CN115851422B - Integrated small-sized nucleic acid detection device and nucleic acid detection method - Google Patents
Integrated small-sized nucleic acid detection device and nucleic acid detection methodInfo
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- CN115851422B CN115851422B CN202211258538.3A CN202211258538A CN115851422B CN 115851422 B CN115851422 B CN 115851422B CN 202211258538 A CN202211258538 A CN 202211258538A CN 115851422 B CN115851422 B CN 115851422B
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Abstract
本发明公开了一种一体化的小型核酸检测装置，包括套筒、外壳和底座组成的封闭卡盒，外壳内壁的上半部分设有两条中心对称的螺旋形轨道；所述套筒壁两侧设有两条对称的垂直导轨；还包括位于套筒内的穿刺吊杆，穿刺吊杆的顶端两端设有两个凸起，其穿过套筒壁上的导轨而嵌入外壳内壁的螺旋形轨道中，使得穿刺吊杆随套筒的旋转而垂直上下移动，所述穿刺吊杆的末端为箭头；还包括试剂仓，所述试剂仓设置于外壳下半部分；所述外壳顶部与套筒、外壳底部与底座之间密封连接。本发明提供了一种集核酸提取、扩增与检测一体化的小型核酸检测装置，可利用所述装置完成样本的核酸提取、靶标序列扩增和检测等全流程，大大减少对检测场所和技术人员的要求。
This invention discloses an integrated small nucleic acid detection device, comprising a closed cartridge consisting of a sleeve, an outer shell, and a base. The upper half of the inner wall of the outer shell has two centrally symmetrical spiral tracks; the sleeve wall has two symmetrical vertical guide rails on both sides; it also includes a puncture rod located inside the sleeve, with two protrusions at its top end, which pass through the guide rails on the sleeve wall and embed into the spiral tracks on the inner wall of the outer shell, allowing the puncture rod to move vertically up and down as the sleeve rotates; the end of the puncture rod is an arrow; it also includes a reagent compartment located in the lower half of the outer shell; the top of the outer shell is sealed to the sleeve, and the bottom of the outer shell is sealed to the base. This invention provides a small nucleic acid detection device integrating nucleic acid extraction, amplification, and detection, enabling the entire process of nucleic acid extraction, target sequence amplification, and detection of samples to be completed using this device, greatly reducing the requirements for testing sites and technical personnel.
Description
Technical Field
The invention belongs to the field of nucleic acid detection, and relates to an integrated small-sized nucleic acid detection device and a nucleic acid detection method.
Background
Nucleic acid detection is the most sensitive and accurate method for pathogen detection, and in recent years, a plurality of pathogen detection technologies based on nucleic acid amplification, such as fluorescent quantitative PCR technology, loop-mediated isothermal amplification technology, nucleic acid sequencing technology and the like, have been developed. However, in the detection of pathogens, the techniques all require steps such as sample nucleic acid extraction, target amplification, amplification product detection, etc. Each step requires specialized instrumentation and requires manipulation by a professional familiar with the skill of molecular biology experimentation. The biggest problem in nucleic acid detection is false positive results caused by cross contamination of amplified products, and usually, the steps of nucleic acid extraction, amplification, detection and the like must be performed in a completely physically isolated laboratory, so that it is difficult to achieve rapid detection on site. In order to realize the on-site detection of pathogens, the most ideal way is to develop an integrated device and instrument integrating nucleic acid extraction, amplification and detection, and overcome the defect that the existing pathogen nucleic acid detection needs to be divided into laboratories.
Disclosure of Invention
The invention discloses an integrated small-sized nucleic acid detection device, which integrates the steps of nucleic acid extraction, amplification and detection into a small-sized device.
The aim of the invention is achieved by the following technical scheme:
the integrated small nucleic acid detection device is characterized by comprising a sealing device consisting of a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell comprises two spiral tracks with central symmetry;
The sleeve comprises a sleeve knob and a sleeve wall which are integrated, the sleeve wall extends into the shell, the sleeve knob is embedded at the upper end of the shell, and two symmetrical vertical guide rails are arranged on two sides of the sleeve wall;
The puncture suspension rod is positioned in the sleeve, two ends of the top end of the puncture suspension rod are provided with two protrusions which penetrate through guide rails on the wall of the sleeve and are embedded into spiral tracks on the inner wall of the shell, so that the puncture suspension rod vertically moves up and down along with the rotation of the sleeve, and the tail end of the puncture suspension rod is an arrow;
the reagent bin is arranged at the lower half part of the shell;
The top of the shell is connected with the sleeve the bottom of the shell is connected with the base in a sealing way.
