CN108456673B - Functionalized capillary for nucleic acid extraction and preparation method and application thereof - Google Patents
Functionalized capillary for nucleic acid extraction and preparation method and application thereof Download PDFInfo
- Publication number
- CN108456673B CN108456673B CN201710090355.8A CN201710090355A CN108456673B CN 108456673 B CN108456673 B CN 108456673B CN 201710090355 A CN201710090355 A CN 201710090355A CN 108456673 B CN108456673 B CN 108456673B
- Authority
- CN
- China
- Prior art keywords
- capillary
- nucleic acid
- solution
- capillary tube
- functionalized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 57
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 57
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 57
- 238000000605 extraction Methods 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 5
- 238000011065 in-situ storage Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 238000004140 cleaning Methods 0.000 claims description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 11
- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 4
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 4
- 230000005484 gravity Effects 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 8
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- NJSSICCENMLTKO-HRCBOCMUSA-N [(1r,2s,4r,5r)-3-hydroxy-4-(4-methylphenyl)sulfonyloxy-6,8-dioxabicyclo[3.2.1]octan-2-yl] 4-methylbenzenesulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)O[C@H]1C(O)[C@@H](OS(=O)(=O)C=2C=CC(C)=CC=2)[C@@H]2OC[C@H]1O2 NJSSICCENMLTKO-HRCBOCMUSA-N 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012418 validation experiment Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a functionalized capillary for nucleic acid extraction and a preparation method and application thereof, belonging to the technical field of biological detection. According to the functionalized capillary, polydiallyldimethylammonium chloride is marked on the inner surface of the capillary, so that the inner surface of the capillary is provided with a large number of positive charges, nucleic acid with electronegativity can be adsorbed, and the purpose of simply and rapidly separating and extracting the nucleic acid is achieved. The invention greatly simplifies the operation process of nucleic acid separation and extraction and shortens the time of nucleic acid separation and extraction. The functionalized capillary tube provided by the invention is used for separating and extracting nucleic acid, is not required to be assisted by additional equipment, is convenient to integrate, can realize automatic extraction of nucleic acid, and is simple to operate and high in efficiency. The separated nucleic acid can be directly amplified and detected in situ.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a functionalized capillary for nucleic acid extraction, and a preparation method and application thereof.
Background
The gene analysis technology plays an important role in the fields of medical care, food safety, environmental monitoring and the like. The separation and extraction of nucleic acid is the first key step in the whole gene analysis process. Nucleic acid is always combined with various proteins in cells, and the separation of the nucleic acid can separate the nucleic acid from biological macromolecular substances such as proteins, polysaccharides, fats and the like, so that the influence of the substances on the amplification and detection of downstream nucleic acid is eliminated, and the accuracy of gene analysis is ensured.
The current commercialized nucleic acid extraction methods mainly include phenol chloroform method and magnetic bead method. The methods need to depend on complex equipment such as a centrifuge, a magnetic frame and the like to work in a matching way, the operation steps are complicated, the used time is long, and some reagents have toxicity, so that the popularization and the application of the methods in clinic are limited. Therefore, there is a need to develop a nucleic acid separation and extraction device that is simple to operate, fast, and does not require additional equipment.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide a functionalized capillary for nucleic acid extraction. According to the functionalized capillary, the polydiallyldimethylammonium chloride is marked on the inner surface of the capillary, so that the inner surface of the capillary is provided with a large number of positive charges, nucleic acid with electronegativity can be adsorbed, and the purpose of simply and rapidly separating and extracting the nucleic acid is achieved.
Another object of the present invention is to provide a method for preparing the functionalized capillary for nucleic acid extraction.
Another object of the present invention is to provide the use of the above functionalized capillary for nucleic acid extraction.
It is still another object of the present invention to provide a kit containing a functionalized capillary for nucleic acid extraction.
