Oligonucleotide fluorescent probe and method for environmental DNA detection using the same

Abstract

The present invention discloses an oligonucleotide fluorescent probe and a method for environmental DNA detection using the same, and relates to the field of DNA detection. The 3' end of the oligonucleotide fluorescent probe contains a rhodamine fluorescent unit, the oligonucleotide fluorescent probe is obtained by modifying a solid phase carrier with a rhodamine fluorescent unit, and the surface modified functional group of the porous glass microsphere is a primary amino group; X1 to X 11 Each is independently selected from H, carbonyl, oxycarbonyl, alkoxy, halogen, amino, substituted or unsubstituted alkyl and ester. The fluorescent probe has good thermal stability and improves the detection efficiency of the fluorescent probe.

Description

Technical Field

[0001] The present invention relates to the field of DNA detection, and in particular to an oligonucleotide fluorescent probe and an environmental DNA detection method using the same. Background Art

[0002] Environmental DNA testing (eDNA) is a technology that extracts DNA of specific biological species from environmental samples such as water and sediment to monitor the abundance of specific aquatic biological species. This technology requires the use of nucleic acid probes to detect the extracted DNA of biological species. Among the various nucleic acid probes, fluorescent probe structures containing fluorescent markers are an important type of probe in DNA detection. The fluorescent unit in the probe controls the generation, quenching or change of detection wavelength of fluorescence by combining with the target sequence to form a double strand or other secondary structure, thereby realizing the detection of the target sequence. Nucleic acid fluorescent probes have the advantages of high sensitivity, high specificity, short response time, simple operation and low cost.

[0003] The fluorescent emission units currently used for nucleic acid probes mainly include rhodamine dyes (as shown in formula a below), cyanine dyes (as shown in formula b below) and coumarin dyes (as shown in formula c below).

[0004]

[0005] However, current fluorescent probes have poor stability, so oligonucleotide fluorescent probes and environmental DNA detection methods still need to be improved. Summary of the invention

[0006] In order to improve the thermal stability of double-stranded nucleic acid probes, the present invention provides an oligonucleotide fluorescent probe and its application. The inventors found that the chemical structure connecting the fluorescent unit and the nucleic acid has an important influence on the thermal stability of the double-stranded nucleic acid probe. Improving the thermal stability of the double-stranded nucleic acid probe, that is, improving the melting point between the fluorescent nucleic acid probe and the detected eDNA, is of great value for improving the detection efficiency of the nucleic acid probe for eDNA. The present application adopts a connecting unit containing a serinol structure, especially a connecting unit containing a serinol structure and a succinic acid structure to connect the solid phase carrier and the fluorescent unit, and connects the rhodamine fluorescent structure to the porous glass microsphere with the chemical structure of an amide bond and an ester bond, so as to obtain an oligonucleotide fluorescent probe with a rhodamine fluorescent unit modified at the 3' end. The melting point of the probe is significantly increased, the thermal stability is improved, and the detection efficiency of the fluorescent probe is improved.

[0007] In order to achieve the above object, the present invention provides an oligonucleotide fluorescent probe on one hand. The oligonucleotide fluorescent probe is used for environmental DNA detection, wherein: the 3' end of the oligonucleotide fluorescent probe contains a rhodamine fluorescent unit, and the oligonucleotide fluorescent probe is obtained by modifying a solid phase carrier with a rhodamine fluorescent unit, and the solid phase carrier includes a porous glass microsphere and has the following structure:

[0008] ,

[0009] Wherein, the surface modified functional groups of the porous glass microspheres are primary amino groups;

[0010] X1 to X 11 Each is independently selected from H, carbonyl, oxycarbonyl, alkoxy, halogen, amino, substituted or unsubstituted alkyl, and ester.

