CN204369906U - A kind of test kit extracting and preserve nucleic acid - Google Patents
A kind of test kit extracting and preserve nucleic acid Download PDFInfo
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- CN204369906U CN204369906U CN201420212643.8U CN201420212643U CN204369906U CN 204369906 U CN204369906 U CN 204369906U CN 201420212643 U CN201420212643 U CN 201420212643U CN 204369906 U CN204369906 U CN 204369906U
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- nucleic acid
- test kit
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- storage vessel
- cracking
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 110
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 110
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 110
- 238000012360 testing method Methods 0.000 title claims abstract description 59
- 239000007788 liquid Substances 0.000 claims abstract description 43
- 238000003860 storage Methods 0.000 claims abstract description 37
- 238000005070 sampling Methods 0.000 claims abstract description 17
- 238000005336 cracking Methods 0.000 claims description 27
- 239000011324 bead Substances 0.000 claims description 10
- 229920003023 plastic Polymers 0.000 claims description 6
- 239000004033 plastic Substances 0.000 claims description 6
- 210000003296 saliva Anatomy 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 39
- 229910052814 silicon oxide Inorganic materials 0.000 abstract description 23
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 17
- 238000000605 extraction Methods 0.000 abstract description 11
- 230000002934 lysing effect Effects 0.000 abstract description 11
- 239000004005 microsphere Substances 0.000 description 75
- 239000000523 sample Substances 0.000 description 47
- 108020004414 DNA Proteins 0.000 description 42
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 38
- 238000000034 method Methods 0.000 description 26
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- FCKYPQBAHLOOJQ-UHFFFAOYSA-N Cyclohexane-1,2-diaminetetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)C1CCCCC1N(CC(O)=O)CC(O)=O FCKYPQBAHLOOJQ-UHFFFAOYSA-N 0.000 description 2
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- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
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- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
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- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
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- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model provides a kind of test kit for extracting and preserve nucleic acid, it is characterized in that, described test kit comprises: one or more storage vessel, and described storage vessel comprises: nucleic acid is in conjunction with phase, and described nucleic acid combines for adsorb and/or in conjunction with nucleic acid to be purified; Cracking-in conjunction with liquid, described cracking-in conjunction with liquid, for lysing cell film, discharges nucleic acid to be purified, and impels nucleic acid to be purified to be incorporated into described nucleic acid in conjunction with phase; And described nucleic acid combination has mutually: (1) kernel; (2) silicon oxide layer on described kernel is positioned at; (3) the carboxyl modified layer on described silicon oxide layer surface is positioned at.Test kit of the present utility model may be used for sampling under non-laboratory condition and nucleic acid extraction, and nucleic acid samples is preserved.
Description
Technical field
The utility model relates to field of biological detection, and particularly, the utility model provides a kind of test kit that can be used in extracting and preserving nucleic acid.
Background technology
Gene is the information storage material in all organisms, and these information comprise the essential characteristic of organism, the moiety of organism and building form etc.Vital movement due to organism is the result of the various matter interactions of composition organism, and therefore, gene information also contains the information of organism vital movement form and mode.Gene test is exactly the information demonstrating gene by experiment, can carry out causing the microbial species alanysis of disease, identity authentication, Clinicopathologic Diagnosis, pathophysiologic features analysis by gene test.
The quality of gene test and level decide primarily of sample preparation, quantity of information and analytical technology, detect sample can be blood, saliva, oral mucosa, body fluid, hair root, tissue etc., to these biological specimens carry out sample process isolating nucleic acid be in molecular biology, medical test must through step.
The blood gathered or other biological sample usually can be delivered in laboratory and carry out nucleic acid extraction, because these biological specimens are potential virus and bacteriological infection source, transport or process these biological samples and bring threat to operator's life and health; Meanwhile, sample needs to store and transport at low temperature usually, and incorrect biological specimen collection, transhipment and treating processes usually cause nucleic acid substances degrade thus cause false negative result.
DNA is attached on the magnetic carriers such as silicon oxide by magnetic bead nucleic acid purification method, and impurity is washed away by selectivity and is widely applied just in the lab.But existing magnetic bead separate nucleic acid technology still comes with some shortcomings: the sample handling processes as complexity: need carry out protease digestion process, need temperature control etc.; These problems bring disadvantageous effect to Rapid nucleic acid sample process, particularly hinder its application in a large amount of sample of nucleic acid gathers safely and detects.
Therefore, need a kind of to combine, to preserve and transhipment biological specimen amplifying nucleic acid, while safe and convenient carry out method and the reagent of subsequent gene detection.
Utility model content
The purpose of this utility model is to provide and a kind of combines, preserve and transhipment biological specimen amplifying nucleic acid, while safe and convenient carry out the nucleic acid extraction kit of subsequent gene detection.
