Nucleic acid probe and method for DNA detection using nucleic acid probe

Abstract

The invention discloses a nucleic acid probe and a method for DNA detection using the nucleic acid probe, and relates to the field of DNA detection. The nucleic acid probe comprises: the 3' end of the nucleic acid probe is modified with azadiphenylcyclooctyne, the nucleic acid probe is obtained by modifying a solid phase carrier with azadiphenylcyclooctyne structure, the solid phase carrier is a porous glass microsphere modified with azadiphenylcyclooctyne structure, and the surface modification functional group of the porous glass microsphere is a primary amino group. This can make up for the problem of lack of nucleic acid 3' end modified azadiphenylcyclooctyne products, provide more probe modification types for nucleic acid detection, and expand the scope of nucleic acid detection.

Description

Technical Field

[0001] The present invention relates to the field of DNA detection, and in particular to a nucleic acid probe and a method for performing DNA detection using the nucleic acid probe. Background Art

[0002] Environmental DNA (eDNA) detection technology is a technology developed in the 1990s. This technology extracts DNA of specific biological species from environmental samples such as water and sediment to monitor the abundance of specific biological species. At present, eDNA technology is used to detect a variety of marine species and determine species abundance. It is increasingly widely used in biological species monitoring and biomass estimation due to its advantages such as simple sampling and high sensitivity. In eDNA detection, a variety of nucleic acid probes are needed to capture and detect the extracted eDNA. In order to meet the needs of high-sensitivity detection, chemical modifications are required at the bases, sugar rings, phosphodiester bonds, 3' ends, 5' ends, and various connection sites between nucleotides of nucleic acids to achieve different functionalization of nucleic acid probes.

[0003] Dibenzoazacyclooctyne (DBCO, as shown in the following formula (1)) compounds have good chemical stability, lower ring strain and higher reactivity. They can react with azide functional groups to undergo efficient click chemistry at room temperature, and thus play an important role in the design of nucleic acid probes for detecting eDNA.

[0004] (1)

[0005] However, there are still no reports on nucleic acid probes modified with azadiphenylcyclooctyne compounds at the 3' end of nucleic acids, so the current nucleic acid probes and methods for DNA detection using probes still need to be improved. Summary of the invention

[0006] In view of the problem that there is no nucleic acid probe with azadiphenylcyclooctyne modified at the 3' end of nucleic acid and the use of the probe for DNA detection, the present invention provides a nucleic acid probe and a DNA detection method. The probe uses a solid phase carrier modified with azadiphenylcyclooctyne to obtain a nucleic acid probe with azadiphenylcyclooctyne modified at the 3' end, and the synthesis effect is good.

[0007] In order to achieve the above object, the present invention provides a nucleic acid probe on the one hand. The nucleic acid probe is used for environmental DNA detection, wherein: the 3' end of the nucleic acid probe is modified with azadiphenylcyclooctyne, the nucleic acid probe is obtained by a solid phase carrier modified with azadiphenylcyclooctyne structure, the solid phase carrier is a porous glass microsphere modified with azadiphenylcyclooctyne structure, and has the following structure:

[0008] ,

[0009] The surface modification functional groups of the porous glass microspheres are primary amino groups.

[0010] The nucleic acid probe of the present invention adopts a solid phase carrier modified with azadiphenylcyclooctyne to obtain a nucleic acid with azadiphenylcyclooctyne modified at the 3' end. The solid phase carrier has good wettability in the solution, thereby improving the synthesis effect of the nucleic acid with azadiphenylcyclooctyne modified at the 3' end, making up for the problem of lack of nucleic acid products with azadiphenylcyclooctyne modified at the 3' end, providing more probe modification types for nucleic acid detection, and expanding the scope of nucleic acid detection.

[0011] According to an embodiment of the present invention, the nucleic acid probe is a polynucleotide composed of one or more nucleotides.