[0011] In one embodiment of the present invention, X1 to X 11each independently selected from the group consisting of a hydrogen atom, an oxycarbonylmethyl group, an oxycarbonylethyl group, an oxycarbonylisopropyl group, an oxycarbonyltert-butyl group, an oxycarbonylphenyl group, an oxycarbonyl4-methylphenyl group, an oxycarbonyl4-chlorophenyl group, a methoxy group, an ethoxy group, an isopropoxy group, a tert-butoxy group, a phenoxy group, a 2-methylphenoxy group, a 3-methylphenoxy group, a 4-methylphenoxy group, a 4-phenylphenoxy group, a fluorine atom, a chlorine atom, a bromine atom, an amino group, an oxycarbonylmethylamino group, a dimethylamino group, a diethylamino group, a N-(dimethylene) group, a N-(dimethylene) group, a N-(trimethylene) group, a N-(trimethylene) group N-(trimethylene), N-(trimethylene)N-(tetramethylene), N-(1-methylmethylene-methylene)N-(1-methylmethylene-methylene), N-(bis-1-methylmethylene)N-(bis-1-methylmethylene), N-(1-methylmethylene-dimethylene)N-(trimethylene), N-(1-methylmethylene-dimethylene)N-(1-methylmethylene-dimethylene), N-(1,1-dimethylmethylene-dimethylene)N-(trimethylene), N-(1,1-dimethylmethylene-dimethylene)N-(1,1-dimethylmethylene-dimethylene) yl-dimethylene), cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, methylthio, ethylthio, methyl carboxylate, ethyl carboxylate, propyl carboxylate, carboxymethyl isopropyl, tert-butyl carboxylate, benzyl carboxylate, carboxymethyl methyl, carboxymethyl ethyl, carboxymethyl propyl, carboxymethyl isopropyl, carboxymethyl tert-butyl, carboxymethyl benzyl, carboxyethyl methyl, carboxyethyl ethyl, carboxyethyl propyl, carboxyethyl isopropyl, carboxyethyl tert-butyl, carboxyethyl benzyl, carboxypropyl methyl, carboxypropyl ethyl, carboxypropyl propyl, carboxypropyl isopropyl, carboxypropyl tert-butyl Butyl, carboxypropylbenzyl, formamide, acetamido, propionamide, butyramide, methyl, ethyl, propyl, sulfonic acid, methylenecarboxymethyl, methylenecarboxyethyl, methylenecarboxypropyl, methylenecarboxytert-butyl, methylenecarboxybenzyl, ethylenecarboxymethyl, ethylenecarboxyethyl, ethylenecarboxypropyl, ethylenecarboxytert-butyl, ethylenecarboxybenzyl, dimethyl phosphate, diethyl phosphate, diphenyl phosphate, di(2-cyanoethyl) phosphate, di(trichloromethyl) phosphate and di(trichloromethylmethylene)methyl phosphate.

[0012] The solid phase carrier modified with rhodamine fluorescent dye using serinol structure as connecting unit has a shorter connecting unit, so the nucleotide of the oligonucleotide fluorescent probe modified at the 3' end is short in distance from the rhodamine fluorescent unit, has a high melting point and good thermal stability, and can effectively improve the detection efficiency of the fluorescent probe.

[0013] As a preferred embodiment of the present invention, the rhodamine fluorescent unit is 6-carboxyfluorescein, and the solid phase carrier has the following structure:

[0014] .

[0015] In one embodiment of the present invention, the nucleotide unit in the oligonucleotide fluorescent probe comprises at least one of the following structures:

[0016] 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-oxoethyl 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, 3' and 5' link flipped ribose;

[0017] 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, N-methylpseudouridine;

[0018] 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, and monooctyl hypophosphite;

[0019] Non-natural structures linking nucleosides and phosphates include 2', 3'-dideoxy-β-D-pyranose glucopyranose, in which the base is at the 6-β position; 2-hydroxymethylmorpholine, in which the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, in which the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, in which the base is at the 3-position; S-1, 3-dihydroxymethyl-2-methylene unit, in which the base is at the 2-position; R-1, 3-dihydroxymethyl-2-methylene unit, in which the base is at the 2-position; and an unlocked nucleic acid unit, which has an RNA-like structure without a chemical bond between 2' and 3', in which the base is at the 1'-β position.

[0020] In one embodiment of the present invention, the length of the oligonucleotide is 2-240 nucleotides.

[0021] In one embodiment of the present invention, the length of the oligonucleotide is 20-160 nucleotides.

