A sealed nucleic acid detection device and use method

Abstract

The present invention discloses a sealed nucleic acid detection device and a method of use, which belongs to the field of nucleic acid detection technology, and solves the problems of high difficulty in detection operation, easy contamination, low detection accuracy and stability of existing nucleic acid detection devices. The sealed nucleic acid detection device of the present invention is provided with a first chamber, a second chamber, a nucleic acid detection device, a magnetic bead channel and nano-magnetic beads and reagents of different shapes, so that each functional area can be physically separated, and the magnetic bead channel is connected by providing a magnetic bead channel, so that the nano-magnetic beads can move between different areas along each magnetic bead channel under the action of a magnetic field, thereby realizing the extraction, purification and nucleic acid post-processing of nucleic acid samples. At the same time, since the entire nano-magnetic bead movement process does not require other treatments such as heating, this can not only simplify the operation of nucleic acid detection, but also improve the stability and flexibility of nucleic acid detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a sealed nucleic acid detection device and a use method thereof. Background Art

[0002] Nucleic acid amplification detection technology is widely used in the rapid detection and identification of bacteria, fungi, viruses and parasites, as well as in the early screening and diagnosis of diseases due to its advantages such as rapidity, sensitivity, specificity and accuracy. Existing nucleic acid detection usually uses solid phase separation to separate the lysis solution, cleaning solution, eluent and nucleic acid amplification solution, and then uses a mechanical device to perform corresponding operations to gradually extract the nucleic acid from the sample and perform the test. However, due to the high sensitivity of nucleic acid amplification detection technology, it is necessary to effectively reduce the sample contamination caused during the sample extraction process and reduce the occurrence of false positives. Therefore, the existing devices and technologies have the problems of cumbersome operation, low detection efficiency and low detection flexibility. Summary of the invention

[0003] In view of the above analysis, the present invention provides a sealed nucleic acid detection device and a method of use, which are used to solve the problems of high difficulty in detection operation, easy contamination, low detection accuracy and stability of existing nucleic acid detection devices.

[0004] The invention provides a sealed nucleic acid detection device, comprising a shell, a sample port, a first chamber, a second chamber, a reagent port, a nucleic acid detection device, a magnetic bead channel, a sealing member and nano-magnetic beads; a first partition is arranged between the first chamber and the second chamber; a liquid-phase hydrophobic liquid and a lysis liquid are filled in the first chamber; a liquid-phase hydrophobic liquid and a first cleaning liquid are filled in the second chamber; the sample port is arranged at the upper end of the first chamber of the shell; reagent ports are respectively arranged at the upper ends of the first chamber and the second chamber; the magnetic bead channel is arranged at the upper end of the first partition, the height of the magnetic bead channel is located in the height area where the liquid-phase hydrophobic liquid is located, and is used to pass through the first chamber and the second chamber; the first chamber is arranged upstream of the second chamber, and the nucleic acid detection device is arranged downstream of the second chamber; the second chamber is connected with the nucleic acid detection device through the magnetic bead channel arranged on the wall plate of the shell; a reagent port is arranged on the nucleic acid detection device, and the nucleic acid detection device is filled with liquid-phase hydrophobic liquid and nucleic acid amplification reaction liquid; the sealing member is used to seal the sample port and the reagent port.

[0005] Optionally, the liquid phase hydrophobic liquid is paraffin oil or mineral oil.

[0006] Optionally, the lysis buffer includes guanidine cyanate isosulfate and disodium ethylenediaminetetraacetic acid.

[0007] Optionally, the first cleaning solution includes alcohol and sodium acetate.

[0008] Optionally, a second partition is provided in the second chamber, and the second chamber is divided into a first cleaning zone and a second cleaning zone by the second partition; the first cleaning zone is provided between the first chamber and the second cleaning zone; the first cleaning zone is filled with liquid-phase hydrophobic liquid and the first cleaning liquid; the second cleaning zone is filled with liquid-phase hydrophobic liquid and the second cleaning liquid; a magnetic bead channel is provided at the upper end of the second partition, and the height of the magnetic bead channel is located in the height area where the liquid-phase hydrophobic liquid is located; the second cleaning zone is connected to the nucleic acid detection device through the magnetic bead channel provided on the wall panel of the shell; reagent ports are provided at the upper end of the second chamber corresponding to the first cleaning zone and the second cleaning zone, respectively.

[0009] Optionally, the second cleaning solution includes tris(hydroxymethyl)aminomethane and sodium acetate.

[0010] Optionally, the nucleic acid detection device includes a solid phase segmentation chamber and a nucleic acid amplification region; the solid phase segmentation chamber is located above the nucleic acid amplification region.

[0011] Optionally, the solid phase separation chamber is triangular in shape, and a plurality of conical diversion blocks are arranged in the solid phase separation chamber.

[0012] Optionally, the sealing member is a cover.

[0013] On the other hand, the present invention also provides a method for using the aforementioned sealed nucleic acid detection device, and the specific steps include: placing the closed nucleic acid detection device vertically and allowing it to stand until the liquid hydrophobic liquid is located above the lysis solution, the first cleaning solution and / or the second cleaning solution and the amplification reaction solution; adding a support and nanomagnetic beads containing a nucleic acid sample to the lysis solution area, and after the cells of the nucleic acid sample are lysed to release the nucleic acid, the nucleic acid is adsorbed to the nanomagnetic beads to complete the extraction of the nucleic acid; then, the nanomagnetic beads are driven by a magnetic field to pass through the liquid hydrophobic liquid and through the magnetic bead channel into the solid phase separation chamber and into the nucleic acid amplification area; finally, the nucleic acid in the nucleic acid amplification area is detected by a detection device. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the sealed nucleic acid detection device of the present invention.

[0015] Reference numerals:

[0016] 1. Shell; 2. Sample port; 3. Liquid-phase hydrophobic liquid; 4. Lysis solution; 5. First cleaning area; 6. Second cleaning area; 7. Reagent port; 8. Diverter block; 9. Nucleic acid amplification area; 10. Nucleic acid detection device; 11. Magnetic bead channel. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0018] A specific embodiment of the present invention is shown in the attached Figure 1, discloses a sealed nucleic acid detection device, including a shell 1, a sample port 2, a first chamber, a second chamber, a reagent port 7, a nucleic acid detection device 10, a magnetic bead channel 11, a seal and nano-magnetic beads; a first partition is arranged between the first chamber and the second chamber; the first chamber is filled with a liquid-phase hydrophobic liquid 3 and a lysis liquid 4; the second chamber is filled with a liquid-phase hydrophobic liquid 3 and a first cleaning liquid; the sample port 2 is arranged at the upper end of the first chamber of the shell 1; the upper ends of the first chamber and the second chamber are respectively provided with reagent ports 7; a magnetic bead channel 11 is arranged at the upper end of the first partition Channel 11, the height of the magnetic bead channel 11 is located in the height area where the liquid-phase hydrophobic liquid 3 is located, and is used to pass through the liquid-phase hydrophobic liquid 3 in the first chamber and the second chamber; the first chamber is arranged upstream of the second chamber, and the nucleic acid detection device 10 is arranged downstream of the second chamber; the second chamber is connected with the nucleic acid detection device 10 through the magnetic bead channel 11 arranged on the wall panel of the shell 1; the nucleic acid detection device 10 is provided with a reagent port 7, and the nucleic acid detection device 10 is filled with the liquid-phase hydrophobic liquid 3 and the nucleic acid amplification reaction liquid; the sealing member is used to seal the sample port 2 and the reagent port 7.

[0019] Optionally, a second partition is provided in the second chamber, and the second chamber is divided into a first cleaning zone 5 and a second cleaning zone 6 by the second partition; the first cleaning zone 5 is provided between the first chamber and the second cleaning zone 6; the first cleaning zone 5 is filled with a liquid-phase hydrophobic liquid 3 and a first cleaning liquid; the second cleaning zone 6 is filled with a liquid-phase hydrophobic liquid 3 and a second cleaning liquid; a magnetic bead channel 11 is provided at the upper end of the second partition, and the height of the magnetic bead channel 11 is located in the height area where the liquid-phase hydrophobic liquid 3 is located; the second cleaning zone 6 is connected to the nucleic acid detection device 10 through the magnetic bead channel 11 provided on the wall panel of the shell 1; reagent ports 7 are respectively provided at the upper end of the second chamber and at positions corresponding to the first cleaning zone 5 and the second cleaning zone 6.

[0020] Optionally, the liquid-phase hydrophobic liquid 3 is paraffin oil or mineral oil; the liquid-phase hydrophobic liquid 3 passes through the various partition areas of the sealed nucleic acid detection device through the magnetic bead channel 11, so that the nanomagnetic beads can effectively move through the magnetic bead channel 11 in each area under the action of the external magnetic field, thereby achieving the effect of nucleic acid extraction, purification and lysis.

[0021] Optionally, the lysis solution 4 includes 4-5 mol / L guanidine cyanide sulfate and 20-50 mmol / L disodium ethylenediaminetetraacetic acid; preferably, the guanidine cyanide sulfate is 4 mol / L; the lysis solution 4 can effectively lyse the cells in the sample to achieve the effect of releasing nucleic acids.

[0022] Optionally, the first cleaning solution includes 60-80% alcohol and 0.1-1 mol / L sodium acetate; the cleaning solution of the second cleaning solution 6 includes 20-50 mmol / L tris(hydroxymethyl)aminomethane and 0.1-1 mol / L sodium acetate. Both the first cleaning solution 5 and the second cleaning solution 6 are water-soluble cleaning agents, which can further remove proteins and water-soluble substances on the surface of the nanomagnetic beads, thereby achieving the purpose of further purification.

[0023] Optionally, the nucleic acid detection device 10 includes a solid phase separation chamber and a nucleic acid amplification area 9; the solid phase separation chamber is located above the nucleic acid amplification area 9; a plurality of nucleic acid amplification areas 9 are provided, the plurality of nucleic acid amplification areas 9 are independent of each other, and a third partition is provided between each nucleic acid amplification area 9; a fourth partition is provided at the upper end of each nucleic acid amplification area 9, and a plurality of magnetic bead channels 11 are provided on the fourth partition corresponding to each nucleic acid amplification area 9; and different types of nucleic acid amplification reaction solutions are filled in the plurality of nucleic acid amplification areas 9.

[0024] Optionally, the first partition plate, the second partition plate and the third partition plate are perpendicular to the bottom edge of the shell 1 , and the fourth partition plate is parallel to the bottom edge of the shell 1 .

[0025] Optionally, after all reagents are filled in the sealed nucleic acid detection device, because the density of paraffin oil or mineral oil is lighter than that of aqueous reagents and they are immiscible, the liquid hydrophobic liquid 3 will float on the surface of the aqueous reagent after filling, thereby producing a boundary, that is, the liquid hydrophobic liquid 3 is located above the lysis solution 4, the first cleaning solution, the second cleaning solution and the amplification reaction solution.

[0026] Optionally, the pore size of the magnetic bead channel 11 is 0.5mm-1mm. After the pore size of the magnetic bead channel 11 is matched with the liquid-phase hydrophobic liquid 3, lysate 4, first cleaning liquid, second cleaning liquid and amplification reaction liquid of the aforementioned components filled in the shell 1 and the nucleic acid detection device 10, the various liquids will form a specific surface tension at the magnetic bead channel 11, thereby avoiding the problem of liquid mixing caused by inversion of the device during transportation.

[0027] Optionally, the solid phase separation chamber is filled with a liquid hydrophobic liquid 3; the solid phase separation chamber is triangular, and a plurality of conical shunt blocks 8 are provided in the solid phase separation chamber to achieve the purpose of effectively and evenly separating the nanomagnetic beads. The nanomagnetic beads sink under the action of gravity, and due to the conical surface of the conical shunt blocks 8, they can be effectively separated. After multiple separations of the conical shunt blocks 8, the nanomagnetic beads can effectively enter the nucleic acid amplification area 9; the solid phase separation chamber and the plurality of nucleic acid amplification areas 9 are provided with reagent ports 7.

[0028] Optionally, multiple rows of conical shunt blocks 8 are arranged, and the number of each row is arranged in the solid phase separation chamber in increasing order from top to bottom, and the conical shunt blocks 8 in the last row adjacent to the nucleic acid amplification area 9 are located directly above the third partition plate; a magnet is arranged at the bottom of the nucleic acid amplification area 9, and the magnet reciprocates parallel to the bottom edge of the shell 1, and with the reciprocating movement, the nano-magnetic beads falling by gravity fall on the fourth partition plate, and can enter each nucleic acid amplification area 9 through the magnetic bead channel 11 on the fourth partition plate under the reciprocating drive of the magnet. Preferably, the magnet is detachably arranged outside the nucleic acid amplification area 9.

[0029] Optionally, the nanomagnetic beads are magnetic iron balls, magnetic balls or steel balls, etc.; the sealing member is a cover.

[0030] Compared with the prior art, the present invention physically separates each functional area by filling the first chamber, the first cleaning area 5, the second cleaning area 6 and the nucleic acid detection device 10 in the housing 1 with oil and water reagents, and connects by setting the magnetic bead channel 11, so that the nanomagnetic beads can move between different areas along each magnetic bead channel 11 under the action of the external magnetic field, thereby realizing the extraction, purification and nucleic acid post-processing of nucleic acid samples. At the same time, since the entire nanomagnetic bead movement process does not require other treatments such as heating, this can not only simplify the operation of nucleic acid detection, but also improve the stability and flexibility of nucleic acid detection. At the same time, due to the high salt solution and low pH value of the nucleic acid lysing solution of the present invention, the released nucleic acid can be effectively combined with the magnetic beads, and the magnetic beads effectively enrich and reduce the volume of the released nucleic acid; when the magnetic beads enter the liquid phase hydrophobic liquid 3 of the lysis zone from the lysis solution 4 under the action of the external magnetic field, since protein substances and the like are all water-soluble substances, they can be effectively isolated in the lysis zone 4, and the nucleic acid is adsorbed on the surface of the magnetic beads. The magnetic beads adsorbed with the nucleic acid enter the liquid phase hydrophobic liquid 3 under the action of the external magnetic field and enter the second chamber through the magnetic bead channel 11; when the first cleaning zone 5 and the second cleaning zone 6 are provided, the method of use is the same. In addition, the nucleic acid cleaning solution filled in the first cleaning zone 5 and the second cleaning zone 6 can effectively remove various impurities on the nanomagnetic beads, thereby achieving the effect of purifying nucleic acid.

[0031] The present invention adds a liquid-phase hydrophobic liquid 3 in the first chamber, the second chamber and the nucleic acid detection device 10. The presence of the liquid-phase hydrophobic liquid 3 is not only conducive to the movement of the nanomagnetic beads between different areas, but also effectively blocks the cross-flow of reagents between the first chamber, the second chamber and the different functional areas of the nucleic acid detection device 10; at the same time, during the movement, the surface of the nanomagnetic beads is coated with a liquid-phase hydrophobic substance, which can not only effectively improve the effect of nucleic acid extraction, but also prevent various inhibitors from entering the nucleic acid amplification area 9.

[0032] The assembly process of various reagents of the present invention is as follows: first, the lysate, the first cleaning solution and / or the second cleaning solution and the multiple nucleic acid amplification reaction solution are respectively filled into each different functional area through the reagent port 7; then, the liquid phase hydrophobic liquid 3 is filled through the reagent port 7, so that each different functional area is connected to each other through the liquid phase hydrophobic liquid 3; finally, the lid is closed to form a sealed nucleic acid detection device. When the detection application is finally performed, the solid phase support (such as a cotton swab) or liquid sample (blood, cell eluate, etc.) stained with the sample and the nanomagnetic beads are added to the area of ​​the lysate 4 through the sample port 2.

[0033] The use process of the present invention is as follows: the closed nucleic acid detection device is placed vertically, and after standing for a period of time, the liquid-phase hydrophobic liquid 3 is located above the lysing liquid 4, the first cleaning liquid, the second cleaning liquid and the amplification reaction liquid; a solid-phase support (such as a cotton swab) or a liquid (blood, cell eluate, etc.) containing a nucleic acid sample and nano-magnetic beads are added to the area of ​​the lysing liquid 4 through the sample port 2, so that the cells of the nucleic acid sample can be fully lysed, and the nucleic acid is released to be effectively adsorbed and combined with the nano-magnetic beads to complete the extraction of the nucleic acid; then, the nano-magnetic beads are driven by an external magnetic field to pass through the liquid-phase hydrophobic liquid 3 and through the magnetic bead channel 11 through the first cleaning area 5, and then through the second cleaning area 6, so as to achieve the purpose of purifying and concentrating the nucleic acid sample; then, the nano-magnetic beads are driven by an external magnetic field to pass through the liquid-phase hydrophobic liquid 3 and through the magnetic bead channel 11 into the solid-phase separation chamber, and the nano-magnetic beads fall into the nucleic acid amplification area 9 under the action of gravity; finally, the nucleic acid sample in the nucleic acid amplification area 9 is subjected to real-time amplification detection by an optical detection device.

[0034] When the solid phase separation chamber is provided with a conical diversion block 8, effective separation can be performed. After multiple separations by the conical diversion block 8, the nanomagnetic beads can effectively enter the nucleic acid amplification area 9; the PCR amplification reaction solution (low salt and neutral pH) in the nucleic acid amplification area 9 achieves the purpose of releasing and amplifying the nucleic acid carried by the nanomagnetic beads.

[0035] When multiple nucleic acid amplification areas 9 are set up and each nucleic acid amplification area 9 is filled with a different type of nucleic acid amplification reaction solution, in order to allow the nanomagnetic beads to smoothly enter the nucleic acid amplification area 9, when the nanomagnetic beads reach the top of the solid phase separation chamber, the external magnetic field driving the nanomagnetic beads to move in the shell 1 is removed, and a magnet is set at the bottom of the nucleic acid amplification area 9 for reciprocating movement. After the nanomagnetic beads fall by gravity and fall on the fourth partition plate, they can enter each nucleic acid amplification area 9 through the magnetic bead channel 11 on the fourth partition plate under the reciprocating drive of the magnet.

[0036] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A sealed nucleic acid detection device, characterized in that: It comprises a shell, a sample port, a first chamber, a second chamber, a reagent port, a nucleic acid detection device, a magnetic bead channel, a seal and nano-magnetic beads; a first partition is arranged between the first chamber and the second chamber; the first chamber is filled with a liquid-phase hydrophobic liquid and a lysis liquid; the second chamber is filled with a liquid-phase hydrophobic liquid and a first cleaning liquid; the sample port is arranged at the upper end of the first chamber of the shell; the upper ends of the first chamber and the second chamber are respectively provided with reagent ports; the magnetic bead channel is arranged at the upper end of the first partition, the height of the magnetic bead channel is located in the height area where the liquid-phase hydrophobic liquid is located, and is used to pass through the first chamber and the second chamber; the first chamber is arranged upstream of the second chamber, and the nucleic acid detection device is arranged downstream of the second chamber; the second chamber is connected with the nucleic acid detection device through a magnetic bead channel arranged on the wall plate of the shell, and the magnetic bead channel is located at the upper end of the wall plate of the shell; a reagent port is arranged on the nucleic acid detection device, and the nucleic acid detection device is filled with a liquid-phase hydrophobic liquid and a nucleic acid amplification reaction liquid; The sealing member is used to seal the sample port and the reagent port; A second partition is arranged in the second chamber, and the second chamber is divided into a first cleaning zone and a second cleaning zone by the second partition; the first cleaning zone is arranged between the first chamber and the second cleaning zone; the first cleaning zone is filled with liquid-phase hydrophobic liquid and the first cleaning liquid; the second cleaning zone is filled with liquid-phase hydrophobic liquid and the second cleaning liquid; a magnetic bead channel is arranged at the upper end of the second partition, and the height of the magnetic bead channel is located in the height area where the liquid-phase hydrophobic liquid is located; the second cleaning zone is connected with the nucleic acid detection device through the magnetic bead channel arranged on the wall panel of the shell; reagent ports are respectively arranged at the upper end of the second chamber and at positions corresponding to the first cleaning zone and the second cleaning zone.

2. The sealed nucleic acid detection device according to claim 1, characterized in that: The liquid phase hydrophobic liquid is paraffin oil or mineral oil.

3. The sealed nucleic acid detection device according to claim 1, characterized in that: The lysis buffer includes guanidine cyanate isosulfate and disodium ethylenediaminetetraacetic acid.

4. The sealed nucleic acid detection device according to claim 1, characterized in that: The first cleaning solution includes alcohol and sodium acetate.

5. The sealed nucleic acid detection device according to any one of claims 1 to 4, characterized in that: The second cleaning solution includes tris(hydroxymethyl)aminomethane and sodium acetate.

6. The sealed nucleic acid detection device according to claim 1, characterized in that: The nucleic acid detection device includes a solid phase segmentation chamber and a nucleic acid amplification area; the solid phase segmentation chamber is located above the nucleic acid amplification area.

7. The sealed nucleic acid detection device according to claim 1, characterized in that: The solid phase separation chamber is triangular in shape and is provided with a plurality of conical flow dividing blocks.

8. The sealed nucleic acid detection device according to claim 1, characterized in that: The sealing member is the lid.

9. A method for using the sealed nucleic acid detection device according to any one of claims 1 to 8, characterized in that: The specific steps include: vertically placing the closed nucleic acid detection device and allowing it to stand until the liquid hydrophobic liquid is located above the lysis liquid, the first cleaning liquid and / or the second cleaning liquid and the amplification reaction liquid; adding a support containing a nucleic acid sample and nanomagnetic beads to the lysis liquid area, and after the nucleic acid is released from the cell lysis of the nucleic acid sample, the nucleic acid is adsorbed to the nanomagnetic beads to complete the extraction of the nucleic acid; then, the nanomagnetic beads are driven by a magnetic field to pass through the liquid hydrophobic liquid and through the magnetic bead channel into the solid phase separation chamber and into the nucleic acid amplification area; finally, the nucleic acid in the nucleic acid amplification area is detected by a detection device.

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