oil was added and the mixture was shaken on a high power vortex shaker to prepare an emulsion.

(3) The emulsion was dispensed into PCR tubes, each of 90. Mu.L, and PCR was cycled 25. The procedure is: 95 ℃ for 2min; the cycle was 95℃1min,60℃1min,72℃1min,25 ℃.

(4) And recovering the ePCR product.

(5) And concentrating the ePCR product by using n-butanol, transferring the ePCR product into a 10ml centrifuge tube, adding the n-butanol to fill up, uniformly mixing, and centrifuging 10000g for 2min. After centrifugation, the layers were separated, the upper clear solution was aspirated, and the lower amplification product was transferred to a small EP tube (about 100. Mu.L). Transfer 90. Mu.L to a small centrifuge tube, add 100. Mu. L Urea loading buffer to mix well and heat the PCR instrument for 10min at 95 ℃.

(6) Separating single chain by denaturing PAGE electrophoresis (electrophoresis, cutting gel, boiling gel).

(7) The ssDNA was concentrated in n-butanol.

(8) The ssDNA was dialyzed overnight against DPBS in a 3.5KD dialysis bag and the concentration was determined by microUV.

1.4 repeating the second to ninth rounds.

1.5Q-PCR detection library affinity experiments

(1) Pool1 was diluted to 500nM 100. Mu.L with DPBS, and lib1S1CS-biotin primer 10. Mu.L 10. Mu.M was added to each, and the mixture was centrifuged and packed in PCR tubes. Subpackaging into PCR 8 row, renaturation of the PCR instrument, 10min at 95deg.C and 1min at 57 deg.C, and cooling to 25deg.C (rate 0.1deg.C). The concentrations of the UV assay library and biotin modified primer mixtures were C1-2, respectively.

(2) 100. Mu.L of SA beads were aspirated, 200. Mu.L of DPBS was washed 5 times, and the final pass was to temporarily store the beads in buffer without allowing them to dry.

(3) The library with good renaturation was added to 100. Mu.L of magnetic beads, respectively, and the mixture was shaken at room temperature for 30min. The magnetic beads are fished by a magnet, the supernatant is removed, and the concentration of the supernatant measured by UV is C2-1 respectively.

(4) 200 μLDPBS was added to the beads and rinsed for a total of 6 washes.

(5) 200 mu LDPBS was added to the SA beads of the previous step, and incubated for 20min with shaking bed, and the beads were fished with magnet.

(6) Dividing the magnetic beads into two equal parts, adding 200 mu LDPBS into one part, incubating for 20min by a shaking table, and fishing the magnetic beads by a magnet. Supernatant was pipetted into an EP tube. Is denoted as elision 1-. Another portion was added with 200. Mu.L of 1mM 5-mTHF and incubated for 20min on a shaker to magnet the beads. The supernatant, eleuth, was aspirated into the EP tube, designated eleuth1+.

(7) Pool6 was diluted to 500nM 100. Mu.L with DPBS, added with 10. Mu.L of lib1S1CS-biotin primer and spun into PCR tubes. Subpackaging into PCR 8 row, renaturation of the PCR instrument, 10min at 95deg.C and 1min at 57 deg.C, and cooling to 25deg.C (rate 0.1deg.C). The concentrations of the UV assay library and biotin modified primer mixtures were C1-2, respectively.

(8) 100. Mu.L of SA beads were aspirated, 200. Mu.L of DPBS was washed 5 times, and the final pass was to temporarily store the beads in buffer without allowing them to dry.

(9) The library with good renaturation was added to 100. Mu.L of magnetic beads, respectively, and the mixture was shaken at room temperature for 30min. The magnetic beads are fished by a magnet, the supernatant is removed, and the concentration of the supernatant is C2-2 respectively in the UV measurement.

(10) 200 μLDPBS was added to the beads and rinsed for a total of 6 washes.

(11) 200 mu LDPBS was added to the SA beads of the previous step, and incubated for 20min with shaking bed, and the beads were fished with magnet.

(12) Dividing the magnetic beads into two equal parts, adding 200 mu LDPBS into one part, incubating for 20min by a shaking table, and fishing the magnetic beads by a magnet. The supernatant was pipetted into an EP tube and designated as "solution 6". Another portion was added with 200. Mu.L of 1mM 5-mTHF and incubated for 20min on a shaker to magnet the beads. The supernatant, eleutherence, was pipetted into an EP tube, designated eleutherence 6+.

(13) Pool9 was diluted to 500nM 100. Mu.L with DPBS, added with 10. Mu.L of lib1S1CS-biotin primer and spun into PCR tubes. Subpackaging into PCR 8 row, renaturation of the PCR instrument, 10min at 95deg.C and 1min at 57 deg.C, and cooling to 25deg.C (rate 0.1deg.C). The concentrations of the UV assay library and biotin modified primer mixtures were C1-3, respectively.

(14) 100. Mu.L of SA beads were aspirated, 200. Mu.L of DPBS was washed 5 times, and the final pass was to temporarily store the beads in buffer without allowing them to dry.