Preferably, the reagent cartridge comprises a plurality of reagent reservoirs and a suction reservoir, typically fewer than the reagent reservoirs. The reagent pool is filled with reagent, and the top surface and the bottom surface of the reagent pool are both films which are easy to puncture. The top of the water absorbing pool is hollow, the center of the water absorbing pool is a hollow round hole, a plurality of fences are arranged around the round hole, and water absorbing cotton is filled in a cavity at the outer side of each fence. Each layer of water absorbing pool is tightly combined below the corresponding reagent pool and is used for collecting waste liquid.
Preferably, the puncture device further comprises a magnet, wherein the magnet is positioned in a column above the arrow at the tail end of the puncture boom.
Preferably, the casing is provided with a sample adding hole and a matched sample adding hole cover at the position between the reagent bin and the sleeve wall.
Preferably, the wall thickness of the upper part, the middle part and the lower part of the shell is different, the wall thickness of the part of the lower part corresponding to the reagent bin is thinnest, the wall thickness of the part of the middle part corresponding to the sample adding hole is thicker, the wall thickness difference forms a limiting effect on the reagent bin, the wall thickness of the part of the upper part corresponding to the sleeve is thickest, and the wall thickness difference forms a limiting effect on the lower edge of the sleeve wall.
Preferably, the reagent reservoirs are plural, and the suspension, the cleaning solution, the eluent and the nucleic acid amplification reaction reagent of the magnetic particles and the cell lysate are sequentially contained from top to bottom.
The invention also discloses a nucleic acid detection method based on the integrated small nucleic acid detection device, which is characterized by comprising the following steps:
(1) Puncturing the top surface film of the first layer reagent pool through a sample adding hole on the cartridge shell, and adding a sample solution of nucleic acid to be extracted into the first layer reagent pool;
(2) The magnet on the suspender moves downwards to the first layer reagent pool by rotating the sleeve, so that all magnetic particles combined with sample nucleic acid are adsorbed on the surface of the column body containing the magnet above the arrow;
(3) Continuing to rotate the sleeve to enable the arrow to puncture the bottom surface film of the first layer of reagent tank, so that the liquid in the reagent tank is completely absorbed by the absorbent cotton after being discharged;
(4) Continuously rotating the sleeve to enable the arrow to sequentially enter the next layer or multiple layers of reagent pools, cleaning magnetic particles on the surface of the magnet by using cleaning liquid in each reagent pool, and absorbing the cleaned waste liquid by absorbent cotton;
(5) Continuing rotating the sleeve to enable the arrow to enter a next layer of reagent pool, and immersing the magnetic particles by eluent in the reagent pool to enable nucleic acid on the magnetic particles to be eluted;
(6) Continuing to rotate the sleeve, injecting the extracted nucleic acid into a reagent pool at the bottom layer, and mixing the extracted nucleic acid with a nucleic acid amplification reagent in the reagent pool;
(7) And continuing to rotate the sleeve, injecting the mixed liquid into the base, and performing amplification detection.
The invention has the following advantages:
The invention provides an integrated small-sized nucleic acid detection device, which integrates the steps of nucleic acid extraction, amplification and detection into one device, thereby greatly reducing the requirements on detection places and technicians. The device is disposable, and each sample to be detected is detected by using a single device, so that cross contamination among different samples can be effectively avoided. In addition, the device adopts a fully-closed design, so that the escape of nucleic acid amplification products can be effectively avoided, and false positive results are prevented. In summary, the present invention provides an integrated small nucleic acid detection device and method of use.
Drawings
FIG. 1 is a schematic view of the apparatus of the present invention, (A) is a whole sectional view, (B) is a schematic view before operation, an arrow is located at an upper portion, and (C) is a schematic view after operation, the arrow is moved into a base reaction tube.
FIG. 2 is a view showing the construction of the housing of the apparatus of the present invention, wherein (A) is a perspective view as a whole and (B) is a sectional view as a whole.
Fig. 3 is a sleeve structure diagram of the device of the present invention.
FIG. 4 is a schematic view of a reagent cartridge of the apparatus of the present invention, (A) is a perspective view of a main body of the reagent cartridge, (B) is a perspective view of a cross section of the reagent cartridge, (C) is a schematic view of a reagent reservoir, and (D) is a schematic view of a water suction reservoir.