First aspect of the present utility model, provide a kind of test kit for extracting and preserve nucleic acid, described test kit comprises:
One or more storage vessel, described storage vessel comprises:
Nucleic acid is in conjunction with phase, and described nucleic acid combines for adsorb and/or in conjunction with nucleic acid to be purified;
Cracking-in conjunction with liquid, described cracking-in conjunction with liquid, for lysing cell film, discharges nucleic acid to be purified, and impels nucleic acid to be purified to be incorporated into described nucleic acid in conjunction with phase;
And described nucleic acid combination has mutually: (1) magnetic kernel; (2) silicon oxide layer on described kernel is positioned at; (3) the carboxyl modified layer on described silicon oxide layer surface is positioned at.
In another preference, described lysis contains following component in conjunction with liquid: (i) chaotropic salt, and concentration is 3-6M; (ii) salts solution (comprising monovalent salt, divalent salts), concentration is 0.1-1.5M; (iii) tensio-active agent, concentration is 2-5% (v/v); (iv) sequestrant, concentration is 0-100mM; (v) alcohol, concentration is 20-60% (v/v).
In another preference, described nucleic acid is selected from lower group in conjunction with the magnetic kernel of phase: polymer materials kernel, polysaccharide material kernel, inorganic carrier material kernel, or its combination.
In another preference, described cracking contains high concentration salt solutions in conjunction with in liquid, and described salt is monovalent salt, divalent salts, ammonium salt, or its combination, and its concentration is 0.1-1.5M.
In another preference, described storage vessel is selected from lower group: test tube, centrifuge tube, eppendorf pipe, culturing bottle.
In another preference, described storage vessel is one-trip container.
In another preference, described storage vessel is plastic containers with a scale.
In another preference, described storage vessel is the plastic containers that quantitative liquid is housed in advance.
In another preference, described storage vessel also has a sealing cover, and described sealing cover can be injected device or kapillary penetrates.
In another preference, described sealing cover is plastic closure or rubber closure.
In another preference, described sealing cover is used for preventing oxygen and/or water from entering storage vessel.
In another preference, described storage vessel has an inner pipe portion and a sampling tube part, and described inner pipe portion is positioned at sampling tube part.
In another preference, described interior pipe is containing magnetic kernel and cracking in conjunction with liquid, and described interior pipe bottom can be penetrated by plastic object, syringe or kapillary or puncture.
In another preference, described interior pipe is the interior pipe of plastic inner pipe or rubber.
In another preference, described interior pipe has one and can to puncture bottom, and its structural integrity is destroyed by puncture in described punctured bottom.
In another preference, described sampling tube also has a piercing portions, and described piercing portions for penetrate or puncture described in pipe, thus destroy the integrity of described interior pipe.
In another preference, the capacity of described storage vessel is 1.5-15ml.
In another preference, it is magnetic microsphere mutually that described nucleic acid combines.
In another preference, the particle diameter of described magnetic microsphere is 50 nanometer-2 microns, is preferably 300-1000 nanometer.
In another preference, it is 0.1-5mg mutually that the described nucleic acid in single described storage vessel combines.
In another preference, described test kit also comprises sample collecting device.
In another preference, described sample collecting device is trace sample gathering device.
In another preference, described trace refers to that the collection capacity of sample is 10ul-5ml liquid sample.
In another preference, described trace refers to the collection capacity≤2ml of sample, is preferably≤1ml, is more preferably≤0.5ml.
In another preference, described sample collecting device is connected with described storage vessel.
In another preference, described sample collecting device is connected with described sealing cover.
In another preference, described sample collecting device is blood-taking device.
In another preference, described test kit also comprises anti-coagulant.
In another preference, described blood-taking device is finger tip blood-taking device, and preferably, described finger tip blood-taking device is selected from lower group: finger tip blood taking needle, swab, thieving paper, smear, kapillary, dropper.
In another preference, described sample collecting device is the scraper or the saliva collector that gather Oral Mucosal Cells.
In another preference, described test kit also comprises washings: described washings is used for combining from nucleic acid washing away non-specific adsorption component mutually; And/or
Described test kit also comprises DNA elutriant, and described DNA elutriant is used for that nucleic acid is combined with described nucleic acid and dissociates mutually.
In another preference, described non-specific adsorption component is selected from lower group: albumen, polysaccharide, lipid, or its combination.
In another preference, described washings is positioned at the second storage vessel.
In another preference, described DNA elutriant is selected from lower group: water, weakly alkaline solution, biological buffer, or its combination.
In another preference, the pH value of described weakly alkaline solution is 7.5-9.
In another preference, described DNA elutriant is positioned at the 3rd storage vessel.