[0022] In one embodiment of the present invention, the oligonucleotide may be of the following structure:

[0023] ,

[0024] Among them, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y 10 , Y 11 The functional groups include, but are not limited to, hydrogen, hydroxy, methoxy, ethoxy, isopropoxy, tert-butoxy, phenoxy, 2-methylphenoxy, 3-methylphenoxy, 4-methylphenoxy, 4-phenylphenoxy, fluorine, chlorine, bromine, amino, methylamino, dimethylamino, diethylamino, N-(dimethylene)N-(dimethylene), N-(dimethylene)N-(trimethylene), N-(trimethylene)N-(trimethylene), N-(trimethylene)N-(tetramethylene), N-(1-methylmethylene-methylene)N-(1-methylmethylene-methylene), N-(bis-1-methylmethylene)N-(bis-1-methylmethylene), N-(1-methylmethylene-dimethylene)N-(trimethylene), N-(1-methylmethylene-dimethylene)N-(1-methylmethylene-dimethylene), N-(1,1-dimethylmethylene-dimethylene)N-(trimethylene), N-(1,1-dimethylmethylene-dimethylene)N-(1,1-dimethylmethylene-dimethylene), cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, methylthio, ethylthio, carboxyl, carboxymethyl, carboxyethyl, carboxypropyl, formamide, acetamido, propionamide, butyramide, methyl ester, ethyl ester, propyl ester, sulfonic acid, methylene carboxyl, ethylene carboxyl, phosphate, and a combination of one or more of the above functional groups.

[0025] In another aspect of the present invention, the present invention proposes a method for detecting environmental DNA using the aforementioned oligonucleotide fluorescent probe. The method comprises: providing an oligonucleotide fluorescent probe, wherein the oligonucleotide fluorescent probe is the aforementioned oligonucleotide fluorescent probe; mixing the sample to be tested and the oligonucleotide fluorescent probe for hybridization reaction; capturing the oligonucleotide fluorescent probe after the hybridization reaction, and calculating and determining the DNA content to be detected in the sample to be tested.

[0026] In one embodiment of the present invention, providing an oligonucleotide fluorescent probe includes designing an oligonucleotide fluorescent probe according to a DNA to be detected in a sample to be detected, and providing an oligonucleotide fluorescent 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 oligonucleotide fluorescent probe according to the marker sequence, synthesizing nucleotides and modifying them, and the synthesizing and modifying nucleotides includes: using a solid phase carrier modified with a rhodamine fluorescent unit to obtain the oligonucleotide fluorescent probe whose 3' end is modified with a fluorescent unit.

[0027] In one embodiment of the present invention, the solid phase carrier is obtained by the following steps: providing a compound containing a rhodamine fluorescent unit; mixing the compound containing a rhodamine fluorescent unit with porous glass microspheres and forming a connecting unit between the porous glass microspheres and the compound containing a rhodamine fluorescent unit, wherein the surface of the porous glass microspheres is modified with amino groups, and the connecting unit includes a serinol structure.

[0028] In one embodiment of the present invention, the particle size of the porous glass microspheres is 10-100 microns, the micropore size is 500-1000 angstroms, and the loading amount of the compound containing rhodamine fluorescent units modified on the surface of the porous glass microspheres is 30-120 micromol / g.

[0029] Preferably, the particle size of the porous glass microspheres is 10-100 microns, the pore size is 500-1000 angstroms, and the loading amount of the compound containing rhodamine fluorescent units modified on the surface of the porous glass microspheres is 30-120 micromol / g.

[0030] Further preferably, the pore size of the porous glass microspheres is 500 angstroms, and the loading amount of the surface-modified compound containing rhodamine fluorescent units is 40-120 micromol / g, preferably 58 micromol / g.

[0031] Preferably, the porous glass microspheres have a pore size of 1000 angstroms, and the loading amount of the surface-modified compound containing a rhodamine fluorescent unit is 35-110 micromol / g, preferably 55 micromol / g.

[0032] Generally speaking, the smaller the micropore diameter, the greater the loading capacity, and the more probes that can be synthesized; the shorter the length, the larger the micropore diameter, and the longer the probes that can be synthesized, but the total amount is less. The above technical solution, by selecting the appropriate microsphere particle size and micropore diameter, makes the probe synthesis yield and loading capacity both at a relatively good level.

[0033] Through the above technical solution, the present invention achieves the following beneficial effects:

[0034] 1. The present invention is a solid phase carrier modified with rhodamine fluorescent dye using serinol structure as a connecting unit, which has a shorter connecting unit and can be used for 3' end modification of DNA, RNA, artificially synthesized nucleotides containing non-natural nucleotide units, and artificially synthesized nucleotides containing natural nucleotide units to obtain an oligonucleotide fluorescent probe. Although the distance between the nucleotide and the rhodamine fluorescent unit of the probe is short, it has a high melting point and good thermal stability, and can effectively improve the detection efficiency of the fluorescent probe.