The purpose of the invention is realized by the following technical scheme:
a functionalized capillary for extracting nucleic acid is characterized in that polydiallyldimethylammonium Chloride (PDDA) is marked on the inner surface of the capillary, so that the inner surface of the capillary is provided with a large amount of positive charges, and nucleic acid with electronegativity can be adsorbed.
The glass capillary contains a large amount of hydroxyl groups and has electronegativity; under alkaline conditions, a polydiallyldimethylammonium chloride solution with electropositivity flows through the interior of the capillary, and is adsorbed on the inner surface of the capillary under the action of charge adsorption, so that the inner surface of the capillary is provided with a large amount of positive charges. In this case, the capillary can be used for adsorptive separation of nucleic acids having electronegativity. The schematic diagram is shown in fig. 1.
The preparation method of the functionalized capillary for extracting the nucleic acid comprises the following steps:
(1) cleaning the interior of the capillary tube with hydrochloric acid;
(2) cleaning the interior of the capillary tube with deionized water;
(3) cleaning the interior of the capillary tube with NaOH solution;
(4) cleaning the interior of the capillary tube with deionized water;
(5) washing the interior of the capillary tube with a Tris-HCl solution;
(6) flowing a Poly (Diallyldimethylammonium Chloride, PDDA) solution through the interior of the capillary;
(7) storing the capillary tube at room temperature;
(8) cleaning the interior of the capillary tube with deionized water;
(9) placing the capillary tube in a nitrogen box for drying; obtaining the functionalized capillary for extracting nucleic acid.
The cleaning time from the step (1) to the step (3) and the steps (5) and (8) is 30-90 min, preferably 60 min;
the cleaning time in the step (4) is 30 min-3 h, preferably 2 h;
the flowing time in the step (6) is 30-90 min, preferably 60 min;
the storage time at room temperature in the step (7) is 10 hours or more, preferably 20 hours;
the drying condition in the step (9) is drying at 30-70 ℃ for 10min or more, preferably drying at 50 ℃ for 1 h;
the flow rate of the liquid in the steps (1) to (6) and (8) is 1. mu.L/min to 100. mu.L/min, preferably 10. mu.L/min. Here, when the flow rate is 10. mu.L/min, the above-mentioned washing time and PDDA flow time are optimum, and when the flow rate is decreased or increased, a prolonged time is required.
The concentration of the hydrochloric acid in the step (1) is 0.5M-5M, and preferably 1M;
the concentration of the NaOH solution in the step (3) is 0.5M-5M, and preferably 1M;
the concentration of the Tris-HCl solution in the step (5) is 1 mM-100 mM, and the concentration is preferably 20 mM;
the pH value of the Tris-HCl solution in the step (5) is 7.0-10.0, and is preferably 8.3.
The concentration of the poly (diallyldimethylammonium chloride) solution in the step (6) is 0.5-10% (w/v), preferably 2%;
the PH value of the solvent of the poly (diallyldimethylammonium chloride) solution in the step (6) is 7.0-10.0, preferably 8.3, and the solvent comprises 1 mM-100 mM Tris-HCl and 0.5M-5M NaCl; preferably, it comprises 20mM Tris-HCl and 1.5M NaCl.
The functionalized capillary for nucleic acid extraction is applied to nucleic acid extraction.
A kit for extracting nucleic acid comprises the functionalized capillary for extracting nucleic acid.
The kit further comprises a TE buffer solution.
The kit is used for simple and rapid nucleic acid extraction, and specifically comprises the following steps:
firstly, centrifuging a bacterial culture solution, removing a supernatant, and collecting thalli;
secondly, re-suspending the collected thalli by using a TE buffer solution, and fully and uniformly mixing;
placing the heavy suspension in a water bath, and heating;
fourthly, taking out and standing at room temperature;
carefully sucking the supernatant to at least 1/4 position in the tube by using the functionalized capillary for extracting nucleic acid, and inclining the capillary left and right to make the solution flow left and right in the capillary; preferably to position 2/3; when the suction range is up to the full suction, the liquid is not allowed to flow after the full suction, and the liquid may be left to stand for 20 seconds or more.
