CN115926961A - Small card box integrating nucleic acid extraction, amplification and detection and nucleic acid extraction method - Google Patents
Small card box integrating nucleic acid extraction, amplification and detection and nucleic acid extraction method Download PDFInfo
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- CN115926961A CN115926961A CN202211258530.7A CN202211258530A CN115926961A CN 115926961 A CN115926961 A CN 115926961A CN 202211258530 A CN202211258530 A CN 202211258530A CN 115926961 A CN115926961 A CN 115926961A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a small card box integrating nucleic acid extraction, amplification and detection, which comprises a closed card box consisting of a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell comprises two centrosymmetric spiral tracks; two symmetrical vertical guide rails are arranged on two sides of the sleeve wall; the liquid taking system comprises a suction ball, a ventilation hose, a suspender and a liquid suction pipe which are sequentially connected to form a communicated air passage, the sleeve knob is hollow and is used for accommodating the suction ball, two protrusions are arranged at two ends of the top end of the suspender, and the protrusions penetrate through a guide rail on the sleeve wall and are embedded into a spiral track on the inner wall of the shell; the device also comprises a reagent bin which is arranged at the lower half part of the shell; the base is connected with the bottom of the shell in a sealing mode. The invention provides a small card box integrating nucleic acid extraction, amplification and detection, which can be used for completing the whole processes of nucleic acid extraction, target sequence amplification, detection and the like of a sample, and greatly reduces the requirements on detection places and technicians.
Description
Technical Field
The invention belongs to the field of nucleic acid detection, and relates to a small card box integrating nucleic acid extraction, amplification and detection.
Background
The nucleic acid detection has wide application in the modern biomedical field, and the nucleic acid detection process comprises the main steps of extracting and purifying a nucleic acid sample, amplifying a target sequence, generating a detection signal and the like. In the conventional detection technology, the steps need to be finished in independent operation spaces respectively, and the requirements on detection sites and personnel are high. Meanwhile, when a plurality of samples are detected, the nucleic acid extraction and amplification steps adopt a parallel processing mode, cross contamination among different samples, dissipation of nucleic acid amplification products and the like are easily caused, and deviation of detection results is caused. The POCT (Point of Care Testing) instant detection technology provides a new means for solving the problems. At present, some devices integrating nucleic acid extraction, amplification and detection have been developed, such as GeneXpert molecular diagnostic system of seepei company, and such integrated detection system integrates the processes of nucleic acid extraction, amplification and detection of samples into a small closed space, so as to effectively avoid cross contamination between samples and dissipation of amplification products, greatly simplify the detection process, and improve the accuracy of detection results. However, the existing integrated detection system has complex structure, high cost and large equipment volume, and is difficult to meet the nucleic acid detection requirements of primary medical institutions, field operation environments and the like.
Disclosure of Invention
The invention discloses a small card box integrating nucleic acid extraction, amplification and detection, which integrates reaction reagents for nucleic acid extraction, amplification and detection and an operation control component into a small card box.
The purpose of the invention is realized by the following technical scheme:
a small-sized card box integrating nucleic acid extraction, amplification and detection is characterized in that: the closed card box comprises a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell comprises two centrosymmetric spiral tracks;
the sleeve comprises an integrated sleeve knob and a sleeve wall, the sleeve wall extends into the shell, the sleeve knob is embedded at the upper end of the shell, two symmetrical vertical guide rails are arranged on two sides of the sleeve wall, and the joint of the shell and the sleeve is kept sealed;
the liquid taking system comprises a suction ball, a vent hose, a hanging rod and a liquid suction pipe which are connected in sequence, the sleeve knob is hollow and is used for accommodating the suction ball, two bulges are arranged at two ends of the top end of the hanging rod and penetrate through a guide rail on the sleeve wall to be embedded into a spiral track on the inner wall of the shell;
the base is connected with the bottom of the shell in a sealing mode.
Preferably, the device also comprises a reagent bin which is arranged at the lower half part of the shell; the reagent storehouse includes reagent storehouse shell, a plurality of reagent pond and cotton absorbs water, the reagent storehouse shell is cylindrical, sets up the layering baffle at the inner wall of reagent storehouse shell for it is spacing in each reagent pond, the cotton packing that absorbs water is between each layer reagent pond and the clearance of reagent pond and reagent storehouse shell, the reagent pond is equipped with the reagent, and its top surface and bottom surface are the film that easily punctures.
