CN115747051A - Nucleic acid extraction kit and extraction method - Google Patents
Nucleic acid extraction kit and extraction method Download PDFInfo
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- CN115747051A CN115747051A CN202310012867.8A CN202310012867A CN115747051A CN 115747051 A CN115747051 A CN 115747051A CN 202310012867 A CN202310012867 A CN 202310012867A CN 115747051 A CN115747051 A CN 115747051A
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- 238000000605 extraction Methods 0.000 title claims abstract description 95
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 79
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 79
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 79
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 175
- 238000007789 sealing Methods 0.000 claims abstract description 76
- 238000005406 washing Methods 0.000 claims abstract description 28
- 238000001179 sorption measurement Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 38
- 239000012528 membrane Substances 0.000 claims description 35
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 22
- 238000010828 elution Methods 0.000 claims description 19
- 238000004108 freeze drying Methods 0.000 claims description 17
- 238000009434 installation Methods 0.000 claims description 15
- 239000006166 lysate Substances 0.000 claims description 14
- 229910052742 iron Inorganic materials 0.000 claims description 11
- 230000009089 cytolysis Effects 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000003825 pressing Methods 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 210000001503 joint Anatomy 0.000 claims description 4
- 230000002093 peripheral effect Effects 0.000 claims description 3
- 238000002156 mixing Methods 0.000 abstract description 7
- 206010011409 Cross infection Diseases 0.000 abstract description 4
- 206010029803 Nosocomial infection Diseases 0.000 abstract description 4
- 239000002699 waste material Substances 0.000 description 11
- 239000011324 bead Substances 0.000 description 10
- 108091005804 Peptidases Proteins 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 6
- 235000019419 proteases Nutrition 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000003032 molecular docking Methods 0.000 description 5
- 238000005086 pumping Methods 0.000 description 4
- 238000004891 communication Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
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- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
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Abstract
The application discloses a nucleic acid extraction kit and an extraction method, and the nucleic acid extraction kit comprises a reagent tube, wherein a lower sealing film is arranged at the bottom end of the reagent tube; the reagent box comprises a box body base, a reagent mounting sleeve is arranged on the box body base, the reagent pipe is mounted in the reagent mounting sleeve, a plurality of independent and non-communicated reagent flow channels are arranged in the box body base, one end of each reagent flow channel is communicated with the corresponding reagent mounting sleeve, and a puncture piece is arranged in the reagent mounting sleeve; the device comprises a box body base, a plunger piece, a reagent mounting sleeve and a reagent flow passage, wherein an adsorption film is arranged in the plunger piece, the plunger piece is mounted on the box body base, and can be selectively communicated with one end of each reagent flow passage far away from the reagent mounting sleeve; the extraction tube, the box body base is seted up and is independently linked the extraction runner of extraction tube and plunger piece. The mixing, transferring, washing, eluting and distributing of the nucleic acid solution are all carried out in a sealed environment, and the risk of cross infection can be effectively reduced.
Description
Technical Field
The application relates to the technical field of nucleic acid detection, in particular to a nucleic acid extraction kit and an extraction method.
Background
In Vitro Diagnosis, i.e., IVD (In Vitro Diagnosis), refers to products and services for determining diseases or body functions by detecting human body samples (blood, body fluids, tissues, etc.) In addition to the human body to obtain clinical Diagnosis information.
At present, in the field of biotechnology, nucleic acid extraction is performed by using a traditional centrifugal column method or a magnetic bead method, and four steps of lysis, binding, rinsing, elution and the like are generally required. Firstly, components such as proteinase K, surfactant and the like are used for destroying cell structures and releasing nucleic acid into a solution; then, adding high-concentration salt, ethanol and other components to combine the nucleic acid with the adsorption film or magnetic beads; rinsing the adsorption film or the magnetic beads to remove inhibitors; finally, the nucleic acid on the adsorption membrane or magnetic beads is eluted. The steps of lysis, binding and rinsing are performed in different centrifuge tubes, and a solution containing nucleic acid or magnetic beads needs to be transferred.
In the related art, the solution or magnetic beads containing nucleic acid is transferred, and if the operation is careless, the possibility of cross contamination caused by splashing of liquid can be caused, and meanwhile, some infectious pathogens can cause infection of experimenters due to careless operation, so that certain risks exist.
Disclosure of Invention
In order to help improve the efficiency and safety of nucleic acid extraction, the application provides a nucleic acid extraction kit and an extraction method.