Sixthly, vertically placing the capillary tube, so that most of liquid flows out of the capillary tube under the action of gravity, and forcibly throwing out the residual small amount of liquid;
seventhly, repeating the steps one time;
the separated nucleic acid is adsorbed inside the capillary tube and may be used directly in-situ PCR amplification and detection.
The heating condition in the step (iii) is heating at 90-100 ℃ for 5-20 min, preferably heating at 95 ℃ for 10 min;
the standing time in the step (iv) is 1min or more, preferably 5 min; the purpose of this step is: in the high concentration bacterial condition, the broken cell wall and other components sufficiently precipitate to improve the purity of the extracted DNA, in the low concentration condition, this step can be omitted.
The time of the left-right flow in the fifth step is 20s or more, preferably 1 min.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention greatly simplifies the operation process of nucleic acid separation and extraction and shortens the time of nucleic acid separation and extraction.
(2) The device is used for separating and extracting nucleic acid, does not need additional equipment for assistance, and is simple to operate and high in efficiency.
(3) The device is convenient to integrate, and can realize automatic extraction of nucleic acid.
(4) The separated nucleic acid can be directly amplified and detected in situ.
Drawings
FIG. 1 is a schematic diagram of the basic principle of the present invention, i.e., a method for marking the inside of a capillary tube.
Fig. 2 is the results of a marker validation experiment inside a capillary.
FIG. 3 is the result of an experiment for extracting nucleic acid using a labeled capillary; wherein the nucleic acid is labeled with SYBER GREEN I dye after extraction.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The reagents used in the examples were purchased from New England Biotechnology, capillary from Western medical university and Vibrio Parahaemolyticus (Vibrio Parahaemolyticus) from institute of microorganisms, Guangdong province.
Example 1
1. Glass capillary internal surface marking
(1) The interior of the capillary was cleaned with 1M hydrochloric acid for 1 hour.
(2) The inside of the capillary was washed with deionized water for 1 hour.
(3) The inside of the capillary was washed with a 1M NaOH solution for 1 hour.
(4) The inside of the capillary was washed with deionized water for 2 hours.
(5) The interior of the capillary was washed with a Tris-HCl solution at a concentration of 20mM for 1 hour.
(6) A2% (w/v) solution of poly (diallyldimethylammonium chloride) was allowed to flow through the interior of the capillary for 1 hour.
(7) The capillary was stored at room temperature for 20 hours.
(8) The inside of the capillary was washed with deionized water for 1 hour.
(9) Placing the capillary tube in a nitrogen box at 50 ℃ for drying for 1 hour; obtaining the functionalized capillary for extracting nucleic acid.
The flow rate of the liquid in the steps (1) to (6) and (8) was 10. mu.L/min.
The pH of the Tris-HCl solution used in step (5) was 8.3.
The solvent used in step (6) was a polydiallyldimethylammonium chloride solution having a pH of 8.3, which included 20mM Tris-HCl and 1.5M NaCl.
2. Verification of inner surface modification result of capillary tube
(1) FITC dye with electronegativity is introduced into the labeled capillary, the capillary is tilted left and right, and the FITC solution flows left and right in the capillary for 2 minutes. The interior of the capillary was then rinsed with deionized water.
(2) FITC dye was passed into the interior of an unlabeled capillary tube in the same manner and washed with water, and the result was used as a control.
(3) The capillary was irradiated with 535nm light and the results were observed with a 475nm filter.
The results are shown in FIG. 2(a), where capillary number 1 is not labeled, nor is FITC dye applied; no. 2 capillary tube is not marked, and FITC dye is introduced; number 3 capillary labeled poly diallyldimethylammonium chloride and the FITC dye was applied.
(4) Mixing DNA with electronegativity with SYBER GREEN I dye, introducing into the labeled capillary, and inclining the capillary left and right to allow the mixed solution to flow in the capillary left and right for 2 minutes. The interior of the capillary was then rinsed with deionized water.
