CN113088514A - Novel efficient trace detection material nucleic acid extraction reagent, kit and use method thereof - Google Patents
Novel efficient trace detection material nucleic acid extraction reagent, kit and use method thereof Download PDFInfo
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Abstract
The invention provides a novel high-efficiency trace detection material nucleic acid extraction reagent, which belongs to the field of molecular biology, and comprises a lysis solution, wherein the lysis solution comprises a bifunctional chelating agent with the concentration of 1-20mmol/L, the bifunctional chelating agent simultaneously has a metal chelating end and a protein anchoring end, and the bifunctional chelating agent is NOTA, MAA-NOTA, p-SCN-Bn-NOTA, p-SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA; the invention adds the bifunctional chelating agent which simultaneously has a metal chelating end and a protein anchoring end into the lysis solution, and the bifunctional chelating agent can be used as a connecting intermediate to connect metal ions with protein molecules which are not easy to be adsorbed and remain in the solution, thereby effectively removing the metal ions in the detection material.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to the field of nucleic acid extraction by a paramagnetic particle method, and specifically relates to a novel efficient trace detection material nucleic acid extraction reagent, a kit and a use method thereof.
Background
Nucleic acids, including both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the most important bioinformatic molecules and genetic material of all organisms. Currently, the biotechnology using nucleic acid as a research object, including a series of technologies such as extraction, cloning, amplification, detection, sequencing and the like of nucleic acid, is rapidly developed and widely applied, and the first step of nucleic acid research is to extract nucleic acid from various biological samples, and the efficiency of nucleic acid extraction becomes a limiting factor for success or failure of downstream nucleic acid research. Because of the importance of nucleic acid extraction, scientists have developed a series of nucleic acid extraction methods, including the traditional method of extraction with organic solvents such as phenol/chloroform, the chelating resin method which is simple and convenient to operate but has low DNA purity, the glass powder adsorption method which is basically eliminated at present, the current mainstream silica gel membrane adsorption column method, the immune affinity method of anti-DNA monoclonal antibody, the magnetic bead method which is widely applied by an automated platform, and the like.
The magnetic bead method nucleic acid extraction method uses superparamagnetic silicon oxide nanometer magnetic beads as a carrier, uses silica gel to adsorb DNA, firstly uses cell lysis solution to lyse cells, and uses the principle that magnetic beads adsorb nucleic acid in high-salt solution and nucleic acid in low-salt solution is desorbed from the surfaces of the magnetic beads, DNA molecules free from the cells are adsorbed to the surfaces of magnetic particles, molecules such as protein and the like are not adsorbed and remain in the solution, under the action of a magnetic field, the magnetic particles are separated from the liquid, the particles are recovered, and then pure water or TE is used for eluting the adsorbed DNA. The magnetic bead method does not need centrifugation, is simple to operate, can be used for extracting in the same reaction tube, does not cause sample loss, and is more suitable for automatic extraction. The magnetic bead method has a high recovery rate compared with the chelex-100 extraction method, and DNA typing can be obtained even in 0.5. mu.l of blood, but DNA typing cannot be obtained in some cases by extracting the same amount of blood DNA with chelex-100.
Under the action of sunlight ultraviolet rays or microbial nucleases in the environment, DNA fragments in the test material are degraded into fragments below 500bp, and a large amount of inhibitors such as tannic acid, heme, protease, humic acid, metal ions and the like exist, so that the subsequent PCR amplification is difficult; the existing magnetic bead method nucleic acid extraction kit only has a certain effect of removing organic inhibitors, but has poor effect of removing metal ions, and neglects the protection of DNA fragments in the extraction process, so that the kit is not suitable for degrading test materials.
Disclosure of Invention
Aiming at the problems, the invention provides a novel high-efficiency trace detection material nucleic acid extraction reagent, a kit and a use method thereof based on a magnetic bead extraction method, which can effectively remove inhibitors in detection materials, effectively protect DNA fragments in the extraction process and prevent further degradation.