The nucleic acid extraction kit and the extraction method provided by the application adopt the following technical scheme:
first aspect of the invention
A nucleic acid extraction kit comprising:
the bottom end of the reagent tube is provided with a lower sealing film;
the reagent box comprises a box body base, a reagent installation sleeve is arranged on the box body base, the reagent pipe is installed in the reagent installation sleeve, a plurality of independent and non-communicated reagent flow channels are arranged in the box body base, one end of each reagent flow channel is communicated with the reagent installation sleeve, and a puncturing piece for puncturing a lower sealing film is arranged in the reagent installation sleeve;
the device comprises a box body base, a plunger piece, a reagent mounting sleeve and a reagent flow passage, wherein an adsorption film is arranged in the plunger piece, the plunger piece is mounted on the box body base, and can be selectively communicated with one end of each reagent flow passage far away from the reagent mounting sleeve;
the extraction tube is installed on the box body base, and the box body base is provided with an extraction flow channel capable of independently communicating the extraction tube with the plunger piece.
By adopting the technical scheme, the reagent solution is pre-stored in the reagent tube, when a test is required, a sample is added into one of the reagent tubes, the reagent tube is pressed, the lower sealing membrane of the reagent tube is penetrated through by the puncture piece, the reagent tube is communicated with the reagent flow channels, then the plunger pieces are controlled, the plunger pieces are sequentially communicated with all the reagent flow channels, the nucleic acid solution is transferred, mixed, cleaned and distributed by the plunger pieces, and finally the required nucleic acid solution is extracted by the extraction tube. The mixing, transferring, washing, eluting and distributing of the nucleic acid solution are all carried out in a sealed environment, so that the risk of cross infection can be effectively reduced; simultaneously, in this application waste liquid can directly get back original reagent pipe, please need not to set up extra waste liquid chamber and retrieve the waste liquid, can effectively practice thrift the cost.
Optionally, the plunger member comprises:
the plunger mounting sleeve is arranged on the box body base and is communicated with one end of the reagent flow channel;
the plunger body is rotatably connected in the plunger mounting sleeve and provided with a plunger through hole communicated with the single reagent flow channel or the extraction flow channel, the plunger through hole is communicated with the inner cavity of the plunger body, and the adsorption film is arranged in the inner cavity of the plunger body and close to the plunger through hole;
the plunger rod is connected with the inner cavity of the plunger body in a sliding manner along the axial direction of the plunger body.
By adopting the technical scheme, the plunger body is rotated, so that the plunger body can be independently communicated with the reagent flow channel or the extraction flow channel, then the plunger rod is used for pumping, so that the nucleic acid solution, the cleaning solution or the eluent respectively enter the plunger body, and the nucleic acid solution is sequentially cleaned and extracted. This application only needs to rotate the plunger body and can realize the intercommunication or the closure of each runner because only need control the plunger body, and the plunger body volume is less, consequently enables the intercommunication or the closure of each runner and easily controls more, and the leakproofness is higher, and more difficult mixing has ensured the nucleic acid extraction effect between the solution.
Optionally, the reagent tube comprises:
the sample tube is movably arranged in the reagent installation sleeve, a middle sealing film is arranged in the sample tube, an upper sealing film is arranged at one end, away from the lower sealing film, of the sample tube, a lysate cavity for storing lysate is formed between the upper sealing film and the middle sealing film, a freeze-drying ball cavity is formed between the middle sealing film and the lower sealing film, and freeze-drying balls are placed in the freeze-drying ball cavity;
the liquid washing pipe is movably arranged in the reagent mounting sleeve and can be communicated with the reagent flow channel;
and the elution pipe is movably arranged in the reagent installation sleeve and can be communicated with the reagent flow channel.
Through adopting above-mentioned technical scheme, reagent pipe can independently make, and reagent pipe and box body base are dismantled and assembled, and direct assembly pierces and can use during the use, can reduce equipment cost. The sample tube, the liquid washing tube and the elution tube are hermetically arranged in the reagent installation sleeve when not used, can be transported along with the box body base, reduces the transportation cost, can also be directly in the reagent installation sleeve when used subsequently, ensures the sealing performance when used, and further reduces the pollution risk.
Optionally, a sample tube sealing cover is detachably mounted at the end part of the sample tube close to the upper sealing membrane, and a puncture conical head for puncturing the upper sealing membrane is arranged on the inner side of the sample tube sealing cover.
By adopting the technical scheme, when the sample liquid needs to be added into the sample tube, the upper sealing film is firstly punctured by the puncturing conical head on the inner side of the sealing cover of the sample tube, and then the sample tube is sealed by the sealing cover of the sample tube after the sample liquid is added into the sample tube. The arrangement improves the convenience of personnel operation, and simultaneously can reduce the contact of the solution and the external environment as much as possible, thereby reducing the infection risk.