[0012] According to an embodiment of the present invention, the nucleotide includes at least one of the following: natural and non-natural nucleic acids modified on the sugar ring, wherein the sugar ring is: natural deoxyribose, natural ribose, 2'-methoxy substituted deoxyribose, 2'-fluoro substituted deoxyribose, 2'-methoxyethyl substituted deoxyribose, 2'-methylamino-2-oxyethyl substituted deoxyribose, 2'-propargyloxy substituted deoxyribose, 2'-butoxy substituted deoxyribose, 2'-hexyloxy substituted deoxyribose, 2'-octyloxy substituted deoxyribose, 2'-decyloxy substituted deoxyribose, 2'-dodecyloxy substituted deoxyribose, 2'-tetradecyloxy substituted deoxyribose. 2'-O-deoxyribose, 2'-hexadecyloxy substituted deoxyribose, 2'-octadecyloxy substituted deoxyribose, 2'-eicosyloxy substituted deoxyribose, 2'-oxy-4' carbonyl methylene lock configuration deoxyribose, 2'-oxy-4' carbonyl vinyl lock configuration deoxyribose, 2'-oxy-4' carbonyl-S-configuration ethyl lock configuration deoxyribose, 2'-oxy-4' carbonyl-R-configuration ethyl lock configuration deoxyribose, 2'-fluoro substituted arabinose, 2'-methoxy substituted arabinose, L-configuration deoxyribose, L-configuration ribose, 3' and 5' link flipped deoxyribose or 3' and 5' link flipped ribose;

[0013] Natural and non-natural nucleic acids modified on bases, wherein the bases are: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine or N-methylpseudouridine;

[0014] Natural and non-natural nucleic acids modified on the linking unit, wherein the linking unit is: a racemic phosphorothioate diester bond, an R-configuration phosphorothioate diester bond, an S-configuration phosphorothioate diester bond, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, or monooctyl hypophosphite;

[0015] Non-natural structure modified nucleic acid connecting nucleosides and phosphates: 2', 3'-dideoxy-β-D-pyranose, wherein the base is at the 6-β position; 2-hydroxymethylmorpholine, wherein the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; unlocked nucleic acid unit, whose structure is an RNA-like structure without a chemical bond between 2' and 3', wherein the base is at the 1'-β position.

[0016] According to an embodiment of the present invention, the length of the polynucleotide is 2-240 nucleotides.

[0017] According to an embodiment of the present invention, the length of the polynucleotide is 20-160 nucleotides.

[0018] In the second aspect of the present invention, the present invention proposes a method for DNA detection using the aforementioned nucleic acid probe. The method comprises: providing a nucleic acid probe, the nucleic acid probe being the aforementioned; mixing the sample to be tested and the nucleic acid probe for hybridization reaction; capturing the nucleic acid probe after the hybridization reaction, and calculating and determining the DNA content to be detected in the sample to be tested.

[0019] According to an embodiment of the present invention, providing a nucleic acid probe includes designing a nucleic acid probe according to a DNA to be detected in a sample to be detected, and providing a nucleic acid probe includes the following steps: determining a detection region of the DNA to be detected, and selecting a marker sequence; determining a sequence of the nucleic acid probe according to the marker sequence, synthesizing nucleotides and modifying them, and synthesizing and modifying nucleotides includes: using a solid phase carrier modified with an azadiphenylcyclooctyne structure to obtain the nucleic acid probe with a 3'-end modified with azadiphenylcyclooctyne.

[0020] According to an embodiment of the present invention, the solid phase carrier is obtained by the following steps: providing a nitrogen-containing diphenyl cyclooctyne structure compound; mixing the nitrogen-containing diphenyl cyclooctyne structure compound with porous glass microspheres and forming a connecting unit between the porous glass microspheres and the nitrogen-containing diphenyl cyclooctyne structure compound, wherein the surface of the porous glass microspheres is modified with amino groups, and the connecting unit is formed by succinamide, 3-amino-1,2-propanediol and diethylene glycol ester.

[0021] According to an embodiment of the present invention, the particle size of the porous glass microspheres is 10-100 microns, and the micropore diameter is 500-2000 angstroms. Generally, the smaller the micropore diameter, the greater the load, the more nucleic acids that can be synthesized, and the shorter the length, the larger the micropore diameter, the longer nucleic acids that can be synthesized, but the total amount is less. The above technical scheme, by selecting the appropriate microsphere particle size and micropore diameter, makes the nucleic acid synthesis yield and load both at a better level.

