CN117568138A - Quick extraction tool for nucleic acid and application method thereof - Google Patents
Quick extraction tool for nucleic acid and application method thereof Download PDFInfo
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- CN117568138A CN117568138A CN202311581144.6A CN202311581144A CN117568138A CN 117568138 A CN117568138 A CN 117568138A CN 202311581144 A CN202311581144 A CN 202311581144A CN 117568138 A CN117568138 A CN 117568138A
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 74
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 74
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 74
- 238000000605 extraction Methods 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000005406 washing Methods 0.000 claims abstract description 43
- 239000007788 liquid Substances 0.000 claims abstract description 38
- 238000005336 cracking Methods 0.000 claims abstract description 33
- 238000001179 sorption measurement Methods 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims description 48
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- 230000009089 cytolysis Effects 0.000 claims description 18
- 239000006166 lysate Substances 0.000 claims description 14
- 238000004321 preservation Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 10
- 238000000197 pyrolysis Methods 0.000 claims description 9
- 239000000377 silicon dioxide Substances 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 4
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 4
- YKTSYUJCYHOUJP-UHFFFAOYSA-N [O--].[Al+3].[Al+3].[O-][Si]([O-])([O-])[O-] Chemical compound [O--].[Al+3].[Al+3].[O-][Si]([O-])([O-])[O-] YKTSYUJCYHOUJP-UHFFFAOYSA-N 0.000 claims description 3
- -1 alkyl silica Chemical compound 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 239000011324 bead Substances 0.000 abstract description 7
- 230000007547 defect Effects 0.000 abstract description 2
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
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- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
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- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
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- 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
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
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- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention discloses a rapid extraction tool for nucleic acid and a use method thereof, and aims to solve the defect that the existing extraction method depends on an instrument. The invention comprises a cracking container, a washing container, an eluting container, a preserving pipe and a filter, wherein the cracking container is provided with a cracking liquid, the washing container is provided with a rinsing liquid, the eluting container is provided with an eluting liquid, the filter is provided with a connecting port for connecting the cracking container or the washing container or the eluting container, the filter head is arranged at the opposite end of the connecting port, the filter head is adapted to the pipe orifice of the preserving pipe, and an adsorption film for adsorbing nucleic acid is arranged in the filter. Under the environment without instruments, the extraction quality similar to that of the existing magnetic bead type extraction mode can be obtained by the method only by simple connection and bare-handed mechanical pressure.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, in particular to a rapid nucleic acid extraction tool and a using method thereof.
Background
Molecular diagnosis techniques such as RT-PCR and sequencing are detection techniques commonly used in laboratory medicine or clinical examination, and sample nucleic acid extraction is the first step of detection and is also one of key methods of molecular diagnosis techniques. Nucleic acid extraction involves two steps, cleavage, which is the process of freeing nucleic acids in a sample in a cleavage system, and purification, which is the process of thoroughly separating nucleic acids from other components in the cleavage system, such as proteins, polysaccharides, lipids, salts, and other impurities.
The common purification method is alcohol precipitation, and the nucleic acid is precipitated by using ethanol to separate the nucleic acid from the salt. And secondly, medium purification, wherein under certain specific conditions, a special solid phase medium can selectively adsorb nucleic acid, but not adsorb protein and salt, so that the separation of the nucleic acid from the protein and other impurities is realized.
The current common nucleic acid extraction methods include liquid phase extraction and solid phase extraction. The liquid phase extraction is to crack cells by physical or chemical method, and uses the chemical property difference of nucleic acid and other cell components such as protein, and add different solvents to repeatedly centrifuge, dissolve and precipitate, and is more commonly used as phenol-chloroform method and thermal cracking method. Phenol and chloroform are both organic solvents, and the principle of extracting nucleic acid is that phenol and chloroform have extremely strong denaturation of proteins bound to nucleic acid, but have no influence on nucleic acid itself. After centrifugation, an upper aqueous phase and a lower phenol/chloroform phase are formed, the denatured protein is dissolved in the phenol/chloroform phase or a gel layer is formed at the junction of the two phases, and the nucleic acid is easily soluble in water but insoluble in an organic solvent to remain in the aqueous phase. Finally, the nucleic acid is precipitated by ethanol to achieve the purpose of extraction. Adding cell lysis promoting chemical substances (SDS, tween 20, etc.) into the sample, and optionally adding proteinase K for digestion for a period of time, and heating at 95deg.C for about 10 min; the DNA in the supernatant can be used for PCR amplification by high-speed centrifugation.