Optionally, a limiting block is fixed on the outer wall of the sample tube, and the limiting block abuts against the top end of the reagent installation sleeve.
By adopting the technical scheme, in the process of piercing the upper sealing film by the piercing cone head, the sample tube has the tendency of moving downwards, at the moment, in order to prevent the piercing member from piercing the lower sealing film to cause the solution to be mixed in advance, the scheme is provided with the limiting block, and only under the action of enough external force, the sample tube can continuously move downwards so that the lower sealing film is pierced by the piercing member.
Optionally, the nucleic acid extraction kit further comprises:
the box body is connected with the box body base, and the reagent tube is positioned in the box body;
the magnetic part is connected to the inner wall of the box body in a sliding mode, an iron ball is placed in the lysis liquid cavity, and the magnetic part and the iron ball are magnetically attracted.
By adopting the technical scheme, the magnetic part is utilized to drive the iron ball to reciprocate, and the iron ball can uniformly mix the sample, the lysate and the protease freeze-drying ball during reciprocating motion, so that the nucleic acid can be effectively released into the solution, and the effective adsorption of the subsequent adsorption film on the nucleic acid is ensured.
Optionally, a tube end housing is connected to the bottom end of the reagent tube, a butt-joint housing is installed in the reagent installation sleeve, the puncturing piece is installed in the butt-joint housing, and the tube end housing is movably and hermetically connected with the butt-joint housing.
Through adopting above-mentioned technical scheme, such setting up makes reagent pipe and reagent runner can sealed butt joint, reduces the flow of solution toward other places, also ensures simultaneously that the piece that punctures can effectively puncture lower membrane, improves the mobility of solution.
Optionally, the puncture member comprises a puncture vertebral body, a vertebral body channel is arranged on the peripheral side of the puncture vertebral body, and the vertebral body channel is communicated with the reagent flow channel.
By adopting the technical scheme, the punctured centrum pierces the lower sealing membrane and then extends into the reagent tube, and the solution can flow into the centrum channel and flow to the reagent channel. The arrangement improves the fluidity of the solution and ensures the effective transfer of the reagent.
Optionally, the cartridge base includes:
the reagent tube is arranged on the box body bottom plate;
the reagent flow channel and the extraction flow channel are arranged at the splicing position of the box body cover plate and the box body bottom plate.
By adopting the technical scheme, the processing difficulty can be reduced and the processing cost is reduced on the one hand, and on the other hand, the cleaning machine is convenient to clean subsequently, the convenience of maintenance is improved, and the maintenance cost is reduced.
Second aspect of the invention
A method for extracting nucleic acid, comprising the steps of:
s1, storing reagents required by reaction in a reagent tube in advance, and placing a kit in a matched control instrument after a user adds a sample into the reagent tube;
s2, pressing the reagent tube by using a matched control instrument to enable liquid in the reagent tube to flow into a corresponding reagent flow channel;
s3, controlling a plunger piece, wherein an inner cavity of the plunger piece is firstly communicated with a reagent flow channel with a sample, the plunger piece is controlled to pull, a mixed solution of the sample and the reagent is extracted into the plunger piece, the plunger piece is controlled to push, and a residual sample and the mixed solution of the reagent are pushed back into an original reagent tube;
s4, sequentially controlling the plunger piece to be communicated with the other reagent flow passages, controlling the plunger piece to pull and push, and cleaning and eluting the mixed solution;
s5, controlling the plunger piece to be communicated with the extraction flow channel, and pressing the extracting solution for eluting the nucleic acid into the extraction tube.
Through adopting above-mentioned technical scheme, mix, shift and distribute solution in sealed environment, reduced the risk of infection. Simultaneously, this simple structure, design benefit, reagent pipe can use with the detection kit fast assembly as independent finished product, and equipment cost is low, uses with supporting control instrument cooperation, can extract nucleic acid fast, improves nucleic acid extraction efficiency.
In summary, the present application includes at least one of the following beneficial technical effects:
1. the mixing, transferring, washing, eluting and distributing of the nucleic acid solution are all carried out in a sealed environment, so that the risk of cross infection can be effectively reduced; meanwhile, the waste liquid can be directly returned to the original reagent tube, and an extra waste liquid cavity is not required to be arranged to recover the waste liquid, so that the cost can be effectively saved.