[0035] 2. In a preferred technical solution of the present invention, by selecting appropriate microsphere particle size and micropore size, the probe synthesis yield and loading capacity are both at a relatively optimal level. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the basic reaction cycle of synthesizing an oligonucleotide modified with a rhodamine fluorescent dye at the 3' end of the present invention;

[0037] Figure 2 is a synthetic route diagram of the solid phase carrier in Example 1 of the present invention;

[0038] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound 2 in Example 1 of the present invention;

[0039] Figure 4 is the NMR mass spectrum of compound 2 in Example 1 of the present invention;

[0040] Figure 5 This is a schematic diagram of the basic reaction cycle for synthesizing an oligonucleotide modified with 6-carboxyfluorescein at the 3' end in Example 2 of the present invention;

[0041] Figure 6 is the mass spectrometry result of the nucleic acid modified with 6-carboxyfluorescein in Example 2 of the present invention;

[0042] 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;

[0043] Figure 8is 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;

[0044] Fig. 9 It is a schematic diagram of the melting point test results of the DNA double-strand in Example 2 of the present invention, the DNA sequence of 6-carboxyfluorescein linked to commercial serinol, and the double-stranded DNA formed by the DNA sequence containing 6-carboxyfluorescein in the present invention and the complementary sequence. DETAILED DESCRIPTION

[0045] 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.

[0046] In one aspect of the present invention, the present invention provides an oligonucleotide fluorescent probe, which is used for environmental DNA detection. The oligonucleotide fluorescent probe is obtained by using a solid phase carrier modified with a rhodamine fluorescent unit. Figure 1 By having a solid phase carrier with a more reasonable connection unit, an oligonucleotide fluorescent probe with better thermal stability can be obtained.

[0047] In another aspect of the present invention, the present invention provides a method for environmental DNA detection using the oligonucleotide fluorescent probe described above. The method includes the step of detecting the DNA content to be detected in the sample to be detected using the aforementioned oligonucleotide fluorescent probe. The method has a high detection efficiency.

[0048] In the following examples, the model of the nucleic acid synthesizer is HJ01201202; the model of the high performance liquid chromatograph is Waters 2695, the model of the mass spectrometer is LCMS-IT / TOF, and the model of the melting point determination equipment is Varian Carry 100.

[0049] Example 1 Synthesis of solid phase carrier

[0050] according to Figure 2 The synthetic route shown is used to synthesize the solid phase carrier, and the specific steps are as follows:

[0051] 1) Synthesis of compound 2

[0052] Weigh 54.4 g of compound 1 (0.1 mol, i.e. 6-carboxyfluorescein), 41.2 g of 3-amino-2-hydroxy-1-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl ether (0.105 mol), 45.6 g of O-benzotriazole-tetramethyluronium hexafluorophosphate (0.12 mol), and 40 g of diisopropylethylamine, add to 1500 ml of ethyl acetate, stir at room temperature for 36 hours, filter to remove insoluble matter, wash the filtrate with 4000 ml of water four times, 1000 ml each time, wash the organic phase with 1000 ml of saturated brine, add anhydrous sodium sulfate and dry for 12 hours.

[0053] The drying agent was removed by filtration, and the reaction solution was concentrated to 200 ml, placed in a refrigerator at 4 degrees Celsius for 12 hours, and then filtered to obtain a crude product. The crude product was added to 1500 ml of ethyl acetate, 10 g of succinic anhydride and 70 g of pyridine were added, and stirred at room temperature for 8 hours, and the insoluble matter was removed by filtration. The filtrate was washed four times with 2000 ml of saturated sodium bicarbonate aqueous solution, 500 ml each time, and the organic phase was washed with 1000 ml of saturated brine, and then dried for 12 hours by adding anhydrous sodium sulfate.

[0054] The drying agent was removed by filtration, and the reaction solution was concentrated to 300 ml and left overnight to precipitate a yellow-green solid. The yellow-green crude product was recrystallized using 300 ml of 4°C methanol, and the crystallization was repeated three times to obtain a yellow-green powder product (i.e., compound 2) with a weight of 28 g and a yield of 27.4%.