The solid phase extraction mainly uses the interaction of solid phase nucleic acid adsorbent such as silicon dioxide, magnetic particles and the like with nucleic acid in different salt solutions, such as static electricity, affinity or hydrogen bond and the like, so as to achieve the purpose of separating nucleic acid. Mainly comprises the steps of cracking, combining, washing and eluting, and a membrane filtration column method and a magnetic bead method are commonly used.
However, the common nucleic acid extraction methods all require high-speed centrifugation or magnetic separation devices, and nucleic acid extraction is performed by means of instruments and equipment; the nucleic acid released by the method adopting the normal temperature release agent is easy to interfere an amplification system, and a nucleic acid detection project suitable for on-site diagnosis or home self-detection is limited by the nucleic acid release agent, so that the invention is significant in a nucleic acid rapid extraction device and an extraction method without instruments.
Disclosure of Invention
The invention overcomes the defect that the existing extraction method depends on instruments, and provides a rapid extraction tool for nucleic acid and a use method thereof, which can extract nucleic acid under the condition of no instrument and adapt to the current diagnosis and home self-checking environment.
In order to solve the technical problems, the invention adopts the following technical scheme:
the utility model provides a quick extraction tool of nucleic acid, includes pyrolysis container, washing container, elution container, save pipe and filter, has the lysate in the pyrolysis container, has the rinse solution in the washing container, has the eluent in the elution container, and the filter is equipped with the connector that is used for connecting pyrolysis container or washing container or elution container, and the filter head setting is at the opposite end of connector, the filter head is adapted with the mouth of pipe of save pipe, is equipped with the adsorption film that is used for adsorbing nucleic acid in the filter. The adsorption membrane is used for adsorbing nucleic acid, so that the separation of nucleic acid from the lysate, the rinsing solution and the separation of protein and other objects can be realized.
Preferably, the lysis vessel, the washing vessel and the elution vessel are flexible tubes. For flexible tubing, pressure can be generated by directly squeezing the outer wall of the container to force the container to deform, causing it to create pressure through the filter.
Preferably, the cleavage, wash or elution vessel is molded or injection molded by one of PE, PP, PA, PC, PPS, PET, PVC and PMMA.
Preferably, the material of the film is at least one of silica, silicon oxide, glass frit, alkyl silica, and aluminum silicate. The above materials can efficiently adsorb nucleic acid.
Preferably, the material of the membrane is silica gel. The silica gel material is a high-efficiency nucleic acid adsorption material.
Preferably, the lysis vessel, the washing vessel and the elution vessel are all pistons. When each container is of a non-flexible material, the piston structure is used to create a pressure that drives the movement of the liquid.
An instrumentalless rapid nucleic acid extraction method comprising a lysis vessel having therein a lysis solution, a washing vessel having therein a rinse solution, an elution vessel having therein an elution solution, a preservation tube, and a filter having therein a filter head, the extraction method comprising:
step one, adding a sample into a cracking container, enabling cracking liquid to fully contact the sample, and standing for a preset time;
step two, abutting the cracking container against a filter to discharge the cracking liquid through the filter;
step three, separating the filter from the cracking container and butting the filter with the washing container so that the rinsing liquid is discharged through the filter;
step four, separating the filter from the elution container and butting the filter with the elution container;
step five, connecting the filter head with the preservation pipe and maintaining for a set time, and then enabling the liquid in the elution container to enter the preservation pipe;
and step six, preserving or sampling and detecting the liquid in the preserving pipe.
The application carries out the processes of cracking, washing and eluting on the sample in sequence. The cells are destroyed by lysis, and nucleic acids, proteins, and the like are separated and purified by washing and eluting solutions. Through the foregoing steps, the sample is immersed in the lysate, the rinse solution, and the eluent one by one. In this process, the transfer of the sample is performed through a filter. The filter is used for adsorbing the cracked nucleic acid, and after separating from cells, the cracked nucleic acid is separated from each liquid one by one and separated from impurities such as protein. The nucleic acid rapid extraction of the equipment-free nucleic acid can be realized through the steps, and the specific effect of the application is similar to that of a magnetic bead method through experiments.