2. This application only needs to rotate the plunger body and can realize the intercommunication or the closure of each runner because only need control the plunger body, and the plunger body volume is less, consequently enables the intercommunication or the closure of each runner and easily controls more, and the leakproofness is higher, and is difficult for mixing between the solution, has ensured the nucleic acid extraction effect.
3. The reagent tube can be used as an independent finished product and can be quickly assembled with the detection box, the equipment cost is low, the reagent tube is matched with a matched control instrument for use, the nucleic acid can be quickly extracted, and the nucleic acid extraction efficiency is improved.
Drawings
Fig. 1 is a schematic view of the overall structure of embodiment 1 of the present application.
Fig. 2 is a sectional view of the entire structure of embodiment 1 of the present application.
Fig. 3 is a schematic view of the overall structure of the cassette not shown in embodiment 1 of the present application.
Fig. 4 is a cross-sectional view showing mainly a piercing member in example 1 of the present application.
Fig. 5 is an enlarged schematic view of a portion a in fig. 2.
FIG. 6 is a schematic view showing the communication between the plug member and the extraction flow channel in example 1 of the present application.
FIG. 7 is a schematic view showing the communication between the elution tube and the reagent flow path in example 1 of the present application.
Fig. 8 is an exploded view of the base of the cartridge in example 1 of the present application.
Fig. 9 is an exploded view of the cartridge base in example 2 of the present application.
Description of reference numerals:
100. a box body base; 110. a reagent flow channel; 111. extracting a flow channel; 120. a reagent mounting sleeve; 130. butting the housings; 140. a piercing member; 141. a vertebral body passage; 150. a box body; 151. operating the through hole; 152. a magnetic member; 160. a box body bottom plate; 170. a box body cover plate;
200. a reagent tube;
210. a sample tube; 211. sealing the film; 212. sealing the middle film; 213. sealing the film; 214. a lysis fluid chamber; 215. a freeze-drying ball cavity; 216. freeze-drying the balls; 217. sealing the sample; 218. piercing the cone head; 219. a pipe end housing; 221. a limiting block; 222. an iron ball;
230. a liquid washing pipe;
240. an elution pipe;
300. a plunger member; 310. the plunger is provided with a sleeve; 320. a plunger body; 321. a plunger through hole; 330. a plunger rod;
400. an extraction tube; 401. small magnetic beads; 500. and (5) adsorbing the film.
Detailed Description
The present application is described in further detail below with reference to figures 1-9.
The embodiment of the application discloses a nucleic acid extraction kit.
Example 1
Referring to fig. 1 and 2, the nucleic acid extraction kit includes a cartridge base 100, a reagent tube 200 mounted on the cartridge base 100, a plunger 300 and an extraction tube 400, a plurality of independent and non-communicated reagent flow channels 110 are formed in the cartridge base 100, one end of each reagent flow channel 110 is communicated with the reagent tube 200, the other end of each reagent flow channel 110 is separately communicated with the plunger 300, an adsorption membrane 500 is mounted in the plunger 300, the extraction tube 400 is mounted on the bottom side of the cartridge base 100, an extraction flow channel 111 (not shown) is formed in the cartridge base 100, one end of the extraction flow channel 111 is communicated with the plunger 300, and the other end is communicated with the extraction tube 400.
The control plunger member 300 sequentially extracts the reagents in the reagent tubes 200 into the plunger member 300, sequentially adsorbs, cleans, elutes, and extracts the nucleic acid by the adsorption film 500, and finally presses the extracted nucleic acid into the extraction tube 400, thereby finally realizing the extraction of the nucleic acid. The nucleic acid extraction process is carried out in a sealed environment, so that the infection risk can be effectively reduced.
Referring to fig. 2 and 3, a reagent mounting sleeve 120 is fixed to the cartridge base 100, an inner cavity of the reagent mounting sleeve 120 communicates with the reagent flow path 110, and a reagent tube 200 is mounted in the reagent mounting sleeve 120.
A sample sealing cover 217 is screwed on the outer periphery of the end part of the sample tube 210 close to the upper sealing membrane 211, and a piercing cone 218 for piercing the upper sealing membrane 211 is fixed on the inner side of the sample sealing cover 217. The sample tube 210 is a single finished product, when a sample needs to be added into the sample tube 210, the sample sealing cover 217 is screwed into the top end of the sample tube 210, the upper sealing membrane 211 is punctured by the puncturing cone head 218, then the sample sealing cover 217 is screwed off, the sample is added into the sample tube 210, and finally the sample sealing cover 217 is screwed in.
In the present embodiment, the size of the tube end housing 219 is larger than that of the docking housing 130, so that the tube end housing 219 can be covered on the outer periphery of the docking housing 130 to realize the sealing connection between the two.
