A nucleic acid detection cartridge and a nucleic acid detection method for non-disease diagnostic purposes

Abstract

The application relates to the field of biological medical technology, and discloses a nucleic acid detection box and a nucleic acid detection method for non-disease diagnosis purposes. The nucleic acid detection box comprises a box body, a piston, a valve body, a chamber sealing piece, an amplification detection chip and a capacitance detection electrode. The box body is provided with a plurality of chambers with openings, the plurality of chambers comprise a mixing chamber, a sample injection chamber and a plurality of reagent chambers, and the openings of the mixing chamber and the reagent chambers are provided with chamber sealing pieces. The plurality of reagent chambers comprise a lysis solution chamber, a washing solution chamber, an eluent chamber and a PCR reaction liquid chamber, the lysis solution chamber contains a lysis solution and a magnetic bead mixed solution, the washing solution chamber contains a washing solution, the eluent chamber contains an eluent, and the PCR reaction liquid chamber contains a PCR reaction liquid; the amplification detection chip is provided with reagent microspheres. The mixing chamber is provided with the piston, and the top end of the piston is provided with a connecting part. The valve body is arranged on the box body, and the valve body is provided with an operation part exposed to the box body. The capacitance detection electrode is arranged on the box body.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nucleic acid detection kit and a nucleic acid detection method for non-disease diagnostic purposes. Background Technology

[0002] With the rapid development of molecular diagnostic technology, nucleic acid testing has become a key means in the field of in vitro diagnostics (IVD) for pathogen screening, genotyping, and diagnosis of infectious diseases. Traditional nucleic acid testing typically involves multiple independent steps, including sample pretreatment, nucleic acid extraction, reaction system construction, and amplification detection.

[0003] However, existing testing solutions lack automation and integration, and the operation process requires a high degree of human intervention, which is not only cumbersome and time-consuming, but also prone to sample contamination during the testing process.

[0004] Therefore, there is an urgent need for a nucleic acid detection kit and a nucleic acid detection method for non-disease diagnosis purposes to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a nucleic acid detection kit and a nucleic acid detection method for non-disease diagnosis purposes, which improves the degree of automation and integration, reduces the risk of sample contamination during the detection process, and improves the accuracy and speed of operation.

[0006] To achieve this objective, the present invention adopts the following technical solution: A nucleic acid detection kit includes a box body, a piston, a valve body, a chamber seal, an amplification detection chip, and a capacitive detection electrode; The box body is provided with multiple chambers with openings, the multiple chambers including a mixing chamber, a sample injection chamber and multiple reagent chambers, and the openings of the mixing chamber and the reagent chambers are provided with chamber seals; The plurality of reagent chambers include a lysis buffer chamber, a washing buffer chamber, an elution buffer chamber, and a PCR reaction chamber. The lysis buffer chamber contains a mixture of lysis buffer and magnetic beads, the washing buffer chamber contains washing buffer, the elution buffer chamber contains elution buffer, and the PCR reaction chamber contains PCR reaction solution. The amplification detection chip is provided with reagent microspheres. The piston is disposed inside the mixing chamber, and a connecting part is provided at the top of the piston. The connecting part is used to connect with an external operating structure to drive the piston. The valve body is disposed on the housing, and the valve body is provided with an operating part exposed outside the housing. The operating part is used to drive the valve body to control the connection and disconnection between the mixing chamber and the sample injection chamber, between the mixing chamber and each of the reagent chambers, and between the mixing chamber and the amplification detection chip. The capacitance detection electrode is disposed on the housing and is used to monitor the capacitance change in the mixing chamber.

[0007] As an improvement to the above technical solution, the capacitance detection electrode is provided with a connecting contact point, which is exposed outside the housing.

[0008] As an improvement to the above technical solution, multiple amplification detection chips are provided, and the multiple amplification detection chips are spaced apart along a first direction. The valve body can control the connection and disconnection between the mixing chamber and each of the amplification detection chips. The multiple chambers are spaced apart along the first direction, which is the length direction of the valve body.