[0055] The H NMR spectrum of compound 2 is as follows Figure 3 , 1 H-NMR (CDCl3) ppm: 12.20 (s, COO H , 1H),8.70-8.12 (d, 1H, Ar H on FAM), 8.57-8.58 (d, 2H, ArH on Pyr), 8.19-8.25 (m,2H, Ar H on FAM), 7.76-7.81 (d, 2H, Ar H on Pyr), 7.37-7.40 (m, 2H, Ar H onDMT), 7.30-7.33 (m, 5H, ArH on Pyr+ Ar H on DMT), 7.18-7.20 (m, 6H, Ar H onDMT), 6.88-7.00 (m, 6H, Ar Hon DMT), 6.80-6.83 (m, 3H, Ar H on FAM), 4.21-4.43(m, C H 2O), 4.11-4.15 (m, 1H, C H N), 3.71 (s, 6H, C H 3O on DMT), 3.00-3.10 (m,C H 2O), 2.39-2.43 (m, 4H, OCC H 2C H 2CO), 1.31 (s, 18H, (C H 3)3CCO).

[0056] Figure 4 is the NMR mass spectrum of compound 2, whose molecular formula is C 64 H 62 N2O 15 , theoretical molecular weight 1098.4150, measured molecular weight 600.2568 (M+Na+2Pyridine) 2+ .

[0057] 2) Synthesis of 6-carboxyfluorescein on porous glass microspheres with a pore size of 500 angstroms

[0058] Weigh 6.0 g of compound 2, dissolve it in 200 ml of anhydrous dimethyl sulfoxide, add 20 g of primary amino-modified porous glass microspheres with a pore size of 500 angstroms, add 8.1 g of carbonyldiimidazole (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, which is a solid phase carrier containing 6-carboxyfluorescein connected to the porous glass microspheres.

[0059] 3) Synthesis of 6-carboxyfluorescein on porous glass microspheres with a pore size of 1000 angstroms

[0060] Weigh 6.0 g of compound 2, dissolve it in 200 ml of anhydrous dimethyl sulfoxide, add 20 g of primary amino-modified porous glass microspheres with a pore size of 1000 angstroms, add 8.1 g of carbonyldiimidazole (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, which is a solid phase carrier containing 6-carboxyfluorescein connected to the porous glass microspheres.

[0061] The loading determination method is as follows:

[0062] Weigh 15 mg of 6-carboxyfluorescein-modified porous glass microspheres and place them in a 15 ml test tube;

[0063] 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;

[0064] Add 1 ml of 0.1 mol / L p-toluenesulfonic acid acetonitrile solution as eluent to the test tube containing 6-carboxyfluorescein-modified porous glass microspheres, mix well, let stand for 5 minutes, centrifuge for 10 seconds, and remove the supernatant;

[0065] 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.

[0066] The loading calculation formula is as follows:

[0067] Loading (μmol / g) = OD λ498 × dilution times × 12 ÷ CPG powder mass (mg)

[0068] The loading capacity of porous glass microspheres with different pore sizes is shown in Table 1.

[0069] Table 1

[0070]

[0071] As can be seen from Table 1, the loading amount and yield of porous glass microspheres with pore diameters of 1000 angstroms and 500 angstroms are similar, but the loading amount and yield of porous glass microspheres with pore diameters of 500 angstroms are slightly higher than those of porous glass microspheres with pore diameters of 1000 angstroms.

[0072] Example 2 Synthesis of nucleic acids

[0073] The nucleic acid was synthesized using the solid phase carrier synthesized in Example 1, and the experimental environment temperature was 25±1° C. and the humidity was 10% to 30%.

[0074] The nucleic acid structure is DNA, and the sequence is T20 composed of 20 thymidine deoxynucleotides, wherein X represents 6-carboxyfluorescein, and the sequence is: 5'- TTTTTTTTTTTTTTTTTTTTTTX-3'.

[0075] See also Figure 5The 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 6-carboxyfluorescein, the concentration of deoxythymidine phosphoramidite monomer is 0.05 mmol / L, the water content of eluting acetonitrile is 15 ppm, the deprotection reagent is a 3% by mass volume solution 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 capping reagent A is a 10% by mass volume solution of acetic anhydride in tetrahydrofuran, and the capping reagent B is a 5% by mass volume solution of 1-methylimidazole in tetrahydrofuran.