In another embodiment, the size of the tube-end housing 219 is smaller than the size of the docking housing 130, and the docking housing 130 covers the outer circumference of the tube-end housing 219.
Referring to fig. 4, the reagent mounting cannula 120 is provided therein with a puncturing member 140 for puncturing the lower sealing film 213 and the middle sealing film 212, in this embodiment, the puncturing member 140 includes a puncturing cone fixed at the center of the docking enclosure 130, a cone passage 141 is formed at the peripheral side of the puncturing cone, and the cone passage 141 is communicated with the reagent flow channel 110. When the sample tube 210 is pushed to move downward by an external force, the lower sealing membrane 213 and the middle sealing membrane 212 are punctured by the punctured cones, so that the sample, the lysate and the protease in the sample tube 210 are mixed, and the nucleic acid in the cells is released.
Referring to fig. 3 and 4, since the sample tube 210 is slidably inserted into the reagent mounting sleeve 120, in order to prevent the occurrence of the phenomenon that the lower sealing membrane 213 is punctured by puncturing the vertebral body due to the fact that a person carelessly pushes the sample tube 210 when screwing in the sample sealing cover 217, a limiting block 221 is fixed on the periphery of the sample tube 210, at this time, a limiting part is integrally formed at the top end of the reagent mounting sleeve 120, the limiting block 221 abuts against the limiting part, and under the condition that no external force is applied, the limiting block 221 can limit the sample tube 210 to move down, thereby improving the transportation safety and reducing the misoperation of the test; when nucleic acid is required to be extracted, sufficient external force is applied, the limiting block 221 is broken, and the sample tube 210 can move downwards smoothly.
Referring to fig. 4, after the sample, the lysate and the protease are mixed, in order to sufficiently release the nucleic acid in the cell, an iron ball 222 is further disposed in the lysate cavity 214, wherein a cartridge 150 is mounted on the cartridge base 100, the reagent tubes 200 are all located in the cartridge 150, an operation through hole 151 convenient for controlling the positions of the reagent tubes 200 is disposed at the top of the cartridge 150, a magnetic member 152 is slidably connected to a side wall of the cartridge 150 along an axial direction of the sample tube 210, and the magnetic member can be a magnet which is magnetically attracted to the iron ball 222. The magnetic member 152 drives the iron ball 222 to move up and down for 3 times within 10s, so that the sample, the lysate and the protease can be fully mixed.
After the sample, the lysate, and the protease are sufficiently mixed, the mixture is drawn into the plug 300 by the plug 300.
Referring to fig. 2 and 5, the plunger member 300 includes a plunger mounting sleeve 310, a plunger body 320 and a plunger rod 330, the plunger mounting sleeve 310 is vertically fixed on the top surface of the cartridge body base 100, the plunger body 320 is rotatably connected in the plunger mounting sleeve 310 by using an axis as a rotation axis, the plunger body 320 is provided with a plunger through hole 321 communicated with the single reagent flow channel 110 or the extraction flow channel 111, the plunger through hole 321 is communicated with an inner cavity of the plunger body 320, the adsorption film 500 is disposed near the plunger through hole 321, and one end of the plunger body 320 extends to the outside of the plunger mounting sleeve 310, so as to control the plunger body 320 and control the plunger body 320 to be communicated with different reagent flow channels 110.
Referring to fig. 2 and 3, two liquid washing tubes 230 are disposed on the cartridge body base 100, each liquid washing tube 230 contains 400ul of liquid washing solution, the liquid washing tubes 230 are all mounted in the reagent mounting sleeve 120, the bottom of the liquid washing tube 230 is also provided with a lower sealing film 213 (not shown), and the reagent mounting sleeve 120 is provided with a puncturing member 140 for puncturing the lower sealing film 213. The upper ends of the washing liquid tubes 230 are respectively provided with a sealing cover for sealing, the washing liquid tubes 230 are used as independent products and are stored in the reagent mounting sleeve 120, when nucleic acid is required to be washed, the liquid suction tubes are pressed downwards, the lower sealing film 213 is punctured by the puncturing pieces 140, the plunger pieces 300 are controlled, the plunger pieces 300 are communicated with the reagent flow channel 110 connected with the washing liquid tubes 230, the washing liquid is extracted by the plunger pieces 300, and waste liquid after the washing liquid is pushed back into the washing liquid tubes 230, so that the washing liquid process for the nucleic acid is realized.