Preferably, the lysis vessel or wash vessel or elution vessel is inverted after docking the filter. By inversion, the nucleic acid to be extracted can be made less adherent to the wall surface of the container during the discharge, resulting in a practically gradual decrease of the nucleic acid during the transfer and affecting the final detection effect.
Preferably, step one achieves sufficient contact of the lysate and sample by squeezing the lysis vessel; in the second, third and fifth steps, the liquid is transferred by squeezing the cracking container, the washing container or the eluting container.
Preferably, the filter is provided with a connection port for connecting a lysis vessel or a washing vessel or an elution vessel, and the filter head is provided at the opposite end of the connection port, said filter head being adapted to the mouth of the holding tube. The preservation tube is connected with the filter port in a butt joint way, and the eluting solution is used for carrying nucleic acid to be separated from the filter and enter the preservation solution to obtain a preservation sample for detection.
Compared with the prior art, the invention has the beneficial effects that:
under the environment without instruments, the extraction quality similar to that of the existing magnetic bead type extraction mode can be obtained by the method only by simple connection and bare-handed mechanical pressure.
Drawings
FIG. 1 is a schematic illustration of the present invention;
FIG. 2 is a diagram showing the quality comparison of nucleic acid extraction by the magnetic bead method according to the present invention
In the figure:
a cracking vessel 1, a washing vessel 2, an elution vessel 3, a preservation tube 4, a filter 5, a filter head 6 and a connection port 7.
Detailed Description
The disclosure is further described below with reference to the drawings and examples.
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In the present disclosure, terms such as "fixedly coupled," "connected," and the like are to be construed broadly and refer to either a fixed connection or an integral or removable connection; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the terms in the disclosure may be determined according to circumstances, and should not be interpreted as limiting the disclosure, for relevant scientific research or a person skilled in the art.
Examples:
the utility model provides a quick extraction tool of nucleic acid, includes pyrolysis container 1, washing container 2, elution container 3, save pipe 4 and filter 5, has the lysate in the pyrolysis container 1, has the rinse solution in the washing container 2, has the eluent in the elution container 3, and filter 5 is equipped with the connector 7 that is used for connecting pyrolysis container 1 or washing container 2 or elution container 3, and filter head 6 sets up at the opposite end of connector 7, filter head 6 and the mouth of pipe adaptation of save pipe 4 are equipped with the adsorption film that is used for adsorbing the nucleic acid in the filter 5.
Wherein, the cracking vessel 1, the washing vessel 2, the elution vessel 3 and the preservation tube 4 can be of a split structure or can be assembled into a whole. The lysate, the rinse solution and the eluent can be purchased and then prepared, or can be directly preset in the lysis container 1, the washing container 2 and the elution container 3 for group use.
The cracking container 1, the washing container 2 and the elution container 3 are containers with the same shape and liquid outlets. The present embodiment uses a tube shape formed as a shape of a container. Corresponding to the shape of the cleavage vessel 1, washing vessel 2, elution vessel 3. The connection port 7 of the filter 5 is detachably connected with at least one of the lysis vessel 1, the washing vessel 2 and the elution vessel 3, and supports at least 3 times of plugging and unplugging.
The components of the lysate, rinse and eluate are common general knowledge in the art, and as previously described, the present application supports the provision of the lysate, rinse and eluate in a manner that is ex-situ from the formulation. Thus, the lysate, rinse and eluate of the present application do not require specific components, in other words, the components of the lysate, rinse and eluate are not the focus of protection of the present application, and it is within the ability of one skilled in the art to select the respective liquids.
The above-mentioned number of times of insertion and extraction means that when the container is connected with one of the cleavage vessel 1, the washing vessel 2 and the elution vessel 3, a certain sealing performance is maintained, so that the liquid in the container will not leak from the connection point of the container and the connection port 7 even when the container is in an inverted state.