[0076] 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:

[0077]

[0078] Figure 6 This is the molecular weight test result of the T20 sequence modified with 6-carboxyfluorescein (MALDI-TOF, MatrixAssistant Laser Desorption Ionization-Time of Flight, MatrixAssisted Laser Ionization Desorption-Time of Flight Mass Spectrometry), which can be used to characterize the molecular weight of biological molecules with larger molecular weights such as nucleic acids, proteins and polysaccharides. The theoretical molecular weight is 6532.1, and the test result is 6530.8, proving that 6-carboxyfluorescein has been successfully modified to the 3' end of the T20 sequence.

[0079] Figure 7 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 1000 angstroms.

[0080] Table 2 shows the HPLC results of the products synthesized with modified carriers of porous glass microspheres with a pore diameter of 1000 angstroms.

[0081] Table 2

[0082]

[0083] from Figure 7 As can be seen from Table 2, the retention time at which the maximum peak area and peak ratio appear is 17.216 min, and the signal is DNA modified with a fluorescent unit.

[0084] 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.

[0085] Table 3 shows the HPLC results of the products synthesized by using modified carriers of porous glass microspheres with a pore diameter of 500 angstroms.

[0086] Table 3

[0087]

[0088] from Figure 8 As can be seen from Table 3, the retention time of the maximum peak area and peak ratio is 16.97 min, and the signal is DNA modified with fluorescent units.

[0089] Melting point determination:

[0090] Measured objects: nucleic acid synthesized in Example 2, double-stranded DNA (sequence: 5'-TTTTTTTTTTTTTTTTTTTTTX-3'), and commercial serinol-linked 6-carboxyfluorescein DNA having the following structure:

[0091] .

[0092] The complementary sequence of the nucleic acid sequence used for melting point measurement is:

[0093] 5'-AAAAAAAAAAAAAAAAAA-3'.

[0094] The determination method is as follows: prepare a solution with a concentration of 2 μmol·L -1 The sample is slowly transferred to a 1 cm sample pool and the sample pool is placed in the instrument for testing. Set the instrument temperature range to 5~95℃ and the temperature rise rate to 1℃·min -1 ; Cooling range is 95~5℃, cooling speed is 1℃·min -1 The buffer solution was 0.1 M phosphate buffer with a pH of 7.4. Fig. 9 And as shown in Table 4.

[0095] Table 4

[0096]

[0097] from Fig. 9It can be seen from Table 4 that after forming a double strand with the corresponding complementary sequence, the melting point of the double-stranded DNA containing 6-carboxyfluorescein of the linking unit of the present invention is significantly increased compared with the melting point of the commercial double-stranded DNA containing 6-carboxyfluorescein. This increase in the thermal stability of the double strand is beneficial to improving the detection efficiency of the fluorescent probe. Compared with the commercial product, the product of the present invention increases the melting point of the double strand by 5.2°C.

[0098] 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 scheme of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0099] 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.

[0100] 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 detecting environmental DNA using oligonucleotide fluorescent probes, characterized in that: include: Providing oligonucleotide fluorescent probes; Mixing the sample to be tested and the oligonucleotide fluorescent probe to perform a hybridization reaction; The oligonucleotide fluorescent probe that has undergone the hybridization reaction is captured, and the DNA content to be detected in the sample to be tested is determined by calculation, wherein: The 3' end of the oligonucleotide fluorescent probe contains a rhodamine fluorescent unit. The oligonucleotide fluorescent probe is obtained by modifying a solid phase carrier with a rhodamine fluorescent unit. The solid phase carrier includes porous glass microspheres and has the following structure: ， Wherein, the surface modified functional groups of the porous glass microspheres are primary amino groups; X1 to X11 are each independently selected from H, carbonyl, oxycarbonyl, alkoxy, halogen, amino, substituted or unsubstituted alkyl, and ester; The providing of the oligonucleotide fluorescent probe comprises designing the oligonucleotide fluorescent probe according to the DNA to be detected in the sample to be detected, and the providing of the oligonucleotide fluorescent probe comprises the following steps: determining the detection region of the DNA to be detected and selecting a marker sequence; determining the sequence of the oligonucleotide fluorescent probe according to the marker sequence, synthesizing and modifying nucleotides, and the synthesizing and modifying nucleotides comprises: using the solid phase carrier modified with the rhodamine fluorescent unit to obtain the oligonucleotide fluorescent probe whose 3' end is modified with the fluorescent unit.