The elution tube 240 is provided with one on the box body base 100, the elution tube 240 contains 200ul of eluent, the elution tube 240 is installed in the reagent installation sleeve 120, and the bottom of the liquid washing tube 230 is also provided with a lower sealing film 213. The structure of the liquid washing tube 230 is similar to that of the elution tube 240, and will not be described herein.
Referring to FIG. 6, after washing nucleic acids, it is necessary to extract nucleic acids into the extraction tube 400 using an eluent, and since the extracted nucleic acid solution does not easily flow into the extraction tube 400 due to the air pressure in the extraction tube 400, it is necessary to vacuumize the extraction tube 400 before extracting nucleic acids into the extraction tube 400.
The vacuum pumping process includes controlling the plunger member 300 to communicate the plunger member 300 with the extraction flow channel 111, pumping air in the extraction tube 400 by using the plunger member 300, then controlling the plunger member 300 to communicate the plunger member 300 with the reagent flow channel 110 connected with the sample tube 210 or the wash solution tube 230, and then pushing the air into the sample tube 210 or the wash solution tube 230 to achieve vacuum pumping of the extraction tube 400.
Referring to fig. 7, next, the plunger member 300 is controlled such that the plunger member 300 is communicated with the reagent flow path 110 to which the elution tube 240 is connected, and the eluate is extracted; referring to FIG. 6, the plug member 300 is then controlled to communicate with the extraction tube 400, and the nucleic acid-containing eluate is collectively pushed into the extraction tube 400, thereby extracting the nucleic acid.
In addition, still store freeze-drying reagent in the extraction tube 400 and placed little magnetic bead 401, after nucleic acid sample got into extraction tube 400, utilize external control instrument control magnet to drive little magnetic bead 401 and reciprocate, make nucleic acid sample and the abundant mixing of freeze-drying reagent to be favorable to the subsequent save of nucleic acid.
Referring to fig. 8, in order to further reduce the equipment cost, the cassette base 100 includes a cassette base plate 160 and a cassette cover plate 170, the reagent tube 200 is installed on the cassette base plate 160, the cassette base plate 160 and the cassette cover plate 170 are attached to each other, the reagent flow channel 110 and the extraction flow channel 111 are both opened on one side of the cassette base plate 160 close to the cassette cover plate 170, the extraction tube 400 is installed on one side of the cassette cover plate 170 away from the cassette base plate 160 and is communicated with the extraction flow channel 111, the cassette base plate 160 is installed in the cassette 150, and the cassette cover plate 170 is located outside the end of the cassette 150.
The implementation principle of the nucleic acid extraction kit in the embodiment of the application is as follows: the present application controls the plunger members 300 such that the plunger members 300 are sequentially communicated with the respective reagent flow paths 110 and the nucleic acid solution is mixed, transferred, washed and dispensed by the plunger members 300, and finally the desired nucleic acid solution is extracted by the extraction tube 400. Nucleic acid extraction is carried out in a sealed environment, so that the risk of cross infection can be effectively reduced; simultaneously, in this application waste liquid can directly get back original reagent pipe 200, please need not to set up extra waste liquid chamber and retrieve the waste liquid, can effectively practice thrift the cost.
Example 2
This embodiment is different from embodiment 1 in the position of the reagent flow path 110 and the extraction flow path 111. Referring to fig. 9, in the present embodiment, the reagent flow channel 110 and the extraction flow channel 111 are both disposed on one side of the cassette cover plate 170 close to the cassette bottom plate 160, and the cassette cover plate 170 is disposed with corresponding through holes to communicate with the reagent flow channel 110 and the extraction flow channel 111.
The purpose that sets up like this reduces to open a hole fluting on box body bottom plate 160, improves box body bottom plate 160 structural strength, ensures nucleic acid extraction stability.
The embodiment of the application also discloses a nucleic acid extraction method, which comprises the following steps:
s1, storing reagents required by reaction in a reagent tube 200 in advance, and placing the kit in a matched control instrument after a user adds a sample into the reagent tube 200.
Specifically, one sample tube 210 is provided, 200ul of lysis solution and lyophilized spheres 216 are pre-stored in the sample tube 210, the lysis solution and the lyophilized spheres 216 are separately packaged, two washing tubes 230 are provided, 400ul of washing solution is pre-stored in each washing tube 230, one elution tube 240 is provided, and 200ul of elution solution is pre-stored in the elution tube 240.
When a sample needs to be added, the sample sealing cover 217 is screwed into the end part of the sample tube 210, the upper sealing film 211 is punctured by the puncturing conical head 218, then the sample sealing cover 217 is unscrewed, the sample is added, and then the sample sealing cover 217 is screwed in, so that the sample tube 210 is sealed.