Should be understood that within the scope of the utility model, above-mentioned each technical characteristic of the present utility model and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of container in a preference of the present utility model;
Fig. 2 is the schematic diagram of test kit in a preference of the present utility model;
Fig. 3 is the schematic diagram of test kit in a preference of the present utility model;
In figure, 1 for storage vessel, 2 for cracking in conjunction with liquid, 3 for nucleic acid in conjunction with phase, 4 be sealing cover, 5 be interior pipe, 6 be sampling tube.
Embodiment
The present inventor, through long-term and deep research, have developed a kind of test kit that can be used in sample collection, DNA extraction and preservation.Described test kit not only may be used for extracting the DNA in biological specimen, can also stablize to preserve and extract complete DNA sample, therefore be suitable for Large Scale Biology sample collection and preservation, such as common lab or field sample collection etc.
Magnetic microsphere
As used herein, term " magnetic microsphere ", " magnetic bead ", " magnetic-particle " are used interchangeably, and all refer to that a kind of preferred nucleic acid for the utility model separate nucleic acid is in conjunction with phase.
In the utility model, the size of magnetic microsphere is not particularly limited, and generally its particle diameter is 50 nanometer-2 microns, is preferably 300-1000 nanometer.
In the utility model, the material of magnetic microsphere is not particularly limited, and can contain or be made up of various magneticsubstance.
In the utility model, as nucleic acid in conjunction with the magnetic microsphere of phase be surface through carboxyl modified or magnetic microsphere that surface is modified through terminal carboxyl(group) oxide/silica.It is terminal carboxyl(group) oxide/silica magnetic microsphere mutually that a kind of preferred nucleic acid combines.
It is the paramagnetism microballoon that undertaken being separated by magnetic field or particle mutually that a kind of preferred nucleic acid combines.
Various different magnetic microsphere (comprising modified magnetic microsphere), prepared by the method by this area routine, or buy by commercially available approach.Representational example includes, but is not limited to: inorganic microspheres, biopolymer-microsphere, polymer microsphere.
Cracking is in conjunction with liquid
In the utility model, term " cracking is in conjunction with liquid " refers to, both for lysing cell film, discharge nucleic acid to be purified, also for impelling nucleic acid to be purified to be incorporated into the solution of described nucleic acid in conjunction with phase.
Cell pyrolysis liquid is used for lysing cell film, discharge nucleic acid to be purified, DNA for providing the environment being applicable to combining in conjunction with liquid, nucleic acid is combined with nucleic acid and combines mutually, and impurity (comprising protein, carbohydrate, RNA etc.) is not then incorporated into nucleic acid in conjunction with phase.
In the utility model, lysis and DNA cohesive process carry out in same solution, contribute to extracting nucleic acid fast, easily.
Nucleic acid is in conjunction with phase
In the utility model, nucleic acid combines for adsorb and/or in conjunction with nucleic acid to be purified, and described nucleic acid combines and has mutually: (1) magnetic kernel; (2) silicon oxide layer on described kernel is positioned at; (3) the carboxyl modified layer on described silicon oxide layer surface is positioned at.
Preferably, it can be terminal carboxyl(group) oxide/silica magnetic microsphere mutually that described nucleic acid combines, or terminal carboxyl(group) oxide/silica plate.
Surface is a kind of solid phase material conventional in DNA extraction purge process with the magnetic microsphere of carboxyl modified layer, under suitable DNA adsorption conditions (as under the polyoxyethylene glycol of high molecular and the effect of high density sodium-chlor), DNA can separate out and be adsorbed onto on carboxylated solid phase material from the aqueous solution.
Nucleic acid extraction kit
The utility model provides a kind of test kit for extracting and preserve nucleic acid, and described test kit comprises:
One or more storage vessel, described storage vessel comprises:
Nucleic acid is in conjunction with phase, and described nucleic acid combines for adsorb and/or in conjunction with nucleic acid to be purified;
Cracking-in conjunction with liquid, described cracking-in conjunction with liquid, for lysing cell film, discharges nucleic acid to be purified, and impels nucleic acid to be purified to be incorporated into described nucleic acid in conjunction with phase;
And described nucleic acid combination has mutually: (1) magnetic kernel; (2) silicon oxide layer on described kernel is positioned at; (3) the carboxyl modified layer on described silicon oxide layer surface is positioned at.
Preferably, described nucleic acid is selected from lower group in conjunction with the kernel of phase: polymer materials kernel, polysaccharide material kernel, inorganic carrier material kernel, or its combination.Described various kernels all can be obtained by commercially available approach, or prepare with reference to prior art.
In another preference, described polymer materials is selected from lower group: polystyrene, polymethacrylate, Mierocrystalline cellulose, polyvalent alcohol are as the multipolymer of polyvinyl alcohol and polyvinyl butyral acetal and these materials, or its combination.
In another preference, described polysaccharide compound comprises the derivative of dextran, agarose, Mierocrystalline cellulose and above-mentioned arbitrary material, or its combination.