2. The method according to claim 1, characterized in that X1 to X11 are each independently selected from the group consisting of a hydrogen atom, an oxycarbonylmethyl group, an oxycarbonylethyl group, an oxycarbonylisopropyl group, an oxycarbonyltert-butyl group, an oxycarbonylphenyl group, an oxycarbonyl4-methylphenyl group, an oxycarbonyl4-chlorophenyl group, a methoxy group, an ethoxy group, an isopropoxy group, a tert-butoxy group, a phenoxy group, a 2-methylphenoxy group, a 3-methylphenoxy group, a 4-methylphenoxy group, a 4-phenylphenoxy group, a fluorine atom, a chlorine atom, a bromine atom, an amino group, an oxycarbonylmethylamino group, a dimethylamino group, a diethylamino group, a N-(dimethylene)N-(dimethylene)N-(trimethylene) group, a N-( trimethylene) N-(trimethylene), N-(trimethylene) N-(tetramethylene), N-(1-methylmethylene-methylene) N-(1-methylmethylene-methylene), N-(bis-1-methylmethylene) N-(bis-1-methylmethylene), N-(1-methylmethylene-dimethylene) N-(trimethylene), N-(1-methylmethylene-dimethylene) N-(1-methylmethylene-dimethylene), N-(1,1-dimethylmethylene-dimethylene) N-(trimethylene), N-(1,1-dimethylmethylene-dimethylene) N-(1,1-dimethylmethylene-dimethylene) 1-Hydroxy-1,1-dimethylene-2,1-dimethylene-3,1-dimethylene-4,1-dimethylene-5,1-dimethylene-6,1-dimethylene-7,1-dimethylene-8,1-dimethylene-9,1-dimethylene-10,1-dimethylene-11,1-dimethylene-12,1-dimethylene-13,1-dimethylene-14,1-dimethylene-15,1-dimethylene-16,1-dimethylene-17,1-dimethylene-18,1-dimethylene-19,1-dimethylene-20,1-dimethylene-11,1-dimethylene-12,1-dimethylene-19,1-dimethylene-11 tert-butyl, carboxypropylbenzyl, formamide, acetamido, propionamide, butyramide, methyl, ethyl, propyl, sulfonic acid, methylenecarboxymethyl, methylenecarboxyethyl, methylenecarboxypropyl, methylenecarboxy-tert-butyl, methylenecarboxybenzyl, ethylenecarboxymethyl, ethylenecarboxyethyl, ethylenecarboxypropyl, ethylenecarboxy-tert-butyl, ethylenecarboxybenzyl, dimethyl phosphate, diethyl phosphate, diphenyl phosphate, di(2-cyanoethyl) phosphate, di(trichloromethyl) phosphate and di(trichloromethylmethylene)methyl phosphate.

3. The method according to claim 2, characterized in that The solid phase carrier has the following structure: 。 4. The method according to any one of claims 1 to 3, characterized in that: The nucleotide unit in the oligonucleotide fluorescent probe comprises at least one of the following structures: 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-oxoethyl 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, 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, 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, and monooctyl hypophosphite; Non-natural structures linking nucleosides and phosphates include 2', 3'-dideoxy-β-D-pyranose glucopyranose, in which the base is at the 6-β position; 2-hydroxymethylmorpholine, in which the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, in which the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, in which the base is at the 3-position; S-1, 3-dihydroxymethyl-2-methylene unit, in which the base is at the 2-position; R-1, 3-dihydroxymethyl-2-methylene unit, in which the base is at the 2-position; and an unlocked nucleic acid unit, which has an RNA-like structure without a chemical bond between 2' and 3', in which the base is at the 1'-β position.

5. The method according to claim 4, characterized in that The length of the oligonucleotides ranged from 2 to 240 nucleotides.

6. The method according to claim 5, characterized in that The length of the oligonucleotides ranged from 20 to 160 nucleotides.

7. The method according to claim 1, characterized in that The solid phase carrier is obtained by the following steps: Providing a compound containing a rhodamine-type fluorescent unit; The compound containing rhodamine fluorescent units is mixed with porous glass microspheres and a connecting unit is formed between the porous glass microspheres and the compound containing rhodamine fluorescent units. The surface of the porous glass microspheres is amino-modified, and the connecting unit includes a serinol structure.

8. The method according to claim 1, characterized in that The particle size of the porous glass microsphere is 10-100 micrometers, the micropore diameter is 500-1000 angstroms, and the loading amount of the compound containing rhodamine fluorescent units modified on the surface of the porous glass microsphere is 30-120 micromol / g.

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