Preferably, the pipette also comprises a magnet, wherein the magnet is positioned in the pipette tube and has a diameter larger than the pipette opening of the pipette tube.
Preferably, the shell is provided with a sample adding hole and a matched sample adding hole cover at a position between the reagent bin and the sleeve wall.
Preferably, the top of shell is equipped with the arch of the even setting in many places, and the bellied design is for when follow-up will block the card box and assemble the instrument in use, play limiting displacement to fixed shell prevents that the shell from rotating.
Preferably, the reagent cabin shell is formed by splicing two halves, and a clamping structure is arranged between the two halves of the reagent cabin shell.
Preferably, the reagent pool is provided with a plurality of reagent pools, wherein the reagent pools sequentially contain suspension of magnetic particles and cell lysis solution, cleaning solution, eluent and nucleic acid amplification reaction reagent from top to bottom.
The invention also discloses a nucleic acid detection method based on the small card box integrating nucleic acid extraction, amplification and detection, which is characterized by comprising the following steps:
(1) Puncturing the top surface film of the first layer of reagent pool through a sample adding hole on the card box shell, and adding a sample solution of nucleic acid to be extracted into the top surface film;
(2) Moving the pipette on the boom down to the first layer of reagent pool by rotating the sleeve; then sucking the solution in the first layer of reagent pool by a silica gel sucker at the top of the pressing/releasing sleeve, so that the magnetic particles combined with the sample nucleic acid are completely adsorbed to the surface of the magnet in the pipette;
(3) Continuing rotating the sleeve to make the tip of the pipette puncture the bottom film of the first layer of reagent pool; pressing the silica gel sucker to ensure that the liquid in the liquid suction pipe is completely absorbed by the absorbent cotton after being discharged;
(4) Continuing rotating the sleeve, enabling the pipette to sequentially enter the next layer or layers of reagent pools, cleaning the magnetic particles on the surface of the magnet by using the cleaning liquid in each reagent pool, and absorbing the cleaned waste liquid by absorbent cotton;
(5) Continuing rotating the sleeve to enable the pipette to enter the next layer of reagent pool, sucking eluent in the reagent pool, and eluting nucleic acid on the magnetic particles;
(6) Continuously rotating the sleeve, injecting the extracted nucleic acid into the reagent pool at the bottom layer, and mixing the nucleic acid with the nucleic acid amplification reagent in the reagent pool;
(7) And continuing rotating the sleeve, and injecting the mixed liquid into the base to perform amplification detection.
The invention has the following advantages:
the invention provides a small card box integrating nucleic acid extraction, amplification and detection, which can be used for completing the whole processes of nucleic acid extraction, target sequence amplification, detection and the like of a sample, thereby greatly reducing the requirements on detection places and technicians. In addition, the card box is disposable, and every sample that awaits measuring uses separate card box to detect, can effectively avoid the cross contamination between the different samples. In addition, the card box adopts a fully-closed design, so that the dissipation of nucleic acid amplification products can be effectively avoided, and false positive results can be prevented. In summary, the cartridge described can provide a simplified integrated nucleic acid detecting apparatus and method.
Drawings
FIG. 1 is a view showing the structure of a cartridge of the present invention, wherein (A) is an overall perspective view and (B) is an overall sectional view.
Figure 2 is a view of the housing of the cartridge of the present invention.
FIG. 3 is a view showing the structure of a cartridge sleeve according to the present invention, wherein (A) is a view showing the structure of a cartridge body, and (B) is a sectional view showing a complete cartridge sleeve.
FIG. 4 is a reagent cartridge structure diagram of the cartridge of the present invention, wherein (A) is a perspective view of a reagent cartridge main body, (B) and (C) are reagent cartridge housing structure diagrams, and (D) is a sectional view of the reagent cartridge.
FIG. 5 is a schematic representation of the cartridge of the present invention before and after operation, (A) before operation with a pipette in the upper part, and (B) after operation with the pipette tip moved into the base reaction tube.
Detailed Description
Example 1
The small-sized card box integrating nucleic acid extraction, amplification and detection is shown in figure 1 and comprises a tubular base 1, a cylindrical shell 2 and a sleeve 3, wherein the sleeve 3 consists of a sleeve knob 3-1 and a sleeve wall 3-2, the sleeve wall extends into the shell, and a sealing ring 20 is arranged between the shell and the top end of the sleeve wall, so that a sealing structure is formed between the shell and the sleeve. Other sealing arrangements may be used for the connection. The sleeve knob is embedded at the upper end of the shell, and meanwhile, the sleeve knob can rotate under the action of external force, so that the sleeve wall is driven to rotate. The junction of the tubular base 1, the cylindrical housing 2 and the sleeve 3 remains closed, forming a closed cavity. The top end of the sleeve 3 is provided with a silica gel suction ball 4 which is connected with a suspender 6 through a section of ventilation hose 5, and the lower end of the suspender 6 is connected with a liquid suction pipe 7. The silica gel suction ball 4, the vent hose 5, the suspender 6 and the liquid suction pipe 7 form a communicated air passage, and the liquid suction pipe 7 can suck liquid by pressing or releasing the silica gel suction ball 4. The lower part of the housing 2 contains a reagent chamber 9 for containing reagents required for nucleic acid extraction and purification. The tubular base 1 is used for containing nucleic acid amplification reagents.
The cylindrical housing 2 is specifically constructed as shown in fig. 2, and two protrusions 10 and 11 are provided on both sides of the top of the housing 2 for fixing the cartridge. The upper half of the inner wall of the housing 2 contains two centrosymmetric helical tracks 12-1 and 12-2 for limiting the movement path of the housing 2 and the sleeve 3 when they move relative to each other. The middle part of the shell 2 comprises a sample adding hole 13 and a sample adding hole cover 14 of the accessory thereof, which are used for adding a sample to be detected, and the sample adding hole 13 is sealed by the sample adding hole cover 14 after the sample is added, so that a closed cavity is formed inside the card box.
The specific construction of the sleeve 3 is shown in fig. 3, the lower part of the sleeve 3 is provided on both sides with two symmetrical vertical guides 15 and 16, and the boom 6 is provided on both sides of its top end with two projections which can be inserted through the guides 15 and 16 in the wall of the sleeve into the helical track 12 in the inner wall of the housing 2. When the liquid sucking device is used, the lower sleeve 3 is driven by external force to rotate relative to the shell 2, the hanging rod 6 vertically moves up and down along with the lower sleeve, and then the liquid sucking pipe 7 is driven to vertically move up and down and is used for sucking liquid at different positions. The length of the breather hose 5 needs to be longer than the vertical movement range of the pipette 7. At least one magnet 8 is arranged in the pipette 7, the magnet 8 having to be larger than the diameter of the tip of the pipette 7. When in use, the magnetic particles for nucleic acid extraction are adsorbed on the magnet 8 and are not discharged with the liquid.
The specific structure of the reagent chamber 9 is shown in fig. 4, and comprises a reagent chamber housing 17, a reagent pool 18 and absorbent cotton 19. The reagent chamber shell is formed by splicing two halves, a clamping structure is arranged between the two halves of the reagent chamber shells 17-1 and 17-2 so as to be convenient for loading the reagent chambers and the absorbent cotton, and the inner wall of the reagent chamber shell 17 is provided with a layered partition plate for limiting the position of each layer of the reagent chambers 18. When the device is used, reagents for nucleic acid extraction, amplification and the like are prestored in each reagent pool 18-1 to 18-6, the top surface and the bottom surface of each reagent pool are sealed by a thin film, and then the reagent pools are respectively installed in the reagent bin shell 17 according to the extraction process, at least one layer of absorbent cotton 19-1 to 19-5 is pre-installed in gaps between each layer of reagent pool and between each reagent pool and the reagent bin shell 17 and used for collecting waste liquid, and the thin film can be common aluminum foil, waterproof paper film or other materials which are easy to puncture.
When in use, an operator punctures the top film of the reagent pool 18-1 through the sample adding hole 13 on the shell of the card box, adds samples comprising nasopharyngeal swab leaching liquid, blood, sputum, urine, tissues, excrement and the like, and covers the sample adding hole cover 14; rotating the sleeve 3 to make the pipette enter each reagent pool in turn to suck and discharge liquid, so as to complete the extraction and purification of sample nucleic acid and the mixing of amplification reaction reagents.