The filter 5 has a corresponding adsorption membrane built therein for adsorbing nucleic acids. The adsorption films are stacked in a stacked manner so that the liquid can be discharged from the filter 5 through the filter 5. The material of the film is at least one of silicon dioxide, silicon oxide, glass powder, alkyl silicon dioxide and aluminum silicate. The above materials can efficiently adsorb nucleic acid. In some preferred embodiments, the material of the membrane is silica gel. The silica gel material is a high-efficiency nucleic acid adsorption material.
The term "removable" is used herein to refer broadly to a removable connection, and is not limited to a specific form of connection, and the present embodiment supports a corresponding connection manner including interference fit, threaded connection, etc. Corresponding sealing rings or other suitable sealing structures are provided at the connection port 7 in some embodiments to meet the tightness requirements, as required.
The filter head 6 is arranged at the opposite end of the connecting drain, and the filter openings are in some embodiments open openings, and the liquid in the cracking vessel 1, the washing vessel 2 and the elution vessel 3 flows out of the filter head 6 after entering from the connecting opening 7. The filter head 6 in this embodiment functions as a connection port for connecting the holding tube 4, and the filter head 6 is also detachably connected to the holding tube 4. In some preferred embodiments, the filter head 6 is sealingly connected to the holding tube 4.
Through the foregoing description, the filter 5 of the present embodiment is a ring-shaped structure having an adsorption film provided inside. The annular structure of the filter 5 is thick at one end and thin at the other end in order to adapt to the sizes of the holding tube 4, the cleavage vessel 1, the washing vessel 2 and the elution vessel 3.
The shape of the cracking container 1, the washing container 2 and the elution container 3 is a test tube with the same orifice size. The cracking container 1, the washing container 2 and the elution container 3 are all flexible pipes. In particular, the container is molded or injection molded by one of PE, PP, PA, PC, PPS, PET, PVC and PMMA.
In other embodiments, the cleavage vessel 1, the washing vessel 2, the elution vessel 3 are provided with a piston at their end remote from the opening.
An instrumental-free rapid nucleic acid extraction method, the extraction method comprising:
step one, adding a sample into a cracking container 1, enabling a cracking liquid to fully contact the sample and standing for a preset time;
step two, abutting the cracking container 1 against the filter 5 to discharge the cracking liquid through the filter 5;
step three, separating the filter 5 from the cracking container 1 and butting the cracking container with the washing container 2 to enable rinsing liquid to be discharged through the filter 5;
step four, separating the filter 5 from the elution container 3 and docking the elution container 3;
step five, after the filter head 6 is connected with the preservation pipe 4 and the set time is maintained, the liquid in the elution container 3 enters the preservation pipe 4;
and step six, preserving or sampling and detecting the liquid in the preserving pipe 4.
In the process, the liquid in the second and third steps is discharged and discarded through the safety channel.
The predetermined time in the first step is preferably 5 minutes.
The time elapsed set in the fifth step is preferably 2 minutes.
The lysis vessel 1 or the washing vessel 2 or the elution vessel 3 is inverted after being abutted against the filter 5. By inversion, the nucleic acid to be extracted can be made less adherent to the wall surface of the container during the discharge, resulting in a practically gradual decrease of the nucleic acid during the transfer and affecting the final detection effect.
Step one, realizing sufficient contact between the lysate and the sample by extruding the lysis vessel 1; in the second, third and fifth steps, the liquid is transferred by squeezing the cracking vessel 1, the washing vessel 2 or the elution vessel 3. The purpose of the flexible tube is that the pressure in the tube can be raised by squeezing and freehand to force the liquid through the filter 5, thereby avoiding the participation of mechanical devices. The cleavage vessel 1, the washing vessel 2 or the elution vessel 3 is molded or injection-molded by one of PE, PP, PA, PC, PPS, PET, PVC and PMMA.
In some embodiments, the cleavage vessel 1, the wash vessel 2, and the elution vessel 3 are all pistons. When each container is of a non-flexible material, the piston structure is used to create a pressure that drives the movement of the liquid. The piston comprises a shell (container) with an opening, an inner piston and a piston rod penetrating the shell and connected to the piston, and the piston rod is pushed by external force to drive the piston to push the inner liquid to move towards the opening.