The purpose of the invention is realized by adopting the following technical scheme:
a novel high-efficiency trace detection material nucleic acid extraction reagent comprises a lysis solution, wherein the lysis solution comprises a bifunctional chelating agent, the bifunctional chelating agent has a metal chelating end and a protein anchoring end, and the bifunctional chelating agent is NOTA, MAA-NOTA, p-SCN-Bn-NOTA, p-SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA; wherein the content of the first and second substances,
the NOTA is 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;
the MAA-NOTA is (2,2' - (7- (2- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -2-oxoethyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;
the p-SCN-Bn-NOTA is 2- (4-isothiocyanatophenyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;
the p-SCN-NODA is 1,4, 7-triazacyclooctane-1, 4-diacetic acid-7-p-isothiocyanatobenzyl;
the MAA-GA-NODA is 2,2' - (7- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;
said MAA-DOTA is 2,2',2 "- (10- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid ];
the DOTA-NHS is 2,2',2 "- (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid;
the iEDTA is 1- (4-isothiocyanatobenzyl) ethylene diamine-N, N, N ', N' -tetraacetic acid;
the p-SCN-Bn-DTPA is 2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid.
Preferably, the concentration of the bifunctional chelating agent in the lysis solution is 1-20 mmol/L.
Preferably, the lysis solution comprises 3-7mol/L guanidine salt, 5-10mmol/L bifunctional chelating agent, 0.1-0.4mol/L nonionic surfactant, 10-100mmol/L pH buffer and 1-4mol/L isopropanol solution.
Preferably, the guanidine salt is guanidine hydrochloride, guanidine isothiocyanate, guanidine nitrate, guanidine phosphate or guanidine carbonate.
Preferably, the nonionic surfactant is ethyl phenyl polyethylene glycol, polyethylene glycol octyl phenyl ether or sorbitan fatty acid ester polyoxyethylene ether.
Another object of the present invention is to provide a novel nucleic acid extraction kit for a high-efficiency micro sample, which comprises the above extraction reagent.
Preferably, the novel efficient trace detection material nucleic acid extraction kit further comprises nano magnetic beads, a binding solution, a first washing solution, a second washing solution, Bind enzyme and an eluent.
Preferably, the binding solution comprises 2-5mol/L guanidine thiocyanate.
Preferably, the binding solution comprises 3-4mol/L guanidine thiocyanate and 50-150mmol/L pH buffer;
the first washing solution comprises the mixed solution of the lysis solution and ethanol in an equal volume ratio, polyvinylpyrrolidone with a final concentration of 1-5 wt.%, and a pH buffer with a final concentration of 50-150 mmol/L;
the second washing solution comprises ethanol solution with volume fraction of 70%;
the eluent is pure water or TE buffer solution.
The invention further aims to provide a use method of the novel high-efficiency trace detection material nucleic acid extraction kit, wherein the kit comprises 1.2ml of nano magnetic beads, 30ml of lysis solution, 30ml of binding solution, 30ml of first washing solution, 30ml of second washing solution, 110 mu g of Bind enzyme and 5ml of eluent;
the using method comprises the following steps:
s1, adding 110 mu l of eluent into 110 mu g of Bind enzyme, uniformly mixing by vortex, and standing for 10min at room temperature for use; adding 80ml of absolute ethyl alcohol into the washing solution for dilution for later use;
s2, preparing a mixed solution by using 320 mu l of lysate and 2 mu l of Bind enzyme of each sample, and adding 10 mu l of dithiothreitol into a single sample when the detected material is bones, teeth or seminal plaques;
s3, adding 330 mu l of the mixed solution into a test material, uniformly mixing by vortex, and cracking for 30min at 95 ℃;
s4, 13000 Xg centrifuging for 2min to collect digestive juice, adding 300. mu.l binding solution and 20. mu.l nanometer magnetic bead, mixing l0-15 times, standing at room temperature for 5min, mixing once every 30S, 6S each time;
s5, transferring the sample to a magnetic frame to be adsorbed for 5min, and then absorbing and discarding the liquid;
s6, adding 300 mu l of first washing liquid, whirling for 10S to resuspend magnetic beads, transferring to a magnetic frame for adsorption for 2min, and discarding the liquid;
s7, adding 500 mu l of second washing liquid, whirling for 10S to resuspend magnetic beads, transferring to a magnetic frame for adsorption for 2min, and discarding the liquid;
s8, repeating the step S7 once or twice;
s9, centrifugally collecting liquid drops on the tube wall, transferring the liquid drops to a magnetic frame for adsorption for 1min, sucking away all solutions, and drying the solution in air for 10 min;
s10, adding 30-50 μ l of eluent, vortex to disperse magnetic beads, and incubating at 55 deg.C for 10 min;
s11, transferring the sample to a magnetic frame for adsorption for 3min, and transferring the liquid to a new centrifugal tube to obtain a nucleic acid extracting solution.