And S2, pressing the reagent tube 200 by using a matched control instrument to enable the liquid in the reagent tube 200 to flow into the corresponding reagent flow channel 110.
Specifically, the reagent tube 200 is pressed by a matching control instrument, the upper sealing film 211 and the middle sealing film 212 of the sample tube 210 are punctured by the puncturing conical head 218, so that the sample, the lysate and the lyophilized pellet 216 are mixed, and then the iron pellet 222 is driven by a magnet to move up and down, so that the sample, the lysate and the lyophilized pellet 216 are fully and uniformly mixed; the lower sealing film 213 of the liquid washing tube 230 and the elution tube 240 is punctured by the puncturing cone 218, so that the washing liquid and the elution liquid flow into the reagent flow channel 110.
S3, controlling the plunger piece 300, wherein the inner cavity of the plunger piece 300 is firstly communicated with the reagent flow channel 110 with the sample, the plunger piece 300 is pulled, the mixed liquid of the sample and the reagent is extracted into the plunger piece 300, the nucleic acid is adsorbed by utilizing the adsorption membrane 500, then the plunger piece 300 is controlled to push, and the redundant mixed liquid of the sample and the reagent is pushed back into the original reagent tube 200.
And S4, sequentially controlling the plug component 300 to be communicated with the rest of the reagent flow channels 110, controlling the plug component 300 to pull and push, and cleaning and eluting the mixed liquid.
Specifically, the control plug member 300 is connected to the reagent flow channel 110 connected to the wash solution pipe 230 in two steps, and the control plug member 300 is pulled and pushed so that the wash solution passes through the adsorption membrane 500 and washes the nucleic acid on the adsorption membrane 500; then, the plunger member 300 is rotated to communicate the inner cavity of the plunger member 300 with the extraction tube 400, the plunger member 300 is pulled to extract air in the extraction tube 400, then, the plunger member 300 is rotated to communicate the inner cavity of the plunger member 300 with the reagent flow channel 110 connected with the wash solution tube 230 or the sample tube 210, the plunger member 300 is pushed, and redundant air is discharged; the plunger element 300 is then rotated so that the inner lumen of the plunger element 300 is in communication with the reagent flow channel 110 to which the elution tubing 240 is connected, and the plunger element 300 is pulled to draw the elution fluid into the plunger element 300.
S5, controlling the plug component 300 to be communicated with the extraction flow channel 111, and pressing the eluted extracting solution into the extraction tube 400.
Specifically, rotatory plunger piece 300 makes plunger piece 300 inner chamber and extraction tube 400 say and is connected, and propelling movement plunger piece 300 is impressed the eluant that carries the nucleic acid in the plunger piece 300 in the extraction tube 400, and supporting control instrument control magnet of recycling drives the interior little magnetic bead 401 of extraction tube 400 and reciprocates, makes the abundant mixing of freeze-drying reactant in the eluant that carries the nucleic acid and the extraction tube 400.
The above are preferred embodiments of the present application, and the scope of protection of the present application is not limited thereto, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.
Claims (10)
1. A nucleic acid extraction kit, comprising:
the reagent tube (200), the bottom of the reagent tube (200) is provided with a lower sealing film (213);
the reagent box comprises a box body base (100), wherein a reagent mounting sleeve (120) is arranged on the box body base (100), the reagent tubes (200) are mounted in the reagent mounting sleeve (120), a plurality of independent and non-communicated reagent flow channels (110) are arranged in the box body base (100), one end of each reagent flow channel (110) is communicated with the corresponding reagent mounting sleeve (120), and a puncturing piece (140) for puncturing a lower sealing film (213) is arranged in the reagent mounting sleeve (120);
the adsorption membrane (500) is arranged in the column plug part (300), the column plug part (300) is installed on the box body base (100), and the column plug part (300) can be selectively communicated with one end, away from the reagent installation sleeve (120), of each reagent flow channel (110);
the extraction tube (400) is installed on the box body base (100), and the box body base (100) is provided with an extraction flow channel (111) capable of independently communicating the extraction tube (400) with the plunger piece (300).
2. The nucleic acid extraction kit according to claim 1, wherein the plug member (300) comprises:
a plunger mounting sleeve (310), the plunger mounting sleeve (310) being disposed on the cartridge base (100) and communicating with one end of the reagent flow channel (110);
the plunger body (320) is rotatably connected into the plunger mounting sleeve (310), a plunger through hole (321) communicated with the single reagent flow channel (110) or the extraction flow channel (111) is formed in the plunger body (320), the plunger through hole (321) is communicated with an inner cavity of the plunger body (320), and the adsorption film (500) is arranged in the inner cavity of the plunger body (320) and close to the plunger through hole (321);
a plunger rod (330), wherein the plunger rod (330) is connected with the inner cavity of the plunger body (320) in a sliding way along the axial direction of the plunger body (320).