Fig. 5 is a view showing the construction of the base of the apparatus of the present invention.
Detailed Description
Examples
The integrated small nucleic acid detection device shown in the invention is shown in figure 1, and comprises a tubular base 1, a cylindrical shell 2 and a sleeve 3, wherein the top of the sleeve 3 is provided with a sleeve knob 4, the sleeve wall extends into the shell, 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 joint of the tubular base 1, the cylindrical shell 2 and the sleeve 3 is kept airtight, forming a closed cavity. The sleeve 3 contains a boom 5 with an arrow-shaped end, and the column above the arrow contains a magnet 6. The lower part of the housing 2 contains a reagent chamber 7 for containing reagents required for nucleic acid extraction and purification. The tubular base 1 is used for performing a nucleic acid amplification reaction. Wherein a specific construction of the cylindrical housing 2 is shown in fig. 2. The upper half of the inner wall of the housing 2 comprises two spiral tracks A8-1 and B8-2 which are symmetrical in the center and are used for limiting the movement track of the housing 2 and the sleeve 3 during the relative movement. The middle part of the shell 2 comprises a sample adding hole 9 and a sample adding hole cover 10 which is a fitting for adding a sample to be tested, and the sample adding hole 9 is sealed by using the sample adding hole cover 10 after the sample is added, so that a closed cavity is formed inside the device. The wall thickness of the upper part, the middle part and the lower part of the shell is different, the wall thickness of the part of the lower part corresponding to the reagent bin is thinnest, the wall thickness of the part of the middle part corresponding to the sample adding hole is thicker, the wall thickness difference forms a limiting effect on the reagent bin, the wall thickness of the part of the upper part corresponding to the sleeve is thickest, and the wall thickness difference forms a limiting effect on the lower edge of the sleeve wall.
The specific structure of the sleeve 3 is shown in fig. 3, two symmetrical vertical guide rails a11 and B12 are arranged on two sides of the lower part of the sleeve 3, two protrusions are arranged on two sides of the top end of the hanging rod 5, and the protrusions can penetrate through the vertical guide rails a11 and B12 on the sleeve wall and be embedded into the spiral track 8 on the inner wall of the shell 2. When in use, the sleeve 3 is driven by external force to rotate relative to the shell 2, the suspender 5 moves vertically upwards and downwards along with the rotation, and the tail end of the suspender 5 is an arrow 13. At least one magnet 6 is provided in the boom cylinder above arrow 13. When in use, the magnetic particles used for extracting nucleic acid are adsorbed on the surface of the suspender corresponding to the magnet 6.
Wherein the specific construction of the reagent vessel 7 is shown in fig. 4, comprising a reagent reservoir 14 and a water suction reservoir 15. The reagent pools A14-1, B14-2, C14-3, D14-4 and E14-5 are distributed with the water suction pool A15-1, B15-2, C15-3 and D15-4 at intervals to form a nested structure. In use, nucleic acid extraction reagents are pre-stored in each of the reagent reservoirs A-E14-1 to 14-5, nucleic acid amplification reagents are pre-stored in the reagent reservoir F14-6, and the top and bottom surfaces of the reagent reservoirs are sealed by films. The top of the water absorbing pool is hollow, the center of the water absorbing pool is a hollow round hole, a plurality of fences 16 are arranged around the round hole, and water absorbing materials such as water absorbing cotton and the like are preloaded in cavities outside the fences and used for collecting waste liquid. The film may be made of common aluminum foil, waterproof paper film or other materials easy to puncture.
The specific structure of the base is shown in FIG. 5, the upper part 1-1 is tightly connected with the bottom of the shell through threads, and the lower part 1-2 is tubular and is used for carrying out nucleic acid amplification detection reaction.
When in use, an operator pierces the top film of the reagent pool A14-1 through the sample adding hole 9 on the cartridge shell, adds samples including nasopharyngeal swab lixivium, blood, sputum, urine, tissue, excrement and the like, covers the sample adding hole cover 10, rotates the sleeve 3 to enable the arrow 13 at the tail end of the hanging rod 5 to pierce the top film and the bottom film of each reagent pool in sequence, magnetic particles for extracting nucleic acid are adsorbed on the surface of the hanging rod corresponding to the magnet 6, and the waste liquid is completely collected by the water absorbing material in the water absorbing pool after being discharged. Finally, the extracted nucleic acid and the amplification reaction reagent flow into the tube of the base 1 together to perform amplification detection. The extraction, purification and mixing of the amplification reagents of the sample nucleic acid are accomplished by rotating the sleeve in both forward and reverse directions.
Examples
The method for detecting nucleic acid by using the device comprises the following steps:
(1) The aluminum foil on the top surface of the reagent pool A14-1 is pierced at the sample adding hole 9 on the shell, a sample to be detected is added, and cell lysis, nucleic acid release and nucleic acid adsorption are carried out by the magnetic beads;
(2) Rotating the sleeve 3 to enable the boom 5 to move downwards until the boom section corresponding to the magnet 6 is immersed in the liquid in the reagent pool A14-1, and adsorbing the magnetic beads on the surface of the boom corresponding to the magnet 6;
(3) Continuing to rotate the sleeve 3, using the arrow 13 at the tail end of the hanging rod to pierce the aluminum foil at the bottom surface of the reagent tank A14-1, discharging the liquid to the water absorbing tank A15-1, and completely absorbing the liquid by the absorbent cotton;
(4) Continuing to rotate the sleeve 3, using a hanging rod end arrow 13 to pierce an aluminum foil on the top surface of the reagent tank B14-2, and immersing the hanging rod section corresponding to the magnet 6 in the cleaning liquid A in the reagent tank B14-2;
(5) Repeatedly rotating the sleeve forwards/backwards for more than 3 times, and cleaning the magnetic beads on the surface of the suspender corresponding to the magnet 6;
(6) Continuing to rotate the sleeve 3, using the arrow 13 at the tail end of the hanging rod to pierce the aluminum foil at the bottom surface of the reagent tank B14-2, discharging the liquid to the water absorbing tank B15-2, and completely absorbing the liquid by the absorbent cotton;
(7) Continuing to rotate the sleeve 3, using a hanging rod end arrow 13 to pierce an aluminum foil on the top surface of the reagent tank C14-3, and immersing the hanging rod section corresponding to the magnet 6 in the cleaning liquid A in the reagent tank C14-3;
(8) Repeatedly rotating the sleeve forwards/backwards for more than 3 times, and cleaning the magnetic beads on the surface of the suspender corresponding to the magnet 6;
(9) Continuing to rotate the sleeve 3, using the arrow 13 at the tail end of the hanging rod to pierce the aluminum foil at the bottom surface of the reagent tank C14-3, discharging the liquid to the water absorbing tank C15-3, and completely absorbing the liquid by the absorbent cotton;
(10) Continuing to rotate the sleeve 3, using a hanging rod end arrow 13 to pierce an aluminum foil on the top surface of the reagent pool D14-4, and immersing the hanging rod section corresponding to the magnet 6 in the cleaning liquid B in the reagent pool D14-4;
(11) Repeatedly rotating the sleeve forwards/backwards for more than 3 times, and cleaning the magnetic beads on the surface of the suspender corresponding to the magnet 6;
(12) Continuing to rotate the sleeve 3, using the arrow 13 at the tail end of the hanging rod to pierce the aluminum foil at the bottom surface of the reagent tank D14-4, discharging the liquid to the water absorbing tank D15-4, and completely absorbing the liquid by the absorbent cotton;
(13) Standing for at least 1min to volatilize residual liquid on the surfaces of the magnetic beads;
(14) Continuing to rotate the sleeve 3, using a hanging rod end arrow 13 to pierce an aluminum foil on the top surface of the reagent tank E14-5, and immersing the hanging rod section corresponding to the magnet 6 in eluent in the reagent tank E14-5;
(15) Standing for at least 2 minutes to dissolve the nucleic acid on the surface of the magnetic beads into the eluent;
(16) Continuing to rotate the sleeve 3, using the end arrow 13 of the hanging rod to pierce the aluminum foil on the top surface of the reagent pool F14-6, discharging the liquid in the reagent pool 14-5, and dissolving the nucleic acid amplification reaction freeze-dried reagent in the reagent pool F14-6;
(17) Repeatedly rotating the sleeve forwards/reversely for more than 3 times, and uniformly mixing the liquid in the reagent pool F14-6;
(18) The sleeve 3 is continuously rotated, and the aluminum foil on the bottom surface of the reagent pool F14-6 is pierced by using the hanging rod end arrow 13, so that the liquid in the reagent pool F14-6 is discharged into the reaction tube in the base 1, and the nucleic acid amplification reaction is performed.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. The integrated small nucleic acid detection device is characterized by comprising a sealing device consisting of a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell is provided with two spiral tracks with central symmetry;
The sleeve comprises a sleeve knob and a sleeve wall which are integrated, the sleeve wall extends into the shell, the sleeve knob is clamped at the upper end of the shell, and two symmetrical vertical guide rails are arranged on two sides of the sleeve wall;
The puncture suspension rod is positioned in the sleeve, two ends of the top end of the puncture suspension rod are provided with two protrusions which penetrate through guide rails on the wall of the sleeve and are embedded into spiral tracks on the inner wall of the shell, so that the puncture suspension rod vertically moves up and down along with the rotation of the sleeve, and the tail end of the puncture suspension rod is an arrow;
the reagent bin is arranged at the lower half part of the shell;
the top of the shell is connected with the sleeve the bottom of the shell is connected with the base in a sealing way;
The reagent bin comprises a plurality of reagent pools and a water absorption pool, wherein the reagent pools are filled with reagents, the top surfaces and the bottom surfaces of the reagent pools are provided with a plurality of films which are easy to puncture, the reagent pools sequentially contain suspension liquid, cleaning liquid, eluent and nucleic acid amplification reaction reagents of magnetic particles and cell lysate from top to bottom, the water absorption pool is arranged below the reagent pools containing the suspension liquid and the cleaning liquid, the top of the water absorption pool is hollow, the center of the water absorption pool is provided with a hollow round hole, a plurality of fences are arranged around the round hole, water absorption cotton is filled in a cavity outside the fences, and the reagent bin further comprises a magnet, wherein the magnet is positioned in a column above an arrow at the tail end of a puncture suspender.
2. The integrated small nucleic acid detecting apparatus according to claim 1, wherein the housing is provided with a sample-adding hole and a sample-adding hole cover between the reagent chamber and the sleeve wall.
3. The integrated small nucleic acid detecting apparatus according to claim 1, wherein the upper, middle and lower portions of the housing have different wall thicknesses, the lower portion has a thinnest wall thickness corresponding to the reagent cartridge, the middle portion has a larger wall thickness corresponding to the sample well than the reagent cartridge, and the upper portion has a thickest wall thickness corresponding to the sleeve.
4. The method for using an integrated small nucleic acid detecting apparatus according to claim 2, comprising the steps of:
(1) Puncturing the top surface film of the first layer reagent pool through a sample adding hole on the cartridge shell, and adding a sample solution of nucleic acid to be extracted into the first layer reagent pool;
(2) The magnet on the suspender moves downwards to the first layer reagent pool by rotating the sleeve, so that all magnetic particles combined with sample nucleic acid are adsorbed on the surface of the column body containing the magnet above the arrow;
(3) Continuing to rotate the sleeve to enable the arrow to puncture the bottom surface film of the first layer of reagent tank, so that the liquid in the reagent tank is completely absorbed by the absorbent cotton after being discharged;
(4) Continuously rotating the sleeve to enable the arrow to sequentially enter the next layer or multiple layers of reagent pools, cleaning magnetic particles on the surface of the magnet by using cleaning liquid in each reagent pool, and absorbing the cleaned waste liquid by absorbent cotton;
(5) Continuing rotating the sleeve to enable the arrow to enter a next layer of reagent pool, and immersing the magnetic particles by eluent in the reagent pool to enable nucleic acid on the magnetic particles to be eluted;
(6) Continuing to rotate the sleeve, injecting the extracted nucleic acid into a reagent pool at the bottom layer, and mixing the extracted nucleic acid with a nucleic acid amplification reagent in the reagent pool;
(7) And continuing to rotate the sleeve, injecting the mixed liquid into the base, and performing amplification detection.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211258538.3A CN115851422B (en) | 2022-10-14 | 2022-10-14 | Integrated small-sized nucleic acid detection device and nucleic acid detection method |
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| CN202211258538.3A CN115851422B (en) | 2022-10-14 | 2022-10-14 | Integrated small-sized nucleic acid detection device and nucleic acid detection method |
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| WO2005012518A1 (en) * | 2003-07-30 | 2005-02-10 | Riken | Kit for nucleic acid detection |
| JP2016198046A (en) * | 2015-04-10 | 2016-12-01 | セイコーエプソン株式会社 | Bio-related substance extraction device, bio-related substance extraction device, and bio-related substance extraction container |
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| WO2021254518A1 (en) * | 2020-06-19 | 2021-12-23 | 中国疾病预防控制中心病毒病预防控制所 | Sample treatment and testing apparatus |
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