Example 2
A method for detecting nucleic acids using the cartridge, comprising the steps of:
(1) Puncturing the aluminum foil on the top surface of the reagent pool 18-1 at the position of a sample adding hole 13 on the shell, adding a sample to be detected, and performing cell lysis, nucleic acid release and nucleic acid adsorption by magnetic particles;
(2) Pressing the silica gel sucker 4 to make the pipette 7 in a state of liquid to be sucked;
(3) Rotating the sleeve 3 to move the pipette 7 downward until the tip is immersed in the liquid in the reagent reservoir 1;
(4) Releasing the silica gel sucker 4, sucking the magnetic particle suspension in the reagent pool 18-1, and adsorbing the magnetic particles on the surface of the magnet 8;
(5) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the bottom surface of the reagent pool 18-1;
(6) Pressing the silica gel sucker 4 to discharge the liquid in the liquid suction pipe 7, and absorbing the liquid by the absorbent cotton;
(7) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to pierce the aluminum foil on the top surface of the reagent pool 18-2, wherein the tip of the pipette is immersed in the cleaning solution 1 in the reagent pool 18-2;
(8) Repeatedly pressing/releasing the silica gel sucker for more than 4 times, repeatedly sucking the cleaning liquid 1 in the reagent pool 18-2 by the pipette, and cleaning the magnetic particles on the surface of the magnet 8;
(9) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the bottom surface of the reagent pool 18-2;
(10) Pressing the silica gel sucker 4 to discharge the liquid in the liquid suction pipe 7, and absorbing the liquid by the absorbent cotton;
(11) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the top surface of the reagent pool 18-3;
(12) Repeatedly pressing/releasing the silica gel sucker for more than 4 times, repeatedly sucking the cleaning liquid 2 in the reagent pool 18-3 by a pipette, and cleaning magnetic particles on the surface of the magnet;
(13) Continuing to rotate the sleeve 3, and using the tip of the pipette to puncture the aluminum foil on the bottom surface of the reagent pool 18-3;
(14) Pressing the silica gel sucker 4 to discharge the liquid in the liquid suction pipe 7, and absorbing the liquid by the absorbent cotton;
(15) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the top surface of the reagent pool 18-4;
(16) Repeatedly pressing/releasing the silica gel sucker for more than 4 times, repeatedly sucking the cleaning liquid 2 in the reagent pool 18-4 by a pipette, and cleaning magnetic particles on the surface of the magnet;
(17) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the bottom surface of the reagent pool 18-4;
(18) Pressing the silica gel sucker 4 to discharge the liquid in the liquid suction pipe 7, and absorbing the liquid by the absorbent cotton;
(19) Repeatedly pressing/releasing the silica gel sucker 4 for more than three times, blowing air by the pipette 7 to volatilize the residual liquid on the surface of the magnetic particle, and keeping the silica gel sucker 4 in a pressing state;
(20) Continuing to rotate the sleeve 3, using the tip of the pipette 7 to puncture the aluminum foil on the top surface of the reagent pool 18-5, and immersing the tip of the pipette 7 in the eluent in the reagent pool 18-5;
(21) Releasing the silica gel suction head 4, sucking the eluent in the reagent pool 18-5, and standing for at least 2 minutes;
(22) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the top surface of the reagent pool 18-6;
(23) Pressing the silica gel tip 4 to discharge the liquid in the pipette 7 and dissolve the nucleic acid amplification reaction lyophilized reagent in the reagent pool 18-6;
(24) Repeatedly pressing/releasing the silica gel suction head for more than 4 times, and uniformly mixing the liquid in the reagent pool 18-6 and then completely sucking the liquid;
(25) Continuing to rotate the sleeve 3, and using the tip of the pipette 7 to puncture the aluminum foil on the bottom surface of the reagent pool 18-6;
(26) The silica gel tip 4 is pressed to discharge the liquid in the pipette tube 7 into the reaction tube in the base 1, and the nucleic acid amplification reaction is performed.
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 (9)
1. A small-sized card box integrating nucleic acid extraction, amplification and detection is characterized in that: the closed card box comprises a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell comprises two centrosymmetric spiral tracks;
the sleeve comprises an integrated sleeve knob and a sleeve wall, the sleeve wall extends into the shell, the sleeve knob is clamped at the upper end of the shell, two symmetrical vertical guide rails are arranged on two sides of the sleeve wall, and the joint of the shell and the sleeve is kept sealed;
the liquid taking system comprises a suction ball, a ventilation hose, a hanging rod and a liquid suction pipe which are sequentially connected to form a communicated air passage, the sleeve knob is hollow and is used for accommodating the suction ball, and two bulges are arranged at two ends of the top end of the hanging rod and penetrate through a guide rail on the sleeve wall to be embedded into a spiral track on the inner wall of the shell;
the base is connected with the bottom of the shell in a sealing mode.