3. The nucleic acid extraction kit according to claim 1, wherein the reagent tube (200) comprises:
the reagent mounting sleeve comprises a sample tube (210), the sample tube (210) is movably mounted in a reagent mounting sleeve (120), a middle sealing membrane (212) is arranged in the sample tube (210), an upper sealing membrane (211) is arranged at one end, far away from a lower sealing membrane (213), of the sample tube (210), a lysate cavity (214) for storing a lysate is formed between the upper sealing membrane (211) and the middle sealing membrane (212), a freeze-drying ball cavity (215) is formed between the middle sealing membrane (212) and the lower sealing membrane (213), and a freeze-drying ball (216) is placed in the freeze-drying ball cavity (215);
the liquid washing pipe (230), the said liquid washing pipe (230) is movably mounted in reagent mounting sleeve (120) and can communicate with reagent runner (110);
and the elution pipe (240) is movably arranged in the reagent installation sleeve (120) and can be communicated with the reagent flow channel (110).
4. The nucleic acid extraction kit according to claim 3, wherein: the end part, close to the upper sealing membrane (211), of the sample tube (210) is detachably provided with a sample sealing cover (217), and the inner side of the sample sealing cover (217) is provided with a piercing conical head (218) for piercing the upper sealing membrane (211).
5. The nucleic acid extraction kit according to claim 4, wherein: a limiting block (221) is fixed on the outer wall of the sample tube (210), and the limiting block (221) is abutted to the top end of the reagent installation sleeve (120).
6. The nucleic acid extraction kit according to claim 3, wherein the nucleic acid extraction kit further comprises:
the box body (150), the box body (150) is connected with the box body base (100), and the reagent tube (200) is positioned in the box body (150);
the magnetic part (152) is connected to the inner wall of the box body (150) in a sliding mode, an iron ball (222) is placed in the lysis solution cavity (214), and the magnetic part (152) and the iron ball (222) are magnetically attracted.
7. The nucleic acid extraction kit according to claim 1, wherein: the bottom end of the reagent tube (200) is connected with a tube end cover shell (219), a butt joint cover shell (130) is installed in the reagent installation sleeve (120), the puncture piece (140) is installed in the butt joint cover shell (130), and the tube end cover shell (219) is movably connected with the butt joint cover shell (130) in a sealing mode.
8. The nucleic acid extraction kit according to claim 1, wherein: the puncture piece (140) comprises a puncture vertebral body, a vertebral body channel (141) is arranged on the peripheral side edge of the puncture vertebral body, and the vertebral body channel (141) is communicated with the reagent flow channel (110).
9. The nucleic acid extraction kit according to claim 1, wherein the cartridge base (100) comprises:
a cassette base plate (160), the reagent tube (200) being mounted on the cassette base plate (160);
the reagent box comprises a box body cover plate (170), the box body cover plate (170) and the box body bottom plate (160) are spliced in a split mode, and the reagent flow channel (110) and the extraction flow channel (111) are arranged at the splicing position of the box body cover plate (170) and the box body bottom plate (160).
10. A method for extracting nucleic acid based on the nucleic acid extraction kit according to any one of claims 1 to 9, comprising the steps of:
s1, storing reagents required by reaction in a reagent tube (200) in advance, and placing a kit in a matched control instrument after a user adds a sample into the reagent tube (200);
s2, pressing the reagent tube (200) by using a matched control instrument to enable liquid in the reagent tube (200) to flow into the corresponding reagent flow channel (110);
s3, controlling a plunger piece (300), wherein the inner cavity of the plunger piece (300) is firstly communicated with a reagent flow channel (110) with a sample, the plunger piece (300) is pulled, a mixed solution of the sample and the reagent is extracted into the plunger piece (300), the plunger piece (300) is pushed, and a surplus mixed solution of the sample and the reagent is pushed back into the original reagent tube (200);
s4, sequentially controlling the plunger piece (300) to be communicated with the rest reagent flow channels (110), controlling the plunger piece (300) to pull and push, and cleaning and eluting the mixed liquid;
s5, controlling the plug piece (300) to be communicated with the extraction flow channel (111), and pressing the extracting solution for eluting the nucleic acid into the extraction tube (400).
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| CN115747051B (en) | 2023-12-05 |
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