The invention has the beneficial effects that:
(1) the invention provides a novel high-efficiency trace detection material nucleic acid extraction kit which is specially designed for forensic DNA (deoxyribonucleic acid) detection and is suitable for processing 1-104DNA in various complex test materials such as individual cells, trace blood stains, human tissues, old blood stains, saliva stains and the like is extracted without phenol and chloroform or alcohol precipitation, cell lysis is rapid, extraction time is short, and DNA release is sufficient; by adopting a superparamagnetic magnetic particle purification technology, the extraction recovery rate of low-content DNA exceeds 90%, the impurity interference resistance is strong, the applicability of a material to be detected is wide, and full-automatic equipment is used for simultaneous detection.
(2) Compared with the existing technology for extracting nucleic acid by a magnetic bead method, the invention adds the bifunctional chelating agent which has a metal chelating end and a protein anchoring end simultaneously into the lysis solution, wherein the bifunctional chelating agent has a macrocyclic group with strong metal chelating capacity and can be covalently connected with a biological protein molecule, the bifunctional chelating agent can be used as a connecting intermediate during lysis, metal ions are connected with the protein molecule which is not easy to be adsorbed and remains in the solution, and the metal ions are fixed in the solution, so that the metal ions in the detection material are effectively removed, and the metal ions as an inhibitor are prevented from influencing the subsequent PCR amplification.
(3) The invention provides a nucleic acid extraction kit which can effectively remove metal ions and effectively protect degraded DNA fragments in the extraction process, which is different from the method for releasing nucleic acid by high chaotropic salt cracking in the prior art.
Detailed Description
The invention is further described with reference to the following examples.
The embodiment of the invention relates to a novel high-efficiency trace detection material nucleic acid extraction kit, which comprises 1.2ml of nano magnetic beads, 30ml of lysis solution, 30ml of binding solution, 30ml of first washing solution, 30ml of second washing solution, 110 mu g of Bind enzyme and 5ml of eluent; wherein:
the nano magnetic beads are superparamagnetic silicon oxide nano magnetic beads with a core-shell structure, a superparamagnetic core and a silicon oxide shell, and come from a commercialized finished product, and the diameter of the magnetic beads is 100-1000 nm;
the lysis solution consists of 3-7mol/L guanidine hydrochloride or guanidine isothiocyanate, 5-10mmol/L bifunctional chelating agent, 0.1-0.4mol/L nonionic surfactant, 10-100mmol/L pH buffer and 1-4mol/L isopropanol solution, and the pH value of the lysis solution is 7-9;
the bifunctional chelating agent is NOTA, MAA-NOTA, p-SCN-Bn-NOTA, p-SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA;
the NOTA is 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;
the MAA-NOTA is (2,2' - (7- (2- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -2-oxoethyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;
the p-SCN-Bn-NOTA is 2- (4-isothiocyanatophenyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;
the p-SCN-NODA is 1,4, 7-triazacyclooctane-1, 4-diacetic acid-7-p-isothiocyanatobenzyl;
the MAA-GA-NODA is 2,2' - (7- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;
said MAA-DOTA is 2,2',2 "- (10- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid ];
the DOTA-NHS is 2,2',2 "- (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid;
the iEDTA is 1- (4-isothiocyanatobenzyl) ethylene diamine-N, N, N ', N' -tetraacetic acid;
the p-SCN-Bn-DTPA is 2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid;
the nonionic surfactant is ethyl phenyl polyethylene glycol, polyethylene glycol octyl phenyl ether or sorbitan fatty acid ester polyoxyethylene ether;
the composition of the binding solution is 3-4mol/L guanidine thiocyanate and 50-150mmol/L pH buffer, and the pH value of the binding solution is 7-9;
the first washing solution consists of the mixed solution of the lysis solution and ethanol in an equal volume ratio, polyvinylpyrrolidone with a final concentration of 1-5 wt.% and a pH buffer with a final concentration of 50-150mmol/L, and the pH value of the first washing solution is 4-6;
the second washing solution is composed of ethanol solution with volume fraction of 70%;
the eluent is composed of pure water or TE buffer solution;
in order to ensure the best efficiency, the high-efficiency nano magnetic beads and the Bind Enhancer are stored for a long time at 2-8 ℃, and other components are stored at room temperature (15-25 ℃).
Example 1
A novel high-efficiency trace detection material nucleic acid extraction kit comprises 1.2ml of nano magnetic beads, 30ml of lysis solution, 30ml of binding solution, 30ml of first washing solution, 30ml of second washing solution, 110 mu g of Bind Enhancer (Bind enzyme) and 5ml of eluent; wherein:
the nano magnetic beads are superparamagnetic silicon oxide nano magnetic beads with a core-shell structure, a superparamagnetic core and a silicon oxide shell, and come from a commercialized finished product, and the diameter of the magnetic beads is 100-1000 nm;
the composition of the lysis solution is 5mol/L guanidine hydrochloride or guanidine isothiocyanate, 8mmol/L bifunctional chelating agent, 0.2mol/L nonionic surfactant, 60mmol/L pH buffer and 2mol/L isopropanol solution, and the pH value of the lysis solution is 7-9;
the bifunctional chelating agent is 2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid;
the nonionic surfactant is ethyl phenyl polyethylene glycol, polyethylene glycol octyl phenyl ether or sorbitan fatty acid ester polyoxyethylene ether;
the composition of the binding solution is 4mol/L guanidine thiocyanate and 100mmol/L pH buffer, and the pH value of the binding solution is 7-9;
the first washing solution consists of the mixed solution of the lysis solution and ethanol in an equal volume ratio, polyvinylpyrrolidone with a final concentration of 3 wt.% and a pH buffer with a final concentration of 100mmol/L, and the pH value of the first washing solution is 4-6;
the second washing solution is composed of ethanol solution with volume fraction of 70%;
the eluent is composed of pure water or TE buffer solution.
The nucleic acid extracted by the nucleic acid extraction method has no inhibitor and has good PCR effect.
Example 2
A novel high-efficiency trace detection material nucleic acid extraction kit comprises 1.2ml of nano magnetic beads, 30ml of lysis solution, 30ml of binding solution, 30ml of first washing solution, 30ml of second washing solution, 110 mu g of Bind Enhancer (Bind enzyme) and 5ml of eluent; wherein:
the nano magnetic beads are superparamagnetic silicon oxide nano magnetic beads with a core-shell structure, a superparamagnetic core and a silicon oxide shell, and come from a commercialized finished product, and the diameter of the magnetic beads is 100-1000 nm;
the composition of the lysis solution is 5mol/L guanidine hydrochloride or guanidine isothiocyanate, 8mmol/L bifunctional chelating agent, 0.2mol/L nonionic surfactant, 60mmol/L pH buffer and 2mol/L isopropanol solution, and the pH value of the lysis solution is 7-9;
the bifunctional chelating agent is 1- (4-isothiocyanatobenzyl) ethylene diamine-N, N, N ', N' -tetraacetic acid;
the nonionic surfactant is ethyl phenyl polyethylene glycol, polyethylene glycol octyl phenyl ether or sorbitan fatty acid ester polyoxyethylene ether;
the composition of the binding solution is 4mol/L guanidine thiocyanate and 100mmol/L pH buffer, and the pH value of the binding solution is 7-9;
the first washing solution consists of the mixed solution of the lysis solution and ethanol in an equal volume ratio, polyvinylpyrrolidone with a final concentration of 3 wt.% and a pH buffer with a final concentration of 10mmol/L, and the pH value of the first washing solution is 4-6;
the second washing solution is composed of ethanol solution with volume fraction of 70%;
the eluent is composed of pure water or TE buffer solution.
The nucleic acid extracted by the nucleic acid extraction method has no inhibitor and has good PCR effect.
Example 3
A nucleic acid extraction kit comprises 1.2ml of nano magnetic beads, 30ml of lysis solution, 30ml of binding solution, 30ml of first washing solution, 30ml of second washing solution, 110 mu g of Bind Enhancer (Bind enzyme) and 5ml of eluent, wherein the binding solution is 100mmol/L of pH buffer, the pH value of the binding solution is 7-9, and the rest is the same as in example 1.
Example 4
A use method of a novel high-efficiency trace detection material nucleic acid extraction kit comprises the following steps:
s1, adding 110 mu l of eluent into a 110 mu g Bind Enhancer tube, uniformly mixing by vortex, and standing at room temperature for 10min for use; when the automatic equipment is used, the mixture can be directly transferred to Buffer FFL for uniform mixing; adding 80ml of absolute ethyl alcohol into the washing solution for dilution for later use, wherein if the absolute ethyl alcohol is not added or the addition amount is insufficient, the DNA yield can be reduced;
s2, preparing a mixed solution according to 320 mu l of lysate and 2 mu l of Bind Enhancer of each sample (preparing a mixed solution according to 16ml of lysate and 100 mu l of Bind Enhancer of each 48 samples, preparing a mixed solution according to 32ml of lysate and 200 mu l of Bind Enhancer of each 96 samples), adding 10 mu l of dithiothreitol into each single sample, and keeping the mixture for no more than 60min after the lysate and the dithiothreitol are mixed;
when the components (such as grease) in the material to be detected and the lysis solution are subjected to chemical reaction, color or turbidity can occur, and if the turbidity is fine particles or flocculent, the detection is not influenced; if the block appears, the supernatant can be taken for inspection after centrifugation, and the result is not influenced;
s3, placing the test material into a centrifugal sleeve or a nucleic acid basket, adding 330 ul of mixed solution, covering a tube cover, uniformly mixing by vortex, and cracking at 80-95 ℃ for 30 min;
s4, 13000 Xg centrifuging for 2min to collect digestive juice, removing the centrifugal sleeve or basket (removing carrier);
s5, adding 300 mu l of binding solution and 20 mu l of high-efficiency nano magnetic beads into the centrifugal tube, uniformly mixing for l0-15 times, standing for 5min at room temperature, and uniformly mixing for a plurality of times (once every 30S, 6S every time) in the period;
s6, transferring the sample to a magnetic frame to be adsorbed for 5min, and then absorbing and discarding the liquid;
s7, adding 300 mu l of first washing liquid, whirling for 10S to resuspend magnetic beads, transferring to a magnetic frame for adsorption for 2min, and then absorbing and discarding the liquid;
s8, adding 500 mu l of second washing liquid, whirling for 10S to resuspend magnetic beads, transferring to a magnetic frame for adsorption for 2min, and then absorbing and discarding the liquid;
s9, repeating S8 once (or twice);
s10, centrifuging for a short time to collect liquid drops on the tube wall, transferring the liquid drops to a magnetic frame to be adsorbed for 1min, absorbing and discarding all solutions, and drying the solution in air for 10 min;
s11, adding 30-50 mu l of eluent, vortex to disperse magnetic beads, and carrying out shaking incubation at 55 ℃ for 10 min;
s12, transferring the sample to a magnetic frame for adsorption for 3min, and transferring the liquid containing the DNA to a new centrifugal tube.
The concentration and purity of DNA were measured by a microspectrophotometer, and the results of extraction were compared with two commercially available conventional kits, and 20 extraction results were measured (the kit described in examples 1 to 3 was measured by the method of example 4, and the commercially available conventional kits were subjected to the instructions), and the average concentration was determined, and the results are shown in the following table.
| Reagent kit | Concentration (μ g/ml) | OD260/280 |
| Example 1 | 103 | 1.93 |
| Example 2 | 106 | 1.95 |
| Example 3 | 81 | 1.73 |
| Control 1 | 72 | 1.67 |
| Control 2 | 74 | 1.67 |
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A novel high-efficiency trace detection material nucleic acid extraction reagent comprises a lysis solution, and is characterized in that the lysis solution comprises a bifunctional chelating agent, the bifunctional chelating agent has a metal chelating end and a protein anchoring end, and the bifunctional chelating agent is NOTA, MAA-NOTA, p-SCN-Bn-NOTA, p-SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA; wherein the content of the first and second substances,
the NOTA is 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;
the MAA-NOTA is (2,2' - (7- (2- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -2-oxoethyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;
the p-SCN-Bn-NOTA is 2- (4-isothiocyanatophenyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;
the p-SCN-NODA is 1,4, 7-triazacyclooctane-1, 4-diacetic acid-7-p-isothiocyanatobenzyl;
the MAA-GA-NODA is 2,2' - (7- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;
said MAA-DOTA is 2,2',2 "- (10- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid ];
the DOTA-NHS is 2,2',2 "- (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid;
the iEDTA is 1- (4-isothiocyanatobenzyl) ethylene diamine-N, N, N ', N' -tetraacetic acid;
the p-SCN-Bn-DTPA is 2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid.
2. The reagent of claim 1, wherein the concentration of the bifunctional chelating agent in the lysis solution is 1-20 mmol/L.
3. The reagent of claim 1, wherein the lysis solution comprises 3-7mol/L guanidine salt, 5-10mmol/L bifunctional chelating agent, 0.1-0.4mol/L nonionic surfactant, 10-100mmol/L pH buffer and 1-4mol/L isopropanol solution.
4. The reagent for extracting nucleic acid as a novel high-efficiency micro-assay material as claimed in claim 3, wherein the guanidine salt is guanidine hydrochloride, guanidine isothiocyanate, guanidine nitrate, guanidine phosphate or guanidine carbonate.
5. The reagent of claim 3, wherein the non-ionic surfactant is ethyl phenyl polyethylene glycol, polyethylene glycol octyl phenyl ether or sorbitan fatty acid ester polyoxyethylene ether.
6. A novel kit for extracting nucleic acid from a high-efficiency micro-detection material, which is characterized by comprising the lysis solution of any one of claims 1 to 5.
7. The kit for extracting nucleic acid from a novel high-efficiency micro-assay material according to claim 6, further comprising nano magnetic beads, a binding solution, a first washing solution, a second washing solution, Bind enzyme and an eluent.
8. The kit for extracting nucleic acid as claimed in claim 7, wherein the binding solution contains 2-5mol/L guanidinium thiocyanate.
9. The kit for extracting nucleic acid from a novel high-efficiency micro-assay material as claimed in claim 7, wherein the binding solution comprises 3-4mol/L guanidine thiocyanate, 50-150mmol/L pH buffer;
the first washing solution comprises the mixed solution of the lysis solution and ethanol in an equal volume ratio, polyvinylpyrrolidone with a final concentration of 1-5 wt.%, and a pH buffer with a final concentration of 50-150 mmol/L;
the second washing solution comprises ethanol solution with volume fraction of 70%;
the eluent is pure water or TE buffer solution.
10. The use method of the novel high-efficiency micro-detection material nucleic acid extraction kit according to claim 9, characterized in that the kit comprises 1.2ml of nano magnetic beads, 30ml of lysis solution, 30ml of binding solution, 30ml of first washing solution, 30ml of second washing solution, 110 μ g of Bind enzyme and 5ml of eluent;
the using method comprises the following steps:
s1, adding 110 mu l of eluent into 110 mu g of Bind enzyme, uniformly mixing by vortex, and standing for 10min at room temperature for use; adding 80ml of absolute ethyl alcohol into the washing solution for dilution for later use;
s2, preparing a mixed solution by using 320 mu l of lysate and 2 mu l of Bind enzyme of each sample, and adding 10 mu l of dithiothreitol into a single sample when the detected material is bones, teeth or seminal plaques;
s3, adding 330 mu l of the mixed solution into a test material, uniformly mixing by vortex, and cracking for 30min at 95 ℃;
s4, 13000 Xg centrifuging for 2min to collect digestive juice, adding 300. mu.l binding solution and 20. mu.l nanometer magnetic bead, mixing l0-15 times, standing at room temperature for 5min, mixing once every 30S, 6S each time;
s5, transferring the sample to a magnetic frame to be adsorbed for 5min, and then absorbing and discarding the liquid;
s6, adding 300 mu l of first washing liquid, whirling for 10S to resuspend magnetic beads, transferring to a magnetic frame for adsorption for 2min, and discarding the liquid;
s7, adding 500 mu l of second washing liquid, whirling for 10S to resuspend magnetic beads, transferring to a magnetic frame for adsorption for 2min, and discarding the liquid;
s8, repeating the step S7 once or twice;
s9, centrifugally collecting liquid drops on the tube wall, transferring the liquid drops to a magnetic frame for adsorption for 1min, sucking away all solutions, and drying the solution in air for 10 min;
s10, adding 30-50 μ l of eluent, vortex to disperse magnetic beads, and incubating at 55 deg.C for 10 min;
s11, transferring the sample to a magnetic frame for adsorption for 3min, and transferring the liquid to a new centrifugal tube to obtain a nucleic acid extracting solution.
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