(5) In the same manner, a mixture of DNA and SYBER GREEN I dye was passed through the inside of an unlabeled capillary and washed with water, and the result was used as a control.
(6) The capillary was irradiated with 535nm light and the results were observed with a 475nm filter.
The experimental results are shown in fig. 2(b), wherein the capillary of number 1 is not labeled and the mixed solution is not introduced; number 2 capillary tubes are not marked, and mixed liquid is introduced; the capillary numbered 3 is marked with polydiallyldimethylammonium chloride and the mixed solution is introduced.
Experimental results show that polydiallyldimethylammonium chloride is successfully marked in the capillary, has a large amount of positive charges and can be used for separating DNA with electronegativity.
Example 2
1. Vibrio parahaemolyticus DNA extraction
(1) 1mL of overnight-cultured bacterial culture was added to a 1.5mL centrifuge tube, centrifuged at 10000rpm for 2min, and the supernatant was discarded to collect the cells.
(2) The collected cells were resuspended in 400mL of TE buffer and mixed well.
(3) Placing the heavy suspension in a water bath at 95 deg.C, and heating for 10 min.
(4) Taking out the centrifuge tube, and standing for 5min at room temperature.
(5) The supernatant was carefully drawn up by capillary tubing to a position 2/3 in the tube, and the capillary tubing was tilted left and right to allow the solution to flow left and right in the capillary tubing for 1 minute.
(6) The capillary tube is positioned vertically so that most of the liquid flows out of the capillary tube under the influence of gravity. And (4) forcibly throwing the residual small amount of liquid to a waste liquid pool.
(7) Repeating the steps (5) and (6) once.
(8) 20 μ L of SYBER GREEN I dye was drawn into the capillary and the capillary was tilted left and right to allow the dye to flow left and right in the capillary 10 times.
(9) The capillary was placed vertically so that most of the remaining solution flowed out of the capillary under gravity. And (4) forcibly throwing the residual small amount of liquid to a waste liquid pool.
(10) The capillary was irradiated with 535nm light and the results were observed with a 475nm filter.
(11) The steps (1) to (10) were repeated using water instead of the bacterial culture as a control experiment.
The results of the experiments are shown in FIG. 3, where 1 is an experiment for DNA extraction of a bacterial culture and 2 is a control experiment group. The experimental result proves that the method can be used for separating and extracting nucleic acid.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (11)
1. Use of a functionalized capillary for nucleic acid extraction in nucleic acid extraction, characterized in that: according to the functionalized capillary for extracting the nucleic acid, the polydiallyldimethylammonium chloride is marked on the inner surface of the capillary, so that the inner surface of the capillary is provided with a large number of positive charges, and the nucleic acid with electronegativity is adsorbed;
the preparation method of the functionalized capillary for extracting the nucleic acid comprises the following steps:
(1) cleaning the interior of the capillary tube with hydrochloric acid;
(2) cleaning the interior of the capillary tube with deionized water;
(3) cleaning the interior of the capillary tube with NaOH solution;
(4) cleaning the interior of the capillary tube with deionized water;
(5) washing the interior of the capillary tube with a Tris-HCl solution;
(6) flowing a polydiallyldimethylammonium chloride solution through the interior of the capillary; the concentration of the poly diallyl dimethyl ammonium chloride solution is 0.5-10%;
(7) storing the capillary tube at room temperature;
(8) cleaning the interior of the capillary tube with deionized water;
(9) placing the capillary tube in a nitrogen box for drying; obtaining a functionalized capillary for nucleic acid extraction;
the drying condition in the step (9) is drying at 30-70 ℃ for 10min or more.
2. Use according to claim 1, characterized in that:
the cleaning time from the step (1) to the step (3) and the steps (5) and (8) is 30-90 min;
the cleaning time in the step (4) is 30 min-3 h;
the flowing time in the step (6) is 30-90 min;
the storage time at room temperature in the step (7) is 10h or more.