In another preference, described inorganic carrier comprises metal, glass, metal oxide and nonmetal oxide, has the carrier of metallic surface, magnetic microsphere, pipe, film, porous plate, chip and microarray etc., or its combination.
In the utility model, adopt specific surface to have the magnetic microsphere of carboxyl modified layer, extract DNA (especially genomic dna) by magnetic microsphere method.
In another preference, described lysis does not contain proteolytic enzyme (as Proteinase K) in conjunction with liquid.
In another preference, described lysis makes impurity (comprising protein, carbohydrate, RNA etc.) not be incorporated into or substantially not be incorporated into nucleic acid in conjunction with phase in conjunction with liquid.
In another preference, described lysis contains following component in conjunction with liquid:
(i) chaotropic salt, usual concentration is 3-6M;
(ii) metal-salt, usual concentration is 0.1-1.5M;
(iii) tensio-active agent, usual concentration is 2-5% (v/v);
(iv) sequestrant, usual concentration is 0.5mM-100mM; With
V () alcohol, usual concentration is 20-60% (v/v).
In another preference, described chaotropic salt comprises guanidinesalt (example hydrochloric acid guanidine).
In another preference, described metal-salt comprises sylvite, sodium salt, lithium salts, magnesium salts, ammonium salt or its combination.
In another preference, described tensio-active agent comprises Triton X-100, Tween20 or other nonionic surface active agent.
In another preference, described sequestrant comprises EDTA, EGTA, CDTA, Citrate trianion, or its combination.
In another preference, described alcohol comprises ethanol or Virahol.
In another preference, it is the silicon oxide magnetic microspheres of end modified carboxylic group or the silicon oxide plate of end modified carboxylic group mutually that described nucleic acid combines.
In another preference, described cracking, in conjunction with metal-containing salt in liquid, comprises monovalent salt, divalent salts, and described salt is sodium salt, lithium salts, sylvite, magnesium salts or its combination.
In another preference, described an alkali metal salt is lithium chloride or sodium-chlor.
In another preference, salt concn is 0.1-3.0M, is preferably 0.2-2.5M.
In a preferred embodiment of the present utility model, described storage vessel is centrifuge tube 1 as shown in Figure 1, and cracking is arranged in centrifuge tube 1 in conjunction with liquid 2 mutually with nucleic acid combination.When extracting nucleic acid with the utility model test kit, sample containing nucleic acid is added storage vessel, namely cracking starts lysing cell in conjunction with liquid, and from cell, free nucleic acid molecule is out adsorbed onto nucleic acid specifically in conjunction with phase surface, and the impurity such as protein are not stayed in the solution by adsorbing.Then, be separated magnetic microsphere under the action of a magnetic field after, namely highly purified DNA can be obtained with elution magnetic microsphere.
In another preferred embodiment of the present utility model, described storage vessel is culturing bottle 1 as shown in Figure 2, and cracking is arranged in centrifuge tube 1 in conjunction with liquid 2 mutually with nucleic acid combination; Especially, described storage vessel also has a sealing cover 4, described sealing cover is used for described storage vessel to seal, thus make the internal medium of described storage vessel and storage vessel outside atmosphere exchange of substance not occur (such as, water, oxygen diffusion etc.), and described sealing cover can be injected device or kapillary penetrates.In use, penetrated by syringe or thrust sealing cover, thus gathered sample is added in described storage vessel, carrying out DNA extraction and preservation.
In another preferred embodiment of the present utility model, described storage vessel is as shown in Figure 3, and product is made up of two portions:
Interior pipe 5, described interior pipe 5 be store magnetic bead kernel and cracking in conjunction with liquid container, 3 be arranged in described pipe 5 mutually for cracking combines in conjunction with liquid 2 and nucleic acid.The bottom of described interior pipe 5 is film or rubber part, can be injected the diaphragm seal of device or other punctures.Sampling tube 6 is sample collection tube with a scale, for gathering blood, saliva and other biological sample, fixed film piercing portions bottom sampling tube, after sample collection, rotated by sealing cover and be fixed on sampling tube, piercing portions at the bottom of sampling tube destroys film integrity, and magnetic bead kernel and cracking are mixed with collecting sample in conjunction with liquid, reach lysing cell, the effect of bind nucleic acid.
Especially, test kit of the present utility model can also stably stored nucleic acid, in use, can add in storage vessel by sample, makes cracking make free nucleic acid molecule be adsorbed to nucleic acid in conjunction with phase surface in conjunction with liquid with example reaction.Meanwhile, can by the test kit of collecting sample integrally, carry out transporting or storing.In a preference of the present utility model, the test kit of described collecting sample can store 1 year at normal temperatures.