[0009] As an improvement to the above technical solution, the box body is further provided with a mounting cavity, the valve body is disposed in the mounting cavity, and the operating part is used to drive the valve body to rotate relative to the box body; Each of the chambers is connected to the mounting cavity. The valve body is provided with a valve cavity extending along the first direction. The valve body is provided with a plurality of first flow holes, a plurality of second flow holes and a plurality of third flow holes. Multiple first flow holes are spaced apart along the first direction and spaced apart along the circumference of the valve body, driving the valve body to rotate relative to the housing, so that the sample injection chamber and each of the reagent chambers can be connected to the valve chamber through the first flow holes respectively; Multiple second flow holes are spaced apart along the first direction and spaced apart along the circumference of the valve body, driving the valve body to rotate relative to the housing, so that each amplification detection chip can communicate with the valve cavity through the second flow holes respectively; The plurality of third flow holes are all disposed at one end of the valve body along the first direction and are spaced apart circumferentially along the valve body, so that when the sample injection chamber, the reagent chamber or the amplification detection chip is connected to the valve chamber, the mixing chamber can be connected to the valve chamber through the third flow holes.

[0010] As an improvement to the above technical solution, the amplification detection chips are all connected to the housing. The amplification detection chip is provided with an amplification detection chamber, a reagent inlet channel and an exhaust channel. The reagent inlet channel is used to connect the amplification detection chamber and the mounting chamber, and the exhaust channel connects the amplification detection chamber and the outside of the amplification detection chip.

[0011] As an improvement to the above technical solution, the reagent entry channel includes a first channel section and a second channel section connected in sequence; One end of the first flow channel is connected to the mounting cavity, and the other end is connected to one end of the second flow channel. The other end of the second flow channel is connected to the amplification detection cavity. The cross-sectional size of the second flow channel is smaller than that of the first flow channel. The reagent microspheres are disposed within the first flow channel.

[0012] As an improvement to the above technical solution, the amplification detection chip also includes a waterproof and breathable plug, and the waterproof and breathable plug is disposed in the exhaust channel.

[0013] As an improvement to the above technical solution, a cover is also included, which is detachably connected to the box body to cover the opening of the sample injection cavity.

[0014] A nucleic acid detection method for non-disease diagnosis purposes, using the nucleic acid detection kit described in any one of the above claims, includes the following steps: S1. Inject the sample to be tested into the sample injection chamber through the chamber seal, insert the external operating structure into the mixing chamber through the chamber seal, and connect it to the piston through the connecting part to puncture the chamber seal at the opening of each reagent chamber. S2. Drive the valve body and the piston, and monitor the liquid transfer process through the capacitance detection electrode, so that the sample to be tested in the sample injection chamber is mixed with the lysis buffer and magnetic bead mixture in the mixing chamber to form a first mixture, and the first mixture is contained in the lysis buffer chamber. Use an ultrasonic device to perform ultrasonic lysis on the first mixture in the lysis buffer chamber so that the magnetic beads in the first mixture adsorb nucleic acid molecules. S3. Using a magnetic device, magnetic beads in the lysis chamber are attracted to the wall of the lysis chamber, driving the valve body and the piston, and monitoring the liquid transfer process through the capacitance detection electrode, so that the liquid in the lysis chamber enters the sample injection chamber through the mixing chamber; S4. Drive the valve body and the piston, and monitor the liquid transfer process through the capacitor detection electrode, so that the cleaning liquid in the cleaning liquid chamber is mixed with the magnetic beads in the lysis liquid chamber to clean the magnetic beads, and drive the waste liquid generated by cleaning into the sample injection chamber, and the cleaned magnetic beads are contained in the lysis liquid chamber. S5. Drive the valve body and the piston, and monitor the liquid transfer process through the capacitive detection electrode to mix the eluent in the elution chamber with the magnetic beads in the lysis chamber to elute the nucleic acid molecules on the magnetic beads. Use a magnetic device to adsorb the magnetic beads onto the wall of the lysis chamber, and drive the eluent containing nucleic acid molecules to mix with the PCR reaction solution in the PCR reaction chamber through the mixing chamber to form a second mixture. S6. Drive the valve body and the piston to allow the second mixture to enter the amplification detection chip and dissolve the reagent microspheres to form a third mixture. Amplify and collect fluorescence in the third mixture within the amplification detection chip.