(15) The library with good renaturation was added to 100. Mu.L of magnetic beads, respectively, and the mixture was shaken at room temperature for 30min. The magnetic beads are fished by a magnet, the supernatant is removed, and the concentration of the supernatant measured by UV is C2-3 respectively.

(16) 200 μLDPBS was added to the beads and rinsed for a total of 6 washes.

(17) 200 mu LDPBS was added to the SA beads of the previous step, and incubated for 20min with shaking bed, and the beads were fished with magnet.

(18) Dividing the magnetic beads into two equal parts, adding 200 mu LDPBS into one part, incubating for 20min by a shaking table, and fishing the magnetic beads by a magnet. Supernatant was pipetted into an EP tube. And is designated as the solution 9-. Another portion was added with 200. Mu.L of 1mM 5-mTHF and incubated for 20min on a shaker to magnet the beads. Supernatant was pipetted into the EP tube and designated as eluon9+.

(19) Taking 8-joint tubes, adding 30 mu L of mix into each hole, adding 1 mu L of each of the solution-and solution+ into the mix, centrifuging and mixing uniformly, and carrying out fluorescent quantitative PCR detection.

Test results

The test utilizes systematic evolution technology (SELEX) of exponential enrichment ligand, takes magnetic beads as a separation medium, takes pentamethyltetrahydrofolate (5-mTHF) as a target, and finally obtains a small molecule nucleic acid aptamer capable of specifically recognizing the 5-mTHF through 9 rounds of screening, wherein the nucleotide sequence of the nucleic acid aptamer is as follows:

5’-CAGCACACGCATAACGGGGTACCCGGTAATGCGTGTGGTGTATCGGTTTGCGAGTTAT-3’ （SEQ ID No .1）。

test example 2 ITC detection of 5-mTHF Small molecules and screening to obtain ssDNA aptamer affinity assay

The small molecule ssDNA aptamer (SEQ ID No. 1) which recognizes 5-mTHF and is screened in test example 1 is subjected to affinity detection by adopting an ITCT200 instrument, and the specific detection method is as follows:

1. sample preparation: 5-mTHF was diluted to 1mM with PBS; after centrifugation of the synthesized aptamer (SEQ ID No. 1), PBS was added to dissolve in 10. Mu.M.

2. The instrument cleaning process comprises the following steps: respectively cleaning the sample cell and the titration needle with ultrapure water and PBS buffer solution for 5min, and drying for 1-2min after cleaning to avoid residue;

3. small molecule drop buffer: repeating the above instrument cleaning process, adding the sample cell into PBS buffer solution, adding 1mM target 5-mTHF into the titration needle, and performing small molecule titration buffer solution for 20 drops, wherein the heat and water drops have no obvious difference;

4. 5-mTHF droplet aptamer: repeating the cleaning process, adding 20uM aptamer (SEQ ID No. 1) into a sample pool, adding 1mM target 5-mTHF into a titration needle, and starting titration for 20 drops;

5. calculating and giving a K value by an instrument;

6. the affinity of the 5-mTHF small molecule ssDNA aptamer is expressed by the dissociation constant Kd, kd=1/K.

The final determined dissociation constant kd= 0.2571e-6±0.0122 e-6M (fig. 1) for the small molecule ssDNA aptamer (SEQ ID No. 1) that specifically binds to 5-mTHF; the measurement result shows that the small molecular ssDNA aptamer (SEQ ID No. 1) screened in the test example 1 has excellent affinity with 5-mTHF.

Test example 3 5-mTHF Small molecule ssDNA aptamer specificity verification test

The small molecule ssDNA aptamer (SEQ ID No. 1) screened in Experimental example 1 was specifically verified using the same ITC technique as in Experimental example 2.

Reverse screening of the major interfering substances Folic Acid (FA) and Tetrahydrofolate (THF) of 5-mTHF was performed using the same nucleic acid aptamer (SEQ ID No. 1), and the test results showed that the nucleic acid aptamer showed similar results to water in ITC titration of Folic Acid (FA) and Tetrahydrofolate (THF) and that the dissociation constant Kd value could not be fitted (FIG. 2), indicating that the nucleic acid aptamer had No affinity for FA and THF, and that the nucleic acid aptamer specifically recognized small molecule 5-mTHF.

Test example 4 detection assay for 5-mTHF in detection samples Using ssDNA aptamer

The small molecular ssDNA aptamer (SEQ ID No. 1) which is screened in test example 1 and recognizes 5-mTHF is combined with an ITO electrode modified by gold nano particles, and the electrochemical sensing detection is carried out on the 5-mTHF in a sample by a CHI660E electrochemical workstation, wherein the specific detection method is as follows:

1. electrochemical sensor preparation: gold nanoparticles were uniformly sprayed onto the surface of the ITO electrode by ion implantation (voltage: 10keV; implantation dose: 1X 10) ¹⁷ ions cm ^﹣2 The method comprises the steps of carrying out a first treatment on the surface of the Ion beam flux: 0.6 mA) and annealing at 300 ℃ for 2 hours to obtain an ITO electrode (T-Au-ITO) modified by the annealed gold nanoparticles;