3. Use according to claim 1, characterized in that:
the flow rate of the liquid in the steps (1) to (6) and (8) is 1-100 muL/min.
4. Use according to claim 1, characterized in that:
the concentration of the hydrochloric acid in the step (1) is 0.5-5M;
the concentration of the NaOH solution in the step (3) is 0.5M-5M;
the concentration of the Tris-HCl solution in the step (5) is 1 mM-100 mM;
the pH value of the Tris-HCl solution in the step (5) is 7.0-10.0.
5. Use according to claim 1, characterized in that:
the concentration of the poly (diallyldimethylammonium chloride) solution in the step (6) is 2%.
6. Use according to claim 1 or 5, characterized in that:
the PH value of the solvent of the poly (diallyldimethylammonium chloride) solution in the step (6) is 7.0-10.0, wherein the solvent comprises 1 mM-100 mM Tris-HCl and 0.5M-5M NaCl.
7. Use of a kit for nucleic acid extraction in nucleic acid extraction, characterized in that the kit comprises a functionalized capillary for nucleic acid extraction according to any one of claims 1 to 6.
8. A method for extracting nucleic acid using the kit of claim 7, comprising the steps of:
firstly, centrifuging a bacterial culture solution, removing a supernatant, and collecting thalli;
secondly, re-suspending the collected thalli by using a TE buffer solution, and fully and uniformly mixing;
placing the heavy suspension in a water bath, and heating;
fourthly, taking out and standing at room temperature;
fifthly, carefully sucking the supernatant to at least 1/4 position in the tube by using the functionalized capillary for nucleic acid extraction as described in claim 1, and inclining the capillary left and right to make the solution flow left and right in the capillary;
sixthly, vertically placing the capillary tube, so that most of liquid flows out of the capillary tube under the action of gravity, and forcibly throwing out the residual small amount of liquid;
seventhly, repeating the steps one time;
the separated nucleic acid is adsorbed inside the capillary tube and may be used directly in-situ PCR amplification and detection.
9. The method of claim 8, wherein:
and the heating condition in the step (III) is heating at 90-100 ℃ for 5-20 min.
10. The method of claim 8, wherein:
in the fifth step, the supernatant is sucked to the position 2/3 in the tube.
11. The method of claim 8, wherein:
the time of the left-right flow in the fifth step is 20s or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710090355.8A CN108456673B (en) | 2017-02-20 | 2017-02-20 | Functionalized capillary for nucleic acid extraction and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710090355.8A CN108456673B (en) | 2017-02-20 | 2017-02-20 | Functionalized capillary for nucleic acid extraction and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108456673A CN108456673A (en) | 2018-08-28 |
| CN108456673B true CN108456673B (en) | 2021-03-26 |
Family
ID=63229123
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710090355.8A Expired - Fee Related CN108456673B (en) | 2017-02-20 | 2017-02-20 | Functionalized capillary for nucleic acid extraction and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108456673B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468313B (en) * | 2019-01-03 | 2020-12-15 | 华南师范大学 | Light-controlled functionalized capillary tube for nucleic acid extraction and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196514A (en) * | 2007-12-28 | 2008-06-11 | 中国人民解放军第二军医大学 | Method for fixing cell in microchannel |
| CN103120864A (en) * | 2013-03-06 | 2013-05-29 | 苏州环球色谱有限责任公司 | Preparation method of graphene-modified capillary-column chromatography |
| CN104814036A (en) * | 2015-03-27 | 2015-08-05 | 华南师范大学 | Poly(diallyldimethylammonium chloride) modified magnetic particle, preparation method thereof, and application thereof in bacterium removal |
| CN104931628A (en) * | 2015-06-02 | 2015-09-23 | 华南师范大学 | Fe3O4-COOH magnetic nano-material modified open tubular column as well as preparation method and application thereof |