[0022] According to an embodiment of the present invention, the loading amount of the compound containing an azadiphenylcyclooctyne structure modified on the surface of the porous glass microspheres is 30-120 μmol / g.

[0023] Through the above technical solution, the present invention achieves the following beneficial effects:

[0024] The present invention realizes the preparation of a nucleic acid probe with 3'-end modified azadiphenylcyclooctyne through a specific solid phase carrier. The solid phase carrier uses succinamide, 3-amino-1,2-propanediol and diethylene glycol ester structure as connecting units, and modifies azadiphenylcyclooctyne on porous glass microspheres. Compared with traditional solid phase carriers, the solid phase carrier has better wettability in a solution during the process of being used for 3'-end modification of DNA, RNA, artificially synthesized nucleotides containing non-natural nucleotide units, and artificially synthesized nucleotides containing natural nucleotide units, thereby improving the synthesis effect of 3'-end modified azadiphenylcyclooctyne nucleic acid, making up for the problem of lack of nucleic acid 3'-end modified azadiphenylcyclooctyne products, providing more probe modification types for nucleic acid detection, and expanding the scope of nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a basic reaction cycle of one nucleotide for nucleic acid synthesis on a porous glass microsphere type solid phase support;

[0026] Figure 2 is a schematic diagram of the synthesis route of the solid phase carrier in Example 1 of the present invention;

[0027] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound 2 synthesized in Example 1 of the present invention;

[0028] Figure 4is the NMR mass spectrum of compound 2 synthesized in Example 1 of the present invention;

[0029] Figure 5 is a molecular weight diagram of the T20 sequence of the modified azadiphenylcyclooctyne synthesized in Example 2 of the present invention;

[0030] Figure 6 is a high performance liquid chromatography spectrum of nucleic acid synthesis using a modified carrier of porous glass microspheres with a pore diameter of 2000 angstroms in Example 2 of the present invention;

[0031] Figure 7 is a high performance liquid chromatography spectrum of nucleic acid synthesis using a modified carrier of porous glass microspheres with a pore diameter of 1000 angstroms in Example 2 of the present invention;

[0032] Figure 8 This is a high performance liquid chromatography spectrum of nucleic acid synthesis using a modified carrier of porous glass microspheres with a pore diameter of 500 angstroms in Example 2 of the present invention. DETAILED DESCRIPTION

[0033] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0034] For ease of understanding, the following briefly describes the principle by which the present invention can achieve the above beneficial effects:

[0035] As mentioned above, nucleic acids and nucleic acid probes used in current eDNA detection need to be synthesized by chemical methods. The main principle of chemical synthesis is solid phase synthesis based on phosphoramidite chemistry (for details, see Figure 1 ), all synthesis reactions are carried out on solid phase carriers. The current mainstream solid phase carrier is controlled pore glass (CPG) with different numbers and pore sizes. Researchers can perform different structures and different numbers of chemical modifications on the 3' and 5' end groups of natural and non-natural nucleic acids to construct various functional nucleic acid probes, providing basic research tools for clinical, agricultural, animal husbandry, breeding, environmental testing and scientific research.

[0036] (2)

[0037] (3)

[0038] (4)

[0039] At present, the products used for nucleic acid modification with dibenzoazacyclooctyne (DBCO) are mainly products containing phosphoramidites (as shown in the above formula (2)), products containing N-hydroxysuccinimide structures (as shown in the above formula (3)), and phosphoramidites connected to nucleosides (as shown in the above formula (4)). Among them, the products containing phosphoramidites are used to modify the 5' end of nucleic acids, the products containing N-hydroxysuccinimide are used to modify nucleic acids that already contain modified amino groups, and the phosphoramidites connected to nucleosides can be used to modify any site on nucleic acids. However, due to the poor solubility of dibenzoazacyclooctyne at room temperature, a film will form on its surface in the solvent in a nucleic acid synthesizer without a heating function, resulting in poor synthesis effect. Nucleic acid probes modified with dibenzoazacyclooctyne compounds at the 3' end of nucleic acids have not yet been reported.

[0040] The present invention can improve the wettability of the azadiphenylcyclooctyne-containing part in the solution by adopting a specific solid phase carrier, so that a nucleic acid probe with azadiphenylcyclooctyne modified at the 3' end can be easily obtained, thereby expanding the scope of DNA detection.

[0041] In the following examples, the model of the nucleic acid synthesizer is HJ01201202; the model of the high performance liquid chromatography is Waters 2695, and the model of the mass spectrometer is LCMS-IT / TOF.

[0042] Example 1 Synthesis of solid phase carrier

[0043] like Figure 2 As shown, the synthesis steps of the solid phase carrier are:

[0044] 1) Synthesis of compound 2

[0045] Take 30.5 g of compound 1 containing azadiphenylcyclooctyne and succinyl (0.1 mol), 41.2 g of 3-amino-2-hydroxy-1- (bis (4-methoxyphenyl) (phenyl) methoxy) propyl ether (for forming 3-amino-1,2-propanediol) (0.105 mol), 24.7 g of dicyclohexylcarbodiimide (0.12 mol), 1 g of 4,4-dimethylaminopyridine, add to 500 ml of ethyl acetate, stir at room temperature for 36 hours, filter to remove insoluble matter, wash the filtrate twice with 1000 ml of water, 500 ml each time, wash the organic phase with 600 ml of saturated brine, add anhydrous sodium sulfate and dry for 12 hours.

[0046] The crude product from the previous step was added to 400 ml of ethyl acetate, and 10 g of diglycolic anhydride (for forming diglycol ester) and 20 g of triethylamine were added. The mixture was stirred at room temperature for 8 hours, and the insoluble matter was removed by filtration. The filtrate was washed twice with 800 ml of water, 400 ml each time. The organic phase was washed with 600 ml of saturated brine, and then dried with anhydrous sodium sulfate for 12 hours.

[0047] The crude product was recrystallized using 100 ml of methanol at 4 degrees Celsius. The crystallization was repeated twice to obtain a white powder product (ie, compound 2) with a weight of 52.8 g and a yield of 58.9%.

[0048] The H NMR spectrum of compound 2 is as follows Figure 3 As shown, 1 H-NMR (CDCl3) ppm: 7.67-7.66 (m, 1H,Ar H ), 7.38-7.43 (m, 3H, Ar H ), 6.97-7.26 (m, 14H, Ar H ), 6.63-6.65 (m, 4H,Ar H ), 4.76-4.79 (d, 1H, C H 2N), 4.76-4.79 (d, 1H, C H 2O), 3.85-3.91 (m, 2H, C H 2O+C H N), 3.79 (s, 2H, C H 2CO), 3.57 (s, 2H.C H 2CO), 3.34-3.37 (d, 1H, C H 2N),3.18-3.23 (m, 1H, C H 2CO), 2.53-2.57 (t. 6H, CH3C H 2N), 2.65-2.71 (m, 1H,C H 2CO), 2.31-2.38 (m, 1H, C H 2CO), 2.01-2.09 (m, 1H, C H 2CO), 1.76-1.83 (m, 1H,C H 2CO), 0.81-0.84 (t, 9H, C H 3CH2N)

[0049] Figure 4 is the NMR mass spectrum of compound 2, whose molecular formula is C 47H 44 N2O9, theoretical molecular weight 897.4200, measured molecular weight 898.4304 (M+H) + .

[0050] The structural formula of compound 2 is shown in the following formula (6). Formula 6 shows a triethylamine salt of a carboxylic acid, and the structural chemical properties of the salt are more stable.

[0051] (6)

[0052] 2) Synthesis of azadiphenylcyclooctyne-containing porous glass microspheres with a pore size of 500 angstroms

[0053] Weigh 6.0 g of compound 2, dissolve it in 200 ml of anhydrous dimethyl sulfoxide, add 20 g of amino-modified porous glass microspheres with a pore size of 500 angstroms, add 10.3 g of dicyclohexylcarbodiimide (0.05 mol) and 1 g of 4,4-dimethylaminopyridine, and stir at room temperature for 24 hours. Filter, wash the filter cake with dimethyl sulfoxide, 50 ml each time, wash three times, and then wash with anhydrous ethanol, 50 ml each time, wash 5 times. Collect the solid, and vacuum dry to obtain the product, whose structure is shown in formula (5).

[0054] (5)

[0055] 3) Synthesis of azadiphenylcyclooctyne-containing porous glass microspheres with a pore size of 1000 angstroms

[0056] Weigh 6.0 g of compound 2, dissolve it in 200 ml of anhydrous dimethyl sulfoxide, add 20 g of amino-modified porous glass microspheres with a pore size of 1000 angstroms, add 10.3 g of dicyclohexylcarbodiimide (0.05 mol) and 1 g of 4,4-dimethylaminopyridine, and stir at room temperature for 24 hours. Filter, wash the filter cake with dimethyl sulfoxide, 50 ml each time, wash three times, and then wash with anhydrous ethanol, 50 ml each time, wash 5 times. Collect the solid and vacuum dry to obtain the product.

[0057] 4) Synthesis of azadiphenylcyclooctyne-containing porous glass microspheres with a pore size of 2000 angstroms

[0058] Weigh 10.0 g of compound 2, dissolve it in 200 ml of anhydrous dimethyl sulfoxide, add 20 g of amino-modified porous glass microspheres with a pore size of 2000 angstroms, add 10.3 g of dicyclohexylcarbodiimide (0.05 mol) and 1 g of 4,4-dimethylaminopyridine, and stir at room temperature for 24 hours. Filter, wash the filter cake with dimethyl sulfoxide, 50 ml each time, wash three times, and then wash with anhydrous ethanol, 50 ml each time, wash 5 times. Collect the solid and vacuum dry to obtain the product.

[0059] Loading determination method:

[0060] Weigh 15 mg of porous glass microspheres modified with azadiphenylcyclooctyne and place them in a 15 ml test tube;

[0061] Weigh 3.55 g of p-toluenesulfonic acid, add it to a clean 250 ml conical flask, and dissolve it to 200 ml with anhydrous acetonitrile to obtain a p-toluenesulfonic acid acetonitrile solution with a concentration of 0.1 mol / L;

[0062] Add 1 ml of 0.1 mol / L p-toluenesulfonic acid acetonitrile solution as eluent to the test tube containing the porous glass microspheres modified with azadiphenylcyclooctyne, mix well, let stand for 5 minutes, centrifuge for 10 seconds, and take out the supernatant;

[0063] The detection wavelength of the UV-visible spectrophotometer was set at 498 nanometers, the eluate was taken as a reference solution, and the absorbance coefficient of the supernatant was measured.

[0064] The loading calculation formula is as follows:

[0065] Loading (μmol / g) = OD λ498 × dilution factor × 12 ÷ CPG powder mass (mg)

[0066] The loading capacity of porous glass microspheres with different pore sizes is shown in Table 1.

[0067] Table 1

[0068]

[0069] It can be seen from Table 1 that the loading amount of large-pore porous glass microspheres is lower, and the corresponding synthesis yield is also lower.

[0070] Example 2 Synthesis of Nucleic Acid Probes

[0071] The three solid phase carriers synthesized in Example 1 were used to synthesize nucleic acids in an experimental environment with a temperature of 25±1° C. and a humidity of 10% to 30%.

[0072] The nucleic acid structure is DNA, and the sequence is T20 composed of 20 thymidine deoxynucleotides, wherein X represents azadiphenylcyclooctyne, and the sequence is: 5'-TTTTTTTTTTTTTTTTTTTTTX-3'.

[0073] See also Figure 1The specific steps are as follows: the amount of nucleic acid synthesis is 200 nanomoles, the synthesis column is a porous glass microsphere powder filling column modified with azadiphenylcyclooctyne, the concentration of deoxythymidine phosphoramidite monomer is 0.05 mmol / L, the water content of eluting acetonitrile is 17 ppm, the deprotection reagent is a 3% mass volume ratio of trichloroacetic acid in dichloromethane, the activator is a 0.45 mol / L acetonitrile solution of 5-ethylthiotetrazole, the oxidant is a 0.05 mol / L tetrahydrofuran-water-pyridine mixed solution (wherein the volume ratio of tetrahydrofuran-water-pyridine is 90:5:5), the end-capping reagent A is a 10% solution of acetic anhydride in tetrahydrofuran, and the end-capping reagent B is a 5% solution of 1-methylimidazole in tetrahydrofuran.