2. The small cartridge integrated with nucleic acid extraction, amplification and detection according to claim 1, wherein: still include the reagent storehouse, the reagent storehouse sets up in the shell the latter half, the reagent storehouse includes reagent storehouse shell, a plurality of reagent pond and the cotton that absorbs water, reagent storehouse shell is cylindrical, sets up the layering baffle at the inner wall of reagent storehouse shell for each reagent pond is spacing, the cotton packing that absorbs water is between each layer reagent pond and the clearance of reagent pond and reagent storehouse shell, the reagent pond is equipped with reagent, and its top surface and bottom surface are the film of easily puncturing.
3. The cartridge according to claim 1 or 2, wherein the cartridge comprises: the pipette also comprises a magnet, wherein the magnet is positioned in the pipette tube, and the diameter of the magnet is larger than that of the pipette opening.
4. The nucleic acid extraction, amplification and detection integrated miniature cartridge of claim 3 wherein: and a sample adding hole and a matched sample adding hole cover are arranged at the position of the shell between the reagent bin and the sleeve wall.
5. The nucleic acid extraction, amplification and detection integrated miniature cartridge of claim 3 wherein: the top of shell is equipped with the arch of many places uniform settings.
6. The integrated nucleic acid extraction, amplification and detection miniature cartridge of claim 3, wherein: the reagent cabin shell is formed by splicing two halves, and a clamping structure is arranged between the two halves of the reagent cabin shell.
7. The nucleic acid extraction, amplification and detection integrated miniature cartridge of claim 3 wherein: the suction ball is a silica gel suction ball.
8. The nucleic acid extraction, amplification and detection integrated miniature cartridge of claim 3 wherein: the reagent pool is provided with a plurality of suspension, cleaning liquid, eluent and nucleic acid amplification reaction reagent which are sequentially filled with magnetic particles and cell lysate from top to bottom.
9. A method for detecting nucleic acid based on the miniaturized cartridge integrated with nucleic acid extraction, amplification and detection as set forth in any one of claims 1 to 8, comprising the steps of:
(1) Puncturing the top surface film of the first layer of reagent pool through a sample adding hole on the card box shell, and adding a sample solution of nucleic acid to be extracted into the top surface film;
(2) Moving the pipette downwards to the first layer of reagent pool on the suspender by rotating the sleeve; then sucking the solution in the first layer of reagent pool by a silica gel sucker at the top of the pressing/releasing sleeve, so that the magnetic particles combined with the sample nucleic acid are completely adsorbed to the surface of the magnet in the pipette;
(3) Continuing to rotate the sleeve to make the tip of the pipette pierce the bottom film of the first layer of reagent pool; pressing the silica gel sucker to ensure that the liquid in the liquid suction pipe is completely absorbed by the absorbent cotton after being discharged;
(4) Continuing rotating the sleeve, enabling the pipette to sequentially enter the next layer or layers of reagent pools, cleaning the magnetic particles on the surface of the magnet by using the cleaning liquid in each reagent pool, and absorbing the cleaned waste liquid by absorbent cotton;
(5) Continuing rotating the sleeve, enabling the pipette to enter the next layer of reagent pool, sucking the eluent in the reagent pool, and eluting the nucleic acid on the magnetic particles;
(6) Continuously rotating the sleeve, injecting the extracted nucleic acid into the reagent pool at the bottom layer, and mixing the nucleic acid with the nucleic acid amplification reagent in the reagent pool;
(7) And continuing rotating the sleeve, and injecting the mixed liquid into the base to perform amplification detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211258530.7A CN115926961A (en) | 2022-10-14 | 2022-10-14 | Small card box integrating nucleic acid extraction, amplification and detection and nucleic acid extraction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211258530.7A CN115926961A (en) | 2022-10-14 | 2022-10-14 | Small card box integrating nucleic acid extraction, amplification and detection and nucleic acid extraction method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115926961A true CN115926961A (en) | 2023-04-07 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211258530.7A Pending CN115926961A (en) | 2022-10-14 | 2022-10-14 | Small card box integrating nucleic acid extraction, amplification and detection and nucleic acid extraction method |
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| Country | Link |
|---|---|
| CN (1) | CN115926961A (en) |
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2022
- 2022-10-14 CN CN202211258530.7A patent/CN115926961A/en active Pending
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