| CN106198780A (en) * | 2016-06-24 | 2016-12-07 | 华南师范大学 | Protein modified open tubular column and the application in monoclonal antibody charge isomer separates |
-
2017
- 2017-02-20 CN CN201710090355.8A patent/CN108456673B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196514A (en) * | 2007-12-28 | 2008-06-11 | 中国人民解放军第二军医大学 | Method for fixing cell in microchannel |
| CN103120864A (en) * | 2013-03-06 | 2013-05-29 | 苏州环球色谱有限责任公司 | Preparation method of graphene-modified capillary-column chromatography |
| CN104814036A (en) * | 2015-03-27 | 2015-08-05 | 华南师范大学 | Poly(diallyldimethylammonium chloride) modified magnetic particle, preparation method thereof, and application thereof in bacterium removal |
| CN104931628A (en) * | 2015-06-02 | 2015-09-23 | 华南师范大学 | Fe3O4-COOH magnetic nano-material modified open tubular column as well as preparation method and application thereof |
| CN106198780A (en) * | 2016-06-24 | 2016-12-07 | 华南师范大学 | Protein modified open tubular column and the application in monoclonal antibody charge isomer separates |
Non-Patent Citations (2)
| Title |
|---|
| 聚二烯丙基二甲基氯化铵与核酸相互作用的共振光散射光谱及分析应用;周颖琳等;《分析化学研究报告》;20040205;第32卷(第2期);第169-173页 * |
| 聚电解质结构及盐浓度对基于DNA和聚阳离子的层层组装型大孔薄膜结构的影响;于永江等;《高等学校化学学报》;20131110;第34卷(第11期);第2644-2649页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108456673A (en) | 2018-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103789198B (en) | Full automatic instrument for extracting nucleic acid | |
| CN103756871B (en) | Single sample nucleic acid enclosed extractor | |
| CN106124282B (en) | A kind of method of lamination centrifugal filtration separation and Extraction excretion body | |
| CN105296327A (en) | Nucleic acid extraction device and extraction method thereof | |
| CN102242115A (en) | Kit for extracting bacterial plasmid DNA (deoxyribonucleic acid) by magnetic beads, and extraction method thereof | |
| JP2016502862A5 (en) | ||
| CN106999928A (en) | Nucleic acid purification box | |
| CN103820431A (en) | Nucleic acid extraction and purification method based on nanometer magnetic beads and kit | |
| CN103215253A (en) | Reagent kit for extracting virus DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) by using paramagnetic particle method and use method of reagent kit | |
| CN101501503A (en) | Device for processing samples | |
| JP2010528643A5 (en) | ||
| CN108076644A (en) | The apparatus and method of automatic nucleic acid extraction | |
| CN108456673B (en) | Functionalized capillary for nucleic acid extraction and preparation method and application thereof | |
| KR102417478B1 (en) | Syringe nucleic acid extraction apparatus, nucleic acid extraction kit and extraction method therewith | |
| CN1369003A (en) | Detection of micro-organisms | |
| CN203715614U (en) | Closed extraction apparatus for single sample nucleic acid | |
| CN203513676U (en) | Extraction device of bacteria DNA (Desoxvribose Nucleic Acid) in enrichment broth | |
| CN105021835A (en) | Magnetically driven biological sample treatment device and treatment method thereof | |
| CN109468313B (en) | Light-controlled functionalized capillary tube for nucleic acid extraction and preparation method and application thereof | |
| CN103243090A (en) | Method for extracting DNA (deoxyribonucleic acid) of insects | |
| JP2010158190A (en) | Method and apparatus for collecting nucleic acid | |
| CN100537591C (en) | Method for purifying small fragment DNA in gel by grinding method and use thereof | |
| CN103146569B (en) | Centrifugal tube structure for extracting DNA (deoxyribonucleic acid) by adopting silicon bead method | |
| CN104130941B (en) | The centrifuge shield structure of separation and Extraction DNA | |
| US20210130807A1 (en) | Kit, system, and method for nucleic acid extraction from samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210326 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |