An integrated small nucleic acid detection device and nucleic acid detection method

Abstract

The application discloses a kind of integrated small nucleic acid detection device, including sleeve, shell and base composition closed card box, the upper half of shell inner wall is equipped with two center symmetrical spiral tracks;The both sides of the sleeve wall are equipped with two symmetrical vertical guide rails;It further includes the puncture boom in sleeve, the top end of puncture boom is equipped with two protrusions, it is embedded in the spiral track of shell inner wall by passing the guide rail on the sleeve wall, so that puncture boom vertically moves up and down with the rotation of sleeve, and the end of the puncture boom is arrowhead;It further includes reagent bin, and the reagent bin is arranged in the lower half of shell;The top of shell is sealingly connected between sleeve, the bottom of shell is sealingly connected between base.The application provides a kind of integrated small nucleic acid detection device of nucleic acid extraction, amplification and detection, can utilize the device to complete the whole process of sample nucleic acid extraction, target sequence amplification and detection etc., greatly reduces the requirement to detection place and technical personnel.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nucleic acid detection, and relates to an integrated small nucleic acid detection device and a nucleic acid detection method. BACKGROUND

[0002] Nucleic acid detection is the most sensitive and accurate method for pathogen detection, and many pathogen detection technologies based on nucleic acid amplification have been developed in recent years, such as fluorescence quantitative PCR technology, loop-mediated isothermal amplification technology, nucleic acid sequencing technology and the like. However, when these technologies are used for pathogen detection, sample nucleic acid extraction, target amplification and amplification product detection need to be performed. Special instruments and equipment are required for each step, and professional personnel skilled in molecular biology experiments need to operate. The biggest problem of nucleic acid detection is false positive results caused by cross contamination of amplification products. Usually, nucleic acid extraction, amplification and detection need to be performed in completely physically isolated partition laboratories, so it is difficult to realize on-site rapid detection. In order to realize on-site detection of pathogens, the most ideal way is to develop an integrated device and instrument for nucleic acid extraction, amplification and detection, so as to overcome the disadvantages of existing pathogen nucleic acid detection which must have partition laboratories. SUMMARY

[0003] The application discloses an integrated small nucleic acid detection device which integrates nucleic acid extraction, amplification and detection steps in a small device.

[0004] The purpose of the application is achieved by the following technical scheme.

[0005] An integrated small nucleic acid detection device, characterized in that the device comprises a closed device composed of a sleeve, a shell and a base, and the upper half of the inner wall of the shell comprises two center-symmetrical spiral tracks.

[0006] The sleeve comprises an integrated sleeve knob and a sleeve wall, the sleeve wall extends into the shell, the sleeve knob is embedded on the upper end of the shell, and two symmetrical vertical guide rails are arranged on the two sides of the sleeve wall.

[0007] The device further comprises a puncture hanger, the puncture hanger is located in the sleeve, two protrusions are arranged at the top ends of the puncture hanger, the two protrusions pass through the guide rails on the sleeve wall and are embedded in the spiral tracks on the inner wall of the shell, so that the puncture hanger vertically moves up and down with the rotation of the sleeve, and the tail end of the puncture hanger is an arrowhead.

[0008] The device further comprises a reagent bin, and the reagent bin is arranged in the lower half of the shell.

[0009] The top of the shell is in sealed connection with the sleeve, and the bottom of the shell is in sealed connection with the base.

[0010] Preferably, the reagent compartment comprises a plurality of reagent pools and a plurality of water absorption pools, and the number of water absorption pools is less than that of reagent pools. The reagent pools are filled with reagents, and the top and bottom surfaces of the reagent pools are both thin membranes which can be easily punctured. The water absorption pools are empty at the top and have a hollow circle in the center, and a plurality of fences are arranged around the hollow circle, and the space outside the fences is filled with water absorption cotton. Each layer of water absorption pool is closely combined with the corresponding reagent pool below, and is used for waste liquid collection.

[0011] Preferably, a magnet is further arranged in the column above the arrowhead of the puncture hanger.

[0012] Preferably, a sample adding hole and a matching sample adding hole cover are arranged on the shell at the position between the reagent compartment and the sleeve wall.

[0013] Preferably, the wall thicknesses of the upper, middle and lower parts of the shell are different, the wall thickness of the lower part corresponding to the reagent compartment is the thinnest, the wall thickness of the middle part corresponding to the sample adding hole is relatively thick, and the difference in wall thickness forms a limiting effect on the reagent compartment; and the wall thickness of the upper part corresponding to the sleeve is the thickest, and the difference in wall thickness forms a limiting effect on the lower edge of the sleeve wall.

[0014] Preferably, the reagent pools are a plurality of reagent pools, and the reagent pools successively contain, from top to bottom, a suspension of magnetic particles and cell lysis solution, a washing solution, an elution solution and a nucleic acid amplification reagent.

[0015] The application further discloses a nucleic acid detection method based on the integrated small nucleic acid detection device.

[0016] (1) A sample solution containing nucleic acid to be extracted is added into the first reagent pool by puncturing the top surface membrane of the first reagent pool through the sample adding hole on the shell of the card box.

[0017] (2) The magnet on the hanger is moved downward to the first reagent pool by rotating the sleeve, so that the magnetic particles which have combined with the nucleic acid of the sample are all adsorbed to the surface of the column containing the magnet above the arrowhead.

[0018] (3) The bottom surface membrane of the first reagent pool is punctured by continuing to rotate the sleeve, so that the liquid in the reagent pool is completely absorbed by the water absorption cotton after being discharged.

[0019] (4) The arrowhead enters the next layer or a plurality of reagent pools in sequence by continuing to rotate the sleeve, the magnetic particles on the surface of the magnet are washed by using the washing solution in each reagent pool, and the waste liquid after washing is absorbed by the water absorption cotton.

[0020] (5) The arrowhead enters the next layer of reagent pool by continuing to rotate the sleeve, and the nucleic acid on the magnetic particles is eluted by immersing the magnetic particles in the elution solution in the reagent pool.

[0021] (6) The extracted nucleic acid is injected into the reagent pool at the bottom by continuing to rotate the sleeve, and is mixed with the nucleic acid amplification reagent in the reagent pool.

[0022] (7) Continue to rotate the sleeve to inject the mixed liquid into the base for amplification detection.

[0023] The present invention has the following advantages:

[0024] This invention provides an integrated, small-scale nucleic acid detection device that integrates nucleic acid extraction, amplification, and detection steps into a single device, significantly reducing the requirements for testing facilities and technical personnel. The device is for single use only; each sample is tested using a separate device, effectively avoiding cross-contamination between different samples. Furthermore, the device employs a fully sealed design, effectively preventing the escape of nucleic acid amplification products and avoiding false positive results. In summary, this invention provides an integrated, small-scale nucleic acid detection device and its usage method. Attached Figure Description

[0025] Figure 1 This is a structural diagram of the device of the present invention. (A) is an overall cross-sectional view, (B) is a schematic diagram before operation with the arrow located in the upper part, and (C) is a schematic diagram after operation with the arrow moved to the base reaction tube.

[0026] Figure 2 These are structural diagrams of the outer casing of the device of the present invention. (A) is an overall perspective view, and (B) is an overall cross-sectional view.

[0027] Figure 3 This is a diagram of the sleeve structure of the device of the present invention.

[0028] Figure 4 Here are structural diagrams of the reagent compartment of the device of the present invention: (A) is a perspective view of the main body of the reagent compartment, (B) is a cross-sectional perspective view of the reagent compartment, (C) is a structural diagram of the reagent pool, and (D) is a structural diagram of the water absorption pool.

[0029] Figure 5 This is a structural diagram of the base of the device of the present invention. Detailed Implementation

[0030] Example

[0031] The integrated miniature nucleic acid detection device shown in this invention is as follows: Figure 1 The system includes a tubular base 1, a cylindrical outer shell 2, and a sleeve 3. The top of the sleeve 3 has a sleeve knob 4. The sleeve wall extends into the outer shell, and the sleeve knob is fitted into the upper end of the outer shell. The sleeve knob can be rotated under external force, thereby rotating the sleeve wall. The connection between the tubular base 1, the cylindrical outer shell 2, and the sleeve 3 remains sealed, forming a closed cavity. The sleeve 3 contains a rod 5 with an arrowhead-shaped end, and a magnet 6 is contained within the cylinder above the arrowhead. The lower part of the outer shell 2 contains a reagent compartment 7 for holding reagents required for nucleic acid extraction and purification. The tubular base 1 is used for nucleic acid amplification reactions. The specific structure of the cylindrical outer shell 2 is as follows...Figure 2 The upper half of the inner wall of the shell 2 contains two center-symmetrical spiral tracks A 8-1 and B 8-2, which limit the movement trajectory of the shell 2 relative to the sleeve 3. The middle part of the shell 2 includes a sample addition hole 9 and its accessory sample addition hole cover 10, which are used for the addition of the sample to be tested. After the sample is added, the sample addition hole cover 10 is used to seal the sample addition hole 9, so that a closed cavity is formed inside the device. The wall thicknesses of the upper, middle and lower parts of the shell are different. The wall thickness of the lower part corresponding to the reagent bin is the thinnest, the wall thickness of the middle part corresponding to the sample addition hole is relatively thick, and the difference in wall thickness here forms a limiting effect on the reagent bin; the wall thickness of the upper part corresponding to the sleeve is the thickest, and the difference in wall thickness here forms a limiting effect on the lower edge of the sleeve wall.

[0032] The specific structure of the sleeve 3 is shown in Figure 3 The lower part of the sleeve 3 contains two symmetrical vertical guide rails A11 and B12 on both sides, and the top of the hanger rod 5 has two protrusions which can pass through the vertical guide rails A11 and B12 on the sleeve wall and be embedded in the spiral tracks 8 of the inner wall of the shell 2. In use, the sleeve 3 rotates relative to the shell 2 under the action of an external force, and the hanger rod 5 moves vertically up and down accordingly. The end of the hanger rod 5 is indicated by an arrow 13. At least one magnet 6 is arranged in the hanger rod column above the arrow 13. In use, the magnetic microparticles used for nucleic acid extraction are adsorbed to the surface of the magnet 6 corresponding to the hanger rod.

[0033] The specific structure of the reagent bin 7 is shown in Figure 4 The reagent bin 7 contains a reagent pool 14 and a water absorption pool 15. The reagent pools A 14-1, B 14-2, C 14-3, D 14-4 and E 14-5 are distributed at intervals with the water absorption pools A 15-1, B 15-2, C 15-3 and D 15-4, forming a nested structure. In use, each reagent pool A-E 14-1 to 14-5 is pre-stored with nucleic acid extraction reagents, and the reagent pool F 14-6 is pre-stored with nucleic acid amplification reagents. The top and bottom surfaces of the reagent pools are sealed with a film. The top of the water absorption pool is empty, and the center is a hollow circular hole. There are multiple fences 16 around the circular hole, and the cavity outside the fences is pre-loaded with water-absorbing materials such as water-absorbing cotton for collecting waste liquid. The film can be made of common aluminum foil, waterproof paper film or other materials that can be easily punctured.

[0034] The specific structure of the base is shown in Figure 5 The upper part 1-1 is tightly connected to the bottom of the shell by threads, and the lower part 1-2 is tubular, which is used for nucleic acid amplification detection reaction.

[0035] In use, the operator punctures the top film of the reagent pool A 14-1 through the sample adding hole 9 on the cartridge shell, adds the sample, including nasopharyngeal swab extract, blood, sputum, urine, tissue and feces, etc., and covers the sample adding hole cover 10; rotates the sleeve 3, so that the arrow 13 at the end of the hanger 5 punctures the top and bottom films of each reagent pool in turn, the magnetic particles used for nucleic acid extraction are adsorbed on the hanger surface corresponding to the magnet 6, and the waste liquid is completely collected by the water absorption material in the water absorption pool after being discharged. Finally, the extracted nucleic acid and the amplification reaction reagent flow into the tube of the base 1 together, and the amplification detection is performed. The forward and reverse rotation of the sleeve is used to complete the extraction and purification of the sample nucleic acid and the mixing of the amplification reaction reagent.