[0015] As an improvement to the above technical solution, the monitoring of the liquid transfer process through the capacitance detection electrode includes: As the piston moves upward to allow liquid from other chambers to enter the mixing chamber, the capacitance change in the mixing chamber is monitored by the capacitance detection electrode. The system determines whether the pipetting is complete based on the capacitance change in the mixing chamber. If the pipetting is complete, the piston is controlled to stop moving upward.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The nucleic acid detection kit and method for non-disease diagnosis of the present invention include a lysis buffer chamber, a washing buffer chamber, an elution buffer chamber, a PCR reaction chamber, and reagent microspheres, all housed within the nucleic acid detection kit. In use, the sample to be tested is injected into the sample injection chamber. An external device passes through the chamber seal and connects to the piston via a connector at the top of the piston, puncturing the chamber seals at the openings of each reagent chamber. Then, the external device operates the piston and valve body according to a specific sequence (operating the valve body via an operating part). Combined with external ultrasonic equipment, magnetic equipment, and fluorescence acquisition equipment, the kit can complete operations such as lysis, washing, nucleic acid molecule elution, amplification, and fluorescence acquisition. It boasts a high degree of automation and integration, and effectively reduces the risk of sample contamination during the detection process.

[0017] By monitoring the capacitance changes within the mixing chamber using capacitance detection electrodes, the liquid transfer process between the mixing chamber and the sample injection chamber, between the mixing chamber and each reagent chamber, and between the mixing chamber and the amplification detection chip can be accurately monitored. This allows for the rapid and accurate completion of each liquid transfer step, improving operational accuracy and speed. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of the nucleic acid detection kit provided in the embodiment of the present invention. Figure 1 ; Figure 2 This is a schematic diagram of the structure of the nucleic acid detection kit provided in the embodiment of the present invention. Figure 2 ; Figure 3 This is a schematic diagram of the structure of the nucleic acid detection kit provided in the embodiment of the present invention. Figure 3 ; Figure 4 This is a cross-sectional view of the nucleic acid detection kit provided in an embodiment of the present invention. Figure 1 ; Figure 5This is a cross-sectional view of the nucleic acid detection kit provided in an embodiment of the present invention. Figure 2 ; Figure 6 yes Figure 5 Enlarged view of point A in the middle; Figure 7 This is a schematic diagram of a partial structure of the nucleic acid detection kit provided in an embodiment of the present invention. Figure 1 ; Figure 8 This is a schematic diagram of a partial structure of the nucleic acid detection kit provided in an embodiment of the present invention. Figure 2 ; Figure 9 This is a schematic diagram of the valve body of the nucleic acid detection kit provided in this embodiment of the invention. Figure 1 ; Figure 10 This is a schematic diagram of the valve body of the nucleic acid detection kit provided in this embodiment of the invention. Figure 2 ; Figure 11 This is a schematic diagram of a partial structure of the nucleic acid detection kit provided in an embodiment of the present invention. Figure 3 ; Figure 12 This is a schematic diagram of the capacitive detection electrode of the nucleic acid detection kit provided in this embodiment of the invention.

[0019] In the picture: X, first direction; 1. Box body; 11. Mixing chamber; 12. Sample injection chamber; 13. Lysis buffer chamber; 14. Washing buffer chamber; 15. Elution buffer chamber; 16. PCR reaction chamber; 17. Slot; 18. Second connecting channel; 2. Piston; 21. Connecting part; 3. Valve body; 31. Operating part; 32. Valve cavity; 33. First flow passage; 34. Second flow passage; 35. Third flow passage; 36. Limiting part; 4. Chamber sealing components; 5. Amplification detection chip; 51. Amplification detection chamber; 52. Reagent inlet channel; 521. First flow channel section; 522. Second flow channel section; 53. Exhaust channel; 54. Chip body; 55. Top cover; 6. Capacitance detection electrode; 61. Connect the contact points; 62. First electrode; 621. First connecting arm; 622. First sheet electrode structure; 623. First comb-shaped teeth; 63. Second electrode; 631. Second connecting arm; 632. Second comb-shaped teeth; 64. Third electrode; 641. Third connecting arm; 642. Second sheet electrode structure; 65. Grassroots level; 7. Cover; 8. Sealing sleeve; 81. Positioning rib; 9. Chip socket; 91. Socket body; 911. First connecting channel; 92. Buckle; 93. Sealing ring. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms "connected," "linked," and "fixed" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0022] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0023] In the description of this embodiment, the terms "upper," "lower," "right," etc., refer to the orientation or positional relationship shown in the accompanying drawings. They are used only for ease of description and simplification of operation, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. In addition, the terms "first" and "second" are used only for distinction in description and have no special meaning.

[0024] like Figures 1-7As shown, this embodiment provides a nucleic acid detection kit, including a box body 1, a piston 2, a valve body 3, a chamber seal 4, an amplification detection chip 5, and a capacitive detection electrode 6. The box body 1 has multiple chambers with openings, including a mixing chamber 11, a sample injection chamber 12, and multiple reagent chambers. Chamber seals 4 are provided at the openings of the mixing chamber 11 and each reagent chamber. The multiple reagent chambers include a lysis buffer chamber 13, a washing buffer chamber 14, an elution buffer chamber 15, and a PCR reaction chamber 16. The lysis buffer chamber 13 contains a mixture of lysis buffer and magnetic beads; the washing buffer chamber 14 contains washing buffer; the elution buffer chamber 15 contains elution buffer; and the PCR reaction chamber 16 contains PCR reaction solution. Reagent microspheres are disposed within the amplification detection chip 5. A piston 2 is disposed within the mixing chamber 11, and a connecting part 21 is provided at the top of the piston 2 for connecting to an external operating structure to drive the piston 2. A valve body 3 is mounted on the housing 1, and the valve body 3 has an exposed operating part 31. The operating part 31 is used to drive the valve body 3 to control the connection and disconnection between the mixing chamber 11 and the sample injection chamber 12, between the mixing chamber 11 and each reagent chamber, and between the mixing chamber 11 and the amplification detection chip 5. A capacitance detection electrode 6 is mounted on the housing 1 and is used to monitor the capacitance change within the mixing chamber 11. In this embodiment, the connecting part 21 is a blind hole with internal threads. The piston 2 can be made of soft materials such as silicone, rubber, or TPU. In this embodiment, silicone is used. The diameter of the piston 2 is slightly larger than the inner diameter of the mixing chamber 11 to provide a good seal.

[0025] The nucleic acid detection kit provided in this embodiment contains a lysis buffer chamber 13, a washing buffer chamber 14, an elution buffer chamber 15, a PCR reaction chamber 16, and reagent microspheres. When using the nucleic acid detection kit, the sample to be tested is injected into the sample injection chamber 12. An external device passes through the chamber seal 4 and is connected to the piston 2 via the connecting part 21 at the top of the piston 2. The device punctures the chamber seal 4 at the opening of each reagent chamber. Then, the external device operates the piston 2 and the valve body 3 in a specific operating sequence (operating the valve body 3 via the operating part 31). With the cooperation of external ultrasonic equipment, magnetic equipment, and fluorescence acquisition equipment, the nucleic acid detection kit can complete operations such as lysis, washing, nucleic acid molecule elution, amplification, and fluorescence acquisition. It has a high degree of automation and integration and can effectively reduce the risk of sample contamination during the detection process.

[0026] By monitoring the capacitance change in the mixing chamber 11 through the capacitance detection electrode 6, the liquid transfer process between the mixing chamber 11 and the sample injection chamber 12, between the mixing chamber 11 and each reagent chamber, and between the mixing chamber 11 and the amplification detection chip 5 can be accurately monitored, so as to complete each liquid transfer step quickly and accurately, thereby improving the accuracy and speed of operation.

[0027] like Figure 1 , Figure 2 and Figure 7 As shown, specifically in this embodiment, both the capacitance detection electrode 6 and the mixing chamber 11 are disposed at one end of the housing 1 along the first direction X (the first direction X is the length direction of the valve body 3, and also the length direction of the housing 1). The capacitance detection electrode 6 is fixed to the side wall of the housing 1 so that the capacitance detection electrode 6 is adjacent to the mixing chamber 11, facilitating accurate monitoring of capacitance changes within the mixing chamber 11. Furthermore, since the capacitance detection electrode 6 does not directly contact the reagent, the risk of corrosion and cross-contamination of the capacitance detection electrode 6 is effectively eliminated. In this embodiment, the chamber seal 4 is made of aluminum foil and is connected to the housing 1 using processes such as bonding, hot pressing, or laser welding. In other embodiments, the chamber seal 4 can also be made of other similarly usable sealing films such as PTFE membrane (polytetrafluoroethylene membrane). A groove is provided on the side wall of the housing 1, and the capacitance detection electrode 6 is disposed in the groove so that the capacitance detection electrode 6 is as close as possible to the mixing chamber 11, thereby improving the detection accuracy of the capacitance detection electrode 6 in detecting capacitance changes within the mixing chamber 11.