The cracking container 1, the washing container 2 and the elution container 3 respectively cooperate with the inside cracking liquid, the rinsing liquid and the elution liquid to realize the functions that:
decomposing the cells by the lysate to form a mixed solution of disintegrated cells, and in the first and second steps, discharging the liquid to leave the tissue fragments of the disintegrated cells in the filter 5;
the washing container 2 is connected with the filter 5, the washing liquid and the disintegrated cells are fully mixed in an inverted mode, the soluble part is dissolved and then discharged through the filter 5, and at the moment, the nucleic acid is adsorbed in an adsorption film of the filter 5;
the nucleic acid is eluted by the eluent in the elution vessel 3, eluted from the adsorption membrane, and passed through the filter 5 into the storage tube 4.
The storage tube 4 is used for detection and storage as needed.
In this process, the transfer of the sample is performed through the filter 5. By the adsorption of the nucleic acid under lysis by the filter 5, the nucleic acid is separated from the cells, and then separated from the respective liquids one by one and from impurities such as proteins. The nucleic acid rapid extraction of the equipment-free nucleic acid can be realized through the steps, and the specific effect of the application is similar to that of a magnetic bead method through experiments.
Through experiments, compared with the mass of the extracted nucleic acid by the traditional magnetic bead method, a comparison chart as shown in figure 2 is obtained
The above-described embodiments are merely preferred embodiments of the present invention, and the present invention is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.
Claims (9)
1. The utility model provides a quick extraction tool of nucleic acid, its characterized in that includes pyrolysis container, washing container, elution container, save pipe and filter, has the lysate in the pyrolysis container, has the rinse solution in the washing container, has the eluent in the elution container, and the filter is equipped with the connector that is used for connecting pyrolysis container or washing container or elution container, and the filter head setting is at the opposite end of connector, the filter head is adapted with the mouth of pipe of save pipe, is equipped with the adsorption film that is used for adsorbing the nucleic acid in the filter.
2. The rapid nucleic acid extraction tool of claim 1, wherein the lysis vessel, wash vessel and elution vessel are flexible tubes.
3. The rapid nucleic acid extraction kit of claim 2, wherein the lysis vessel, wash vessel or elution vessel is molded or injection molded from one of PE, PP, PA, PC, PPS, PET, PVC and PMMA.
4. The rapid nucleic acid extraction tool according to claim 1, wherein the adsorption film is made of at least one of silica, glass frit, alkyl silica, and aluminum silicate.
5. The rapid nucleic acid extraction tool according to claim 4, wherein the adsorption membrane is made of silica gel.
6. The rapid nucleic acid extraction tool of claim 2, wherein the lysis vessel, wash vessel and elution vessel are all pistons.
7. An instrumental-free rapid nucleic acid extraction method comprising the rapid nucleic acid extraction tool of any one of claims 1 to 6, the extraction method comprising:
step one, adding a sample into a cracking container, enabling cracking liquid to fully contact the sample, and standing for a preset time;
step two, abutting the cracking container against a filter to discharge the cracking liquid through the filter;
step three, separating the filter from the cracking container and butting the filter with the washing container so that the rinsing liquid is discharged through the filter;
step four, separating the filter from the elution container and butting the filter with the elution container;
step five, connecting the filter head with the preservation pipe and maintaining for a set time, and then enabling the liquid in the elution container to enter the preservation pipe;
and step six, preserving or sampling and detecting the liquid in the preserving pipe.
8. The method for rapid extraction of nucleic acid without instrument according to claim 7, wherein the lysis vessel or washing vessel or elution vessel is inverted after being abutted against the filter.
9. The method for rapid extraction of nucleic acid without instrument according to claim 7, wherein the step one is to achieve sufficient contact between the lysate and the sample by squeezing the lysis vessel; in the second, third and fifth steps, the liquid is transferred by squeezing the cracking container, the washing container or the eluting container.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311581144.6A CN117568138A (en) | 2023-11-24 | 2023-11-24 | Quick extraction tool for nucleic acid and application method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311581144.6A CN117568138A (en) | 2023-11-24 | 2023-11-24 | Quick extraction tool for nucleic acid and application method thereof |
Publications (1)
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