[0036] Embodiment

[0037] The method for detecting nucleic acid using the above device, the steps of which include:

[0038] (1) puncture the aluminum foil on the top of the reagent pool A 14-1 through the sample adding hole 9 on the shell, add the sample to be detected, perform cell lysis, release nucleic acid, and adsorb nucleic acid with magnetic beads;

[0039] (2) rotate the sleeve 3, so that the hanger 5 moves downward to immerse the hanger section corresponding to the magnet 6 in the liquid in the reagent pool A 14-1, and the magnetic beads are adsorbed on the hanger surface corresponding to the magnet 6;

[0040] (3) continue to rotate the sleeve 3, and use the arrow 13 at the end of the hanger to puncture the aluminum foil on the bottom of the reagent pool A 14-1, so that the liquid is discharged to the water absorption pool A 15-1 and is completely absorbed by the water absorption cotton;

[0041] (4) continue to rotate the sleeve 3, and use the arrow 13 at the end of the hanger to puncture the aluminum foil on the top of the reagent pool B 14-2, so that the hanger section corresponding to the magnet 6 is immersed in the washing liquid A in the reagent pool B 14-2;

[0042] (5) repeatedly rotate the sleeve 3 forward and backward more than three times to wash the magnetic beads on the hanger surface corresponding to the magnet 6;

[0043] (6) continue to rotate the sleeve 3, and use the arrow 13 at the end of the hanger to puncture the aluminum foil on the bottom of the reagent pool B 14-2, so that the liquid is discharged to the water absorption pool B 15-2 and is completely absorbed by the water absorption cotton;

[0044] (7) continue to rotate the sleeve 3, and use the arrow 13 at the end of the hanger to puncture the aluminum foil on the top of the reagent pool C 14-3, so that the hanger section corresponding to the magnet 6 is immersed in the washing liquid A in the reagent pool C 14-3;

[0045] (8) repeatedly rotate the sleeve 3 forward and backward more than three times to wash the magnetic beads on the hanger surface corresponding to the magnet 6;

[0046] (9) Continue to rotate the sleeve 3, use the arrow 13 at the end of the hanger to pierce the aluminum foil at the bottom of the reagent pool C 14-3, and the liquid is discharged to the water absorption pool C 15-3 and is completely absorbed by the water absorption cotton;

[0047] (10) Continue to rotate the sleeve 3, use the arrow 13 at the end of the hanger to pierce the aluminum foil at the top of the reagent pool D 14-4, and the magnet 6 corresponding to the hanger section is immersed in the cleaning liquid B in the reagent pool D 14-4;

[0048] (11) Rotate the sleeve 3 forward and backward repeatedly more than three times to clean the magnetic beads on the surface of the magnet 6 corresponding to the hanger;

[0049] (12) Continue to rotate the sleeve 3, use the arrow 13 at the end of the hanger to pierce the aluminum foil at the bottom of the reagent pool D 14-4, and the liquid is discharged to the water absorption pool D 15-4 and is completely absorbed by the water absorption cotton;

[0050] (13) Stand still for at least 1 min to evaporate the residual liquid on the surface of the magnetic beads;

[0051] (14) Continue to rotate the sleeve 3, use the arrow 13 at the end of the hanger to pierce the aluminum foil at the top of the reagent pool E 14-5, and the magnet 6 corresponding to the hanger section is immersed in the elution liquid in the reagent pool E 14-5;

[0052] (15) Stand still for at least 2 minutes to dissolve the nucleic acid on the surface of the magnetic beads into the elution liquid;

[0053] (16) Continue to rotate the sleeve 3, use the arrow 13 at the end of the hanger to pierce the aluminum foil at the top of the reagent pool F 14-6, discharge the liquid in the reagent pool 14-5, and dissolve the nucleic acid amplification reaction freeze-dried reagent in the reagent pool F 14-6;

[0054] (17) Rotate the sleeve 3 forward and backward repeatedly more than three times to mix the liquid in the reagent pool F 14-6;

[0055] (18) Continue to rotate the sleeve 3, use the arrow 13 at the end of the hanger to pierce the aluminum foil at the bottom of the reagent pool F 14-6, discharge the liquid in the reagent pool F 14-6 to the reaction tube in the base 1, and perform the nucleic acid amplification reaction.

[0056] The above embodiment is a preferred embodiment of the present application, but the embodiment of the present application is not limited to the above embodiment, and any change, modification, replacement, combination, simplification made without departing from the spirit and principle of the present application should be an equivalent replacement mode, which is included in the protection scope of the present application.

Claims

1. An integrated small nucleic acid detection device, characterized by: The application relates to a closed device composed of a sleeve, a shell and a base, wherein the upper half of the inner wall of the shell is provided with two center-symmetrical spiral tracks; The sleeve comprises an integral sleeve knob and a sleeve wall, the sleeve wall extends into the shell, the sleeve knob is clamped on the upper end of the shell, and two symmetrical vertical guide rails are arranged on the two sides of the sleeve wall; The device further comprises a puncture suspender, the puncture suspender is located in the sleeve, two protrusions are arranged at the top end of the puncture suspender, the two protrusions are embedded into the spiral tracks of the inner wall of the shell through the guide rails on the sleeve wall, the puncture suspender vertically moves up and down along with the rotation of the sleeve, and the end of the puncture suspender is an arrowhead; The device further comprises a reagent bin, the reagent bin is arranged at the lower half of the shell; The top of the shell is in sealed connection with the sleeve, the bottom of the shell is in sealed connection with the base, and the bottom of the shell is in sealed connection with the base; The reagent bin comprises a plurality of reagent pools and a water absorption pool, the reagent pools are filled with reagents, the top surface and the bottom surface of the reagent pools are both thin membranes which can be easily punctured, the reagent pools are arranged in sequence from top to bottom to successively hold a suspension liquid of magnetic microparticles and cell lysate, a washing liquid, an eluent and a nucleic acid amplification reagent, a water absorption pool is arranged below the reagent pools for holding the suspension liquid and the washing liquid, the top of the water absorption pool is empty, the center of the water absorption pool is a hollow circular hole, a plurality of fences are arranged around the circular hole, and the cavity outside the fences is filled with water absorption cotton; the device further comprises a magnet, and the magnet is located in a column above the arrowhead end of the puncture suspender.

2. The integrated small nucleic acid detection device according to claim 1, wherein: The shell 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.

3. The integrated small nucleic acid detection device according to claim 1, wherein: The wall thicknesses of the upper, middle and lower parts of the shell are different, the wall thickness of the lower part corresponding to the reagent bin is the thinnest, the wall thickness of the middle part corresponding to the sample adding hole is larger than that of the part corresponding to the reagent bin, and the wall thickness of the upper part corresponding to the sleeve is the thickest.

4. The method of using the integrated small nucleic acid detection device according to claim 2, characterized in that The steps of the device include the following steps: (1) a sample solution of nucleic acid to be extracted is added into the first layer of reagent pools by puncturing the top surface thin membrane of the first layer of reagent pools through the sample adding hole on the shell of the card box; (2) the magnet on the suspender is moved downwards to the first layer of reagent pools by rotating the sleeve, so that the magnetic microparticles which have combined with the sample nucleic acid are all adsorbed to the surface of the column containing the magnet above the arrowhead; (3) the arrowhead punctures the bottom surface thin membrane of the first layer of reagent pools by continuously rotating the sleeve, so that the liquid in the reagent pools is discharged and is completely absorbed by the water absorption cotton; (4) the arrowhead enters the next layer or layers of reagent pools by continuously rotating the sleeve, the magnetic microparticles on the surface of the magnet are cleaned by using the washing liquid in the reagent pools, and the waste liquid after cleaning is absorbed by the water absorption cotton; (5) the arrowhead enters the next layer of reagent pools by continuously rotating the sleeve, the nucleic acid on the magnetic microparticles is eluted by immersing the magnetic microparticles in the eluent in the reagent pools; (6) the extracted nucleic acid is injected into the reagent pool at the bottom by continuously rotating the sleeve, and is mixed with the nucleic acid amplification reagent in the reagent pool; (7) the mixed liquid is injected into the base by continuously rotating the sleeve, and amplification detection is carried out.

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