In a kind of preferred embodiment of the present utility model, after described test kit collecting sample, test kit is integrally transported in the laboratory of having ready conditions and detecting and carries out high-throughput nucleic acid detection.Described test kit may be used for hospital's conventional blood collection, or field sample collection etc. needs the situation detecting nucleic acid.Preferably, when described test kit is used for blood sample collection, in described test kit, also anti-coagulant is comprised.Described anti-coagulant can make an addition in storage vessel, or is arranged in other containers, adds as required when sampling.
The biomaterial (as cell) that the utility model test kit can detect is not particularly limited, and representative example comprises (but being not limited to): virus, chlamydozoan, bacterium, actinomycetes, yeast, fungi, vegetable cell and zooblast (as mammiferous cell) etc.Described test kit is specially adapted to the nucleic acid extraction of virus, the mankind and animal, and e.g., a preferred embodiment of the present utility model extracts genomic dna from hemocyte.
In another preferred embodiment, when described sample is the biomaterial containing cell wall constituent, also first can carries out suitable process (broken wall) to biomaterial, then add in described test kit.
Because the extraction efficiency of described test kit is high, being therefore not only applicable to from multiple sample, as extracted nucleic acid in whole blood, cell, blood plasma and tissue etc., being also particularly suitable for extracting genomic dna from a small amount of cell.In the utility model, a small amount of cell refers generally to single or 2-1000 cell (preferably 1-100 cell, more preferably (as 1-20 cell).
Nucleic acid for the utility model method purifying may reside in body fluids such as blood, urine, ight soil, saliva, sputum, or is present in tissue and organ sample.Sample of nucleic acid can obtain from the solid support material such as cotton swab, smear preparation, also can obtain from other liquid samples.
Use cracking of the present utility model in conjunction with liquid, can be used for purification of nucleic acid (comprising DNA).Representational example comprises (but being not limited to) genomic dna, cDNA, total serum IgE etc.
In a preference, the utility model adopts above-mentioned test kit, and without the need to protease K digesting process, cell membrane lysis, nucleic acid and nucleic acid combine to combine and uses same solution, thus from multiple sample, extract nucleic acid fast, efficiently, easily, and can stably stored sample of nucleic acid.
Should be understood that test kit of the present utility model also can comprise other optional general component, comprising (but being not limited to): washing reagent, damping fluid, cell walls lysate etc.
Major advantage of the present utility model comprises:
(1) test kit of the present utility model extracts quick, easy and simple to handle.Adopt test kit of the present utility model, lysis in conjunction with liquid one step lysing cell and specific by nucleic acid absorption in nucleic acid in conjunction with phase, the aqueous solution also directly can carry out nucleic acid and the dissociate elution process of nucleic acid in conjunction with phase, without the need to Proteinase K process, nucleic acid can be combined the upper Nucleic Acid Elution combined mutually after only needing simple rinse step to get off, method steps is simple.
(2) mild condition.In the utility model, nucleic acid under mild conditions (pH6.5-8.0) is combined with nucleic acid and combines, and carries out wash-out under water or weakly alkaline (pH7.5-8.5) condition, and operation is at room temperature carried out.
(3) yield of nucleic acid is high.1mg magnetic microsphere in conjunction with the genomic dna up to 10ug, or can extract the people's whole blood genome DNA up to more than 90%.
(4) nucleic acid purity extracted is high.Compared with current nucleic acid extraction kit and working method, this test kit extracts nucleic acid and has the advantages such as purity is high, impurity is low, and the nucleic acid of extraction is applicable to the multiple downstream experiment such as PCR detection, nucleic acid hybridization, without the need to being further purified.
(5) level of automation is high.
(6) test kit of the present utility model can add at sample and fashionablely namely starts leaching process, carry out at test kit can completing nucleic acid extraction in the process of transporting or preserving, and the process of preservation amplifying nucleic acid is not perishable, thus may be used for the collecting work of great amount of samples.
Below in conjunction with specific embodiment, set forth the utility model further.Should be understood that these embodiments are only not used in restriction scope of the present utility model for illustration of the utility model.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
The preparation of embodiment 1 different surfaces modified magnetic microballoon
1) preparation of amination silicon oxide magnetic microspheres: take commercially available silanol base silicon oxide magnetic microspheres appropriate, add dehydrated alcohol, water, strong aqua, finally add 3 '-aminopropyl triethoxysilane, stirring at normal temperature reaction 3h after mixing, product is used dehydrated alcohol and distilled water wash successively, obtains the magnetic microsphere of surface bond amino.
2) preparation of carboxylated silicon oxide magnetic microspheres: take commercially available silanol base silicon oxide magnetic microspheres appropriate, add dehydrated alcohol, water, strong aqua, finally add 3 '-glycydoxy Trimethoxy silane, stirring at normal temperature reaction 3h after mixing, product is used dehydrated alcohol and distilled water wash successively, obtains the magnetic microsphere of surface bond epoxy group(ing).The magnetic microsphere of 4-Aminobutanoicacid and surface bond epoxy group(ing) is reacted, obtains the silicon oxide microsphere of surface carboxyl groups.