2. aptamer modification process: washing an electrode plate (T-Au-ITO) with ultrapure water and absolute ethyl alcohol respectively, placing the washed electrode plate in a 200nM solution of nucleic acid aptamer (SEQ ID No. 1) and placing the solution on an oscillator for oscillation, wherein the oscillator is set at 300rpm and at 24+/-2 ℃, and incubating for 8 hours; taking an electrode after 8h incubation, respectively flushing for three times by using 5% SDS solution and PBS buffer solution, and drying with nitrogen for later use;

3. sample preparation process: preparing three samples of egg, quail egg and pigeon egg respectively; separating yolk, freeze-drying the yolk to obtain yolk powder, and storing at-180deg.C; mixing 1.5g of yolk powder with 15mL of PBS buffer solution (0.01M, pH=7.4) under shaking for 5min, and performing ultrasonic water bath for 10min; adding 30ml of methanol, mixing for 10min, centrifuging at 12000rpm at 4deg.C for 30min, and collecting supernatant; the collected supernatant was dried with nitrogen and added to 16mL of PBS (0.01 m, ph=7.4) buffer solution for ultrasonic dissolution for 10min; filtering by a 0.22 mu M microporous filter membrane filter to obtain a solution to be tested.

4. Electrochemical sensing detection process: the electrode plate modified by the aptamer (SEQ ID No. 1) is used as a working electrode, a silver-silver chloride electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and the electrochemical detection is carried out on the sample solution to be detected through a CHI660E electrochemical workstation under a three-electrode system.

5. The electrochemical sensing detection method comprises the following steps of: cyclic Voltammetry (CV) was chosen as the sweep potential from 0.2V to 1.2V at a sweep rate of 100mV/s;

finally, the small molecular ssDNA aptamer (SEQ ID No. 1) which is screened in test example 1 and recognizes the 5-mTHF is successfully modified on an ITO electrode, and the detection range of the electrochemical sensor based on the aptamer (SEQ ID No. 1) on the 5-mTHF can reach 7000nm from 10nm by an electrochemical sensing detection method, and the detection limit is 1nm; the method is practically applied to detection of 5-mTF in poultry egg samples, and found that eggs contain 5-mTF 195.26 mu g/100g, quail eggs contain 5-mTF 52.43 mu g/100g, and pigeon eggs contain 5-mTF 75.61 mu g/100g.

Claims (9)

1. A nucleic acid aptamer recognizing pentamethyltetrahydrofolate, characterized in that said nucleic acid aptamer is selected from any one of the nucleotide sequences set forth in (a) - (d) below:

(a) A polynucleotide sequence shown in SEQ ID No. 1;

or (b) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID No.1, which polynucleotide sequence is still capable of specifically binding pentamethyltetrahydrofolate;

or (c) an RNA sequence transcribed from the polynucleotide sequence shown in SEQ ID No. 1;

or (d) a polynucleotide sequence having at least 60% homology with the polynucleotide sequence of SEQ ID No.1, and which is still capable of specifically binding pentamethyltetrahydrofolate.

2. The aptamer of claim 1, wherein the aptamer has at least 80% homology to the polynucleotide sequence of SEQ ID No.1, and wherein the polynucleotide sequence still specifically binds pentamethyltetrahydrofolate.

3. The aptamer of claim 1, wherein the aptamer has at least 90% homology to the polynucleotide sequence of SEQ ID No.1, and wherein the polynucleotide sequence still specifically binds pentamethyltetrahydrofolate.

4. A nucleic acid aptamer according to any one of claims 1 to 3, wherein the nucleic acid aptamer sequence is modified, the modification comprising phosphorylation, methylation, amination, carboxylation, sulfhydrylation or isotopic modification; or the modification is to connect the 5 'end or the 3' end of the sequence of the nucleic acid aptamer with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxin, a nano luminescent material or an enzyme.

5. A nucleic acid aptamer sensor for detecting pentamethyltetrahydrofolate, the nucleic acid aptamer sensor comprising PCR primers, a nucleic acid aptamer that specifically binds pentamethyltetrahydrofolate; characterized in that the nucleic acid aptamer specifically binding to pentamethyltetrahydrofolate is the nucleic acid aptamer of any one of claims 1-3.

6. The nucleic acid aptamer sensor of claim 5, wherein the nucleic acid aptamer sequence is modified, the modification comprising phosphorylation, methylation, amination, carboxylation, sulfhydrylation, or isotopic modification; or the modification is to connect the 5 'end or the 3' end of the sequence of the nucleic acid aptamer with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxin, a nano luminescent material or an enzyme.

7. A kit for detecting pentamethyltetrahydrofolate comprises a PCR tube modification solution, a PCR system solution and a nucleic acid aptamer; the nucleic acid aptamer according to any one of claims 1 to 3.

8. The kit of claim 7, wherein the nucleic acid aptamer sequence is modified, the modification comprising phosphorylation, methylation, amination, carboxylation, sulfhydrylation or isotopic modification; or the modification is to connect the 5 'end or the 3' end of the sequence of the nucleic acid aptamer with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxin, a nano luminescent material or an enzyme.

9. Use of a nucleic acid aptamer according to any one of claims 1-3 for detecting pentamethyltetrahydrofolate.