[0074] The nucleic acid synthesis was performed using the DMT-off method. After the synthesis was completed, the porous glass microsphere carrier was removed and placed in 1 ml of 28% ammonia water for aminolysis. The aminolysis temperature was 60 degrees Celsius and the aminolysis time was 2 hours. After the aminolysis solution was drained, it was analyzed using high-performance liquid chromatography. The conditions were: mobile phase A was chromatographic-grade acetonitrile, mobile phase B was 10% triethylamine acetate in deionized water, the analytical column model was C18 Waters X-Bridge, particle size 5 μm, size 250 mm×4.6 mm, flow rate 0.2 mL / min, column temperature 25°C. The synthesized nucleic acid has the following structure:

[0075] (7)

[0076] Figure 5 is the molecular weight of the T20 sequence modified with azadiphenylcyclooctyne. The theoretical molecular weight is 6462.1, and the detection result is 6463.3, proving that azadiphenylcyclooctyne has been successfully modified to the 3' end of the T20 sequence.

[0077] Figure 6 This is the HPLC spectrum of the product after nucleic acid is synthesized using a modified carrier of porous glass microspheres with a pore diameter of 2000 angstroms.

[0078] Table 2 shows the HPLC results of the products synthesized with modified carriers of porous glass microspheres with a pore diameter of 2000 angstroms.

[0079] Table 2

[0080]

[0081] from Figure 6 As can be seen from Table 2, the retention time at which the maximum peak area and peak ratio appear is 32.228 min, and the signal there is the DNA sequence modified with DBCO units.

[0082] Figure 7This is the HPLC spectrum of the product after nucleic acid is synthesized using a modified carrier of porous glass microspheres with a pore diameter of 1000 angstroms.

[0083] Table 3 shows the HPLC results of the products synthesized by using modified carriers of porous glass microspheres with a pore diameter of 1000 angstroms.

[0084] Table 3

[0085]

[0086] from Figure 7 As can be seen from Table 3, the retention time at which the maximum peak area and peak ratio appear is 32.093 min, where the signal is the DNA sequence modified with DBCO units.

[0087] Figure 8 This is the HPLC spectrum of the product after nucleic acid is synthesized using a modified carrier of porous glass microspheres with a pore diameter of 500 angstroms.

[0088] Table 4 shows the HPLC results of the products synthesized with modified carriers of porous glass microspheres with a pore diameter of 500 angstroms.

[0089] Table 4

[0090]

[0091] from Figure 8 As can be seen from Table 4, the retention time at which the maximum peak area and peak ratio appear is 32.171 min, and the signal there is the DNA sequence modified with DBCO units.

[0092] The preferred implementation modes of the present invention are described in detail above in combination with the embodiments; however, the present invention is not limited to the specific details in the above implementation modes. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0093] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0094] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for preparing a nucleic acid probe, characterized in that: The nucleic acid probe is used for environmental DNA detection, wherein: The 3' end of the nucleic acid probe is modified with azadiphenylcyclooctyne, and the nucleic acid probe is obtained by solid phase synthesis of phosphoramidite chemistry. The solid phase synthesis includes using a solid phase carrier modified with azadiphenylcyclooctyne structure, and the solid phase carrier is a porous glass microsphere modified with azadiphenylcyclooctyne structure and has the following structure: ， The surface modification functional groups of the porous glass microspheres are primary amino groups.

2. The method for preparing a nucleic acid probe according to claim 1, characterized in that: The nucleic acid probe is a polynucleotide composed of one or more nucleotides.

3. The method for preparing a nucleic acid probe according to claim 2, characterized in that: The nucleotides include at least one of the following: Natural and non-natural nucleic acids modified on the sugar ring, wherein the sugar ring is: natural deoxyribose, natural ribose, 2'-methoxy substituted deoxyribose, 2'-fluoro substituted deoxyribose, 2'-methoxyethyl substituted deoxyribose, 2'-methylamino-2-oxyethyl substituted deoxyribose, 2'-propargyloxy substituted deoxyribose, 2'-butoxy substituted deoxyribose, 2'-hexyloxy substituted deoxyribose, 2'-octyloxy substituted deoxyribose, 2'-decyloxy substituted deoxyribose, 2'-dodecyloxy substituted deoxyribose, 2'-tetradecyloxy substituted deoxyribose, 2'-hexadecyloxy substituted deoxyribose, Substituted deoxyribose, 2'-octadecyloxy substituted deoxyribose, 2'-eicosyloxy substituted deoxyribose, 2'-oxy-4' carbonyl methylene lock configuration deoxyribose, 2'-oxy-4' carbonyl vinyl lock configuration deoxyribose, 2'-oxy-4' carbonyl-S-configuration ethyl lock configuration deoxyribose, 2'-oxy-4' carbonyl-R-configuration ethyl lock configuration deoxyribose, 2'-fluoro substituted arabinose, 2'-methoxy substituted arabinose, L-configuration deoxyribose, L-configuration ribose, 3' and 5' link flipped deoxyribose or 3' and 5' link flipped ribose; Natural and non-natural nucleic acids modified on bases, wherein the bases are: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine or N-methylpseudouridine; Natural and non-natural nucleic acids modified on the linking unit, wherein the linking unit is: a racemic phosphorothioate diester bond, an R-configuration phosphorothioate diester bond, an S-configuration phosphorothioate diester bond, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, or monooctyl hypophosphite; Non-natural structure modified nucleic acid connecting nucleosides and phosphates: 2',3'-dideoxy-β-D-pyranose, wherein the base is at the 6-β position; 2-hydroxymethylmorpholine, wherein the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; unlocked nucleic acid unit, whose structure is an RNA-like structure without a chemical bond between 2' and 3', wherein the base is at the 1'-β position.

4. The method for preparing a nucleic acid probe according to claim 2 or 3, characterized in that: The polynucleotide has a length of 2-240 nucleotides.

5. The method for preparing a nucleic acid probe according to claim 4, characterized in that: The polynucleotide has a length of 20-160 nucleotides.

6. A method for DNA detection, characterized in that: include: Prepare a nucleic acid probe using the method according to any one of claims 1 to 5; Mixing the sample to be tested and the nucleic acid probe to perform a hybridization reaction; The nucleic acid probes that have undergone the hybridization reaction are captured, and the DNA content to be detected in the sample to be tested is calculated and determined.

7. The method according to claim 6, characterized in that The providing of the nucleic acid probe comprises designing the nucleic acid probe according to the DNA to be detected in the sample to be tested, and the providing of the nucleic acid probe comprises the following steps: Determine the detection region of the DNA to be detected and select a marker sequence; Determine the sequence of the nucleic acid probe according to the marker sequence, synthesize nucleotides and modify them, The synthesizing and modifying nucleotides comprises: using the solid phase carrier to obtain the nucleic acid probe with a 3'-end modified with azadiphenylcyclooctyne.

8. The method according to claim 7, characterized in that The solid phase carrier is obtained by the following steps: Providing a nitrogen-containing diphenyl cyclooctyne structure compound; The nitrogen-containing diphenylcyclooctyne structure compound is mixed with porous glass microspheres and a connecting unit is formed between the porous glass microspheres and the nitrogen-containing diphenylcyclooctyne structure compound. The surface of the porous glass microspheres is modified with amino groups. The connecting unit is formed by succinamide, 3-amino-1,2-propanediol and diethylene glycol ester.

9. The method according to claim 8, characterized in that The particle size of the porous glass microspheres is 10-100 micrometers, and the micropore size is 500-2000 angstroms.

10. The method according to claim 9, characterized in that The loading amount of the compound containing azadiphenylcyclooctyne structure modified on the surface of the porous glass microsphere is 30-120 micromol / g.

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