Embodiment 2 kits nucleic acid
The test kit specific configuration example extracting DNA described in this test kit comprises:
Magnetic microsphere: the carboxylated silicon oxide magnetic microspheres of above-mentioned self-control
Cracking is in conjunction with liquid: 4M Guanidinium hydrochloride, 2%Triton X-100,0.1%SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH5.5), 1mM EDTA, 25% Virahol
Washings I:200mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris damping fluid (pH6.5)
Cleaning solution II: 70% ethanol
Elutriant: 1mM EDTA, 10mM Tris-HCl (pH8.0)
Purification step:
1) get 200 μ L anticoagulations in 1.5ml centrifuge tube, add 750 μ L cracking adsorption liquid vibrations and be mixed evenly lysing cell, leave standstill 5min;
2) add 1mg magnetic microsphere, gentleness shakes up 10min;
3) with Magneto separate frame absorption magnetic microsphere, supernatant is abandoned;
4) wash 2 times with 850 μ L washings I, gentleness shakes up 2min, and absorption magnetic microsphere, abandons supernatant;
5) wash 2 times by 850 μ L cleaning solution II, gentleness shakes up 2min, and absorption magnetic microsphere, abandons supernatant;
6) the ethanol volatilization that 5min makes to remain is left standstill;
7) add 100 μ L elutriants, after being mixed evenly, place 5min in room temperature;
8) magnetic field absorption magnetic microsphere, reclaims elutriant in centrifuge tube, carries out DNA detection and subsequent experimental.
The comparison of several finishing magnetic microsphere of embodiment 3 purifying Whole Blood Genomic DNA
Sample: the mouse anticoagulation of heparin sodium process
Material:
Cracking is in conjunction with liquid: 4M Guanidinium hydrochloride, 2%Triton X-100,0.1%SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH5.5), 1mM EGTA, 25% Virahol
Washings I:100mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris damping fluid (pH6.5)
Cleaning solution II: 70% ethanol
Elutriant: 1mM EDTA, 10mM Tris-HCl (pH8.0)
Magnetic microsphere:
Magnetic microsphere A: commercially available silanol base silicon oxide magnetic microspheres
Magnetic microsphere B: amination silicon oxide magnetic microspheres
Magnetic microsphere C: carboxylated silicon oxide magnetic microspheres
Use experimental procedure purified genomic dna in embodiment 2, result is as following table:
The ratio of A260/A280, closer to 1.80, shows to extract the DNA purity using carboxylated silicon oxide magnetic microspheres to extract higher, the pollution of less protein and RNA; The DNA that the carboxylated silicon oxide magnetic microspheres used extracts survey the ratio of A260/A230 about 1.50, the residual salts contg of the high DNA showing to extract of ratio is relatively less.
The above results is pointed out, and during the carboxylated silicon oxide magnetic microspheres of use, the nucleic acid extracted significantly is better than silanol base magnetic microsphere and amination magnetic microsphere.
Embodiment 4 Nucleic acid purification kits extracts the result of Mouse Blood genomic dna
Sample: the mouse anticoagulation of anticoagulant heparin process
Material:
Magnetic microsphere: terminal carboxyl(group) oxide/silica magnetic microsphere
Cracking is in conjunction with liquid: 4M Guanidinium hydrochloride, 2%Triton X-100,0.1%SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH5.5), 1mM CDTA, 25% Virahol
Washings I:100mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris damping fluid (pH6.5)
Cleaning solution II: 70% ethanol
Elutriant: water
Experimental procedure:
1, use experimental procedure purified genomic dna in embodiment 2: get 25,50,100,200,300,400 μ L anticoagulations respectively in 1.5ml centrifuge tube, add 750 μ L cracking adsorption liquid vibrations and be mixed evenly lysing cell, leave standstill 5min; Add 1mg magnetic microsphere, gentleness shakes up 10min; Magnetic field absorption magnetic microsphere, abandons supernatant; 850 μ L washings I wash magnetic microsphere twice, and absorption magnetic microsphere, abandons supernatant; Wash 2 times by 850 μ L cleaning solution II, absorption magnetic microsphere, abandons supernatant, and room temperature places 5min volatilization ethanol; Add 100 μ L elutriants, after being mixed evenly, place 5min in room temperature; Magnetic field absorption magnetic microsphere, reclaims elutriant in centrifuge tube, carries out DNA content and purity detecting and nucleic acid electrophoresis and detects.
2, after purifying, the PCR of sample detects: PCR reaction system is: mouse gene group DNA after 1ul purifying, 2X PCR master buffer, glyceraldehyde 3-phosphate dehydro-genase (GAPDH) the upstream and downstream primer of 200I, 50pM, finally adding sterilizing ultrapure water to final volume is 50uL.Upstream and downstream primer sequence is respectively 5 '-AGAGTCCATGCCATCACTGCC-3 ', 5 '-GCCTGCTTCACCACCTTCTTG-3 '.PCR reaction conditions is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, and 55 DEG C of annealing 30s, stretch 1min for 72 DEG C, circulate 30 times, and 72 DEG C extend 10min, get PCR primer and carry out electrophoresis detection after reaction terminates.
As shown in Figure 1, when using the blood of different volumes as raw material, test kit described in the utility model all can extract DNA with good yield to result; Use test kit of the present utility model, 1mg magnetic microsphere can in conjunction with the mouse gene group DNA up to 15ug.
Embodiment 5 Nucleic acid purification kits extracts the result of human blood genomic dna
Material:
Sample: the whole blood sample of EDTA anti-freezing process
Magnetic microsphere: terminal carboxyl(group) oxide/silica magnetic microsphere
Cracking is in conjunction with liquid: 4M Guanidinium hydrochloride, 2%Triton X-100,0.1%SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH5.5), 1mM Trisodium Citrate, 25% Virahol
Washings I:100mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris damping fluid (pH6.5)
Cleaning solution II: 70% ethanol
Elutriant: 1mM EDTA, 10mM Tris-HCl (pH8.0)
The theoretical initial value of gDNA (each white corpuscle is containing 6.6pgDNA) is recorded by leukocyte cell counting.
Purification step:
Use experimental procedure purified genomic dna in embodiment 2: get 50,100,200,300,400 μ L anticoagulations respectively in 1.5ml centrifuge tube, add 750 μ L cracking adsorption liquid vibrations and be mixed evenly lysing cell, leave standstill 5min; Add 1mg magnetic microsphere, gentleness shakes up 10min; Magnetic field absorption magnetic microsphere, abandons supernatant; 850 μ L washings I wash magnetic microsphere twice, and absorption magnetic microsphere, abandons supernatant; Wash 2 times by 850 μ L cleaning solution II, absorption magnetic microsphere, abandons supernatant, and room temperature places 5min volatilization ethanol; Add 100 μ L elutriants, after being mixed evenly, place 5min in room temperature; Magnetic field absorption magnetic microsphere, reclaims elutriant in centrifuge tube, carries out DNA content and purity detecting.
As shown in Figure 3, result shows the extraction effect of Nucleic acid purification kits purify DNA, uses the test kit of this utility model can extract genome DNA up to more than 90%.
Embodiment 6 nucleic acid extraction kit preserves the result of genomic dna for a long time
Sample: people's anticoagulation of EDTA anti-freezing process
Material:
Magnetic bead: terminal carboxyl(group) oxide/silica magnetic microsphere
Magnetic bead-cracking is in conjunction with liquid: preparation 4M Guanidinium hydrochloride, 2%Triton X-100,0.1%SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH7.5), 45% Virahol, to cumulative volume 1L, adds above-mentioned 1g terminal carboxyl(group) oxide/silica magnetic microsphere.
Washings I:100mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris damping fluid (pH7.5)
Cleaning solution II: 70% ethanol
Elutriant: TE elutriant (pH8.0)
Experimental procedure:
1, get 200 μ L anticoagulations in 1.5ml centrifuge tube, add the 750 μ L cracking adsorption liquids containing magnetic bead, put upside down mixed 5min, storage at room temperature;
2, different time sampling (0 day, 1 day, 3 days, 10 days, January, March, June, September, December), use rotary mixer mixing 20min, magnetic field absorption magnetic microsphere, abandons supernatant;
3,850 μ L washings I wash magnetic microsphere twice, and absorption magnetic microsphere, abandons supernatant;
4, wash 2 times by 850 μ L cleaning solution II, absorption magnetic microsphere, abandons supernatant, and room temperature places 5min volatilization ethanol;
5, add 100 μ L elutriants, after being mixed evenly, place 5min in room temperature; Magnetic field absorption magnetic microsphere, reclaims elutriant in centrifuge tube, carries out DNA content detection.
Table: DNA long-term stable experiment
5 parts of whole blood samples add cracking containing carboxyl magnetic bead in conjunction with liquid, after storage at room temperature, and different time sampling washing, 100 μ l elution DNA, use nanodrop2000 to carry out assay, nucleic acid concentration unit is ng/ μ l, and detected result is as follows:
From the above experimental results, use test kit processing sample of the present utility model, the shelf time of sample can reach 1 year.
The all documents mentioned at the utility model are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the utility model after having read above-mentioned teachings of the present utility model, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (8)
1. for extracting and preserve a test kit for nucleic acid, it is characterized in that, described test kit comprises:
One or more storage vessel, described storage vessel has one for storing magnetic bead kernel and cracking in conjunction with the inner pipe portion of liquid and a sampling tube part, and described inner pipe portion is positioned at sampling tube part.
2. test kit as claimed in claim 1, is characterized in that, described and storage vessel be selected from lower group: test tube, centrifuge tube, eppendorf pipe, culturing bottle; And
Described interior pipe bottom can be penetrated by plastic object, syringe or kapillary or puncture.
3. test kit as claimed in claim 1, it is characterized in that, described sampling tube also has a piercing portions, and described piercing portions for penetrate or puncture described in pipe, thus destroy the integrity of described interior pipe.
4. test kit as claimed in claim 1, it is characterized in that, the capacity of described storage vessel is 1.5-15ml.
5. test kit as claimed in claim 1, it is characterized in that, described storage vessel is made up of two portions:
Interior pipe (5), described interior pipe (5) is store magnetic bead kernel and cracking to be arranged in described pipe (5) in conjunction with liquid (3) and nucleic acid in conjunction with phase (3) in conjunction with the container of liquid, cracking; The bottom of described interior pipe (5) is film or rubber part;
Sampling tube (6), described sampling tube is sample collection tube with a scale, and bottom fixed film piercing portions.
6. test kit as claimed in claim 1, it is characterized in that, described test kit also comprises sample collecting device.
7. test kit as claimed in claim 6, it is characterized in that, described sample collecting device is blood-taking device.
8. test kit as claimed in claim 7, is characterized in that, described sample collecting device is the scraper or the saliva collector that gather Oral Mucosal Cells.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420212643.8U CN204369906U (en) | 2014-04-28 | 2014-04-28 | A kind of test kit extracting and preserve nucleic acid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420212643.8U CN204369906U (en) | 2014-04-28 | 2014-04-28 | A kind of test kit extracting and preserve nucleic acid |
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| CN204369906U true CN204369906U (en) | 2015-06-03 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105349530A (en) * | 2015-12-11 | 2016-02-24 | 杭州优思达生物技术有限公司 | New type nucleic acid detection method and detector tube |
| CN105670915A (en) * | 2016-04-05 | 2016-06-15 | 苏州英芮诚生化科技有限公司 | Magnetic bead presorting blood collection tube and pretreatment technology of blood DNA using same |
| CN106893721A (en) * | 2017-02-16 | 2017-06-27 | 广东氪生物技术有限公司 | A kind of kit screened for nucleic acid purification or fragment and its application method |
| CN113249433A (en) * | 2021-05-31 | 2021-08-13 | 安康（上海）生物科技有限公司 | Gene long-term storage method and kit |
| CN113430193A (en) * | 2021-06-30 | 2021-09-24 | 上海农林职业技术学院 | Nucleic acid collection kit and use method thereof |
| CN115261183A (en) * | 2022-05-31 | 2022-11-01 | 李治国 | Instant gene detection kit |
| CN115667508A (en) * | 2021-01-15 | 2023-01-31 | 株式会社Gene2Us | Buffer composition for nucleic acid separation for single-tube polymerase chain reaction in column mode and application thereof |
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2014
- 2014-04-28 CN CN201420212643.8U patent/CN204369906U/en not_active Expired - Lifetime
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349530A (en) * | 2015-12-11 | 2016-02-24 | 杭州优思达生物技术有限公司 | New type nucleic acid detection method and detector tube |
| CN105349530B (en) * | 2015-12-11 | 2018-02-27 | 杭州优思达生物技术有限公司 | A kind of nucleic acid detection method and detection pipe |
| CN105670915A (en) * | 2016-04-05 | 2016-06-15 | 苏州英芮诚生化科技有限公司 | Magnetic bead presorting blood collection tube and pretreatment technology of blood DNA using same |
| CN106893721A (en) * | 2017-02-16 | 2017-06-27 | 广东氪生物技术有限公司 | A kind of kit screened for nucleic acid purification or fragment and its application method |
| CN115667508A (en) * | 2021-01-15 | 2023-01-31 | 株式会社Gene2Us | Buffer composition for nucleic acid separation for single-tube polymerase chain reaction in column mode and application thereof |
| EP4130261A4 (en) * | 2021-01-15 | 2024-04-03 | Gene2us Corp. | Buffer composition for nucleic acid isolation for column-based one-tube pcr, and use thereof |
| CN113249433A (en) * | 2021-05-31 | 2021-08-13 | 安康（上海）生物科技有限公司 | Gene long-term storage method and kit |
| CN113249433B (en) * | 2021-05-31 | 2021-10-26 | 安康（上海）生物科技有限公司 | Gene long-term storage method and kit |
| CN113430193A (en) * | 2021-06-30 | 2021-09-24 | 上海农林职业技术学院 | Nucleic acid collection kit and use method thereof |
| CN115261183A (en) * | 2022-05-31 | 2022-11-01 | 李治国 | Instant gene detection kit |
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