[0028] Furthermore, such as Figure 2 and Figure 12 As shown, the capacitive detection electrode 6 is provided with a connection contact point 61, which is exposed outside the housing 1, so that the detection circuit of the external instrument can be quickly connected to the capacitive detection electrode 6 when the nucleic acid detection box is used.

[0029] Optionally, such as Figure 2 and Figure 12 As shown, the capacitive detection electrode 6 has a comb-shaped measurement area and a top reference area, with the top reference area located above the preset highest liquid level in the mixing chamber 11. During the use of the nucleic acid detection kit, the top reference area is always located above the liquid in the mixing chamber 11, used to capture ambient temperature and humidity and external electromagnetic interference in real time, serving as a reference for the background noise of the capacitive detection electrode 6. When the piston 2 moves upward to drive the liquid into the mixing chamber 11 and when the piston 2 moves downward to drive the liquid in the mixing chamber 11 into the amplification detection chip 5, the control system uses a differential algorithm (i.e., the signal from the comb-shaped measurement area minus the signal from the top reference area) to eliminate environmental drift and improve the accuracy of the capacitive detection electrode 6 in detecting the liquid level in the mixing chamber 11 during sample aspiration (i.e., driving the liquid into the mixing chamber 11) and dispensing the liquid to the amplification detection chip 5.

[0030] The comb-shaped measurement area is located below the top reference area. The comb-shaped measurement area adopts an interleaved comb-shaped electrode structure. By increasing the electric field density at the electrode edge, the capacitance detection electrode 6 can have extremely high sensing sensitivity to minute liquid level changes and microscopic disturbances in the medium within the mixing chamber 11.

[0031] like Figure 2 and Figure 12As shown, in this embodiment, the capacitance detection electrode 6 includes a first electrode 62, a second electrode 63, and a third electrode 64. The first electrode 62 includes a first connecting arm 621, a first sheet electrode structure 622, and a plurality of first comb teeth 623. The second electrode 63 includes a second connecting arm 631 and a plurality of second comb teeth 632. The third electrode 64 includes a third connecting arm 641 and a second sheet electrode structure 642.

[0032] Both the second connecting arm 631 and the first connecting arm 621 extend along the height direction of the housing 1, and the second connecting arm 631 and the first connecting arm 621 are spaced apart along the width direction of the housing 1. Each first comb tooth 623 is connected to the side of the first connecting arm 621 facing the second connecting arm 631, and each second comb tooth 632 is connected to the side of the second connecting arm 631 facing the first connecting arm 621. The first comb teeth 623 and the second comb teeth 632 are alternately and spaced apart along the height direction of the housing 1. Each first comb tooth 623 of the first electrode 62 and each second comb tooth 632 of the second electrode 63 cooperate to form the comb-shaped measurement area of ​​the capacitance detection electrode 6.

[0033] The first sheet electrode structure 622 is connected to the top of the first connecting arm 621 and is located on the side of the first connecting arm 621 facing the second connecting arm 631. The third connecting arm 641 is located on the side of the second connecting arm 631 away from the first connecting arm 621 and extends along the height direction of the housing 1. The second sheet electrode is connected to the top of the third connecting arm 641 and is located on the side of the third connecting arm 641 facing the first connecting arm 621. The second sheet electrode structure 642 and the first sheet electrode structure 622 are both located above each of the first comb teeth 623 and each of the second comb teeth 632, and the second sheet electrode structure 642 and the first sheet electrode structure 622 are spaced apart along the height direction of the housing 1. The first sheet electrode structure 622 of the first electrode 62 and the second sheet electrode structure 642 of the third electrode 64 cooperate to serve as the top reference area of ​​the capacitance detection electrode 6. Both the first sheet electrode structure 622 and the second sheet electrode structure 642 are solid square sheets. The bottom ends of the first connecting arm 621, the second connecting arm 631 and the third connecting arm 641 are all provided with connecting contact points 61.

[0034] Furthermore, such as Figure 12 As shown, the capacitance detection electrode 6 also includes a base layer 65, which is attached to the housing 1. The first electrode 62, the second electrode 63, and the third electrode 64 are all disposed on the side of the base layer 65 away from the housing 1. The base layer 65 connects the first electrode 62, the second electrode 63, and the third electrode 64 into a single unit. The base layer 65 is made of an insulating material and is in the form of a thin film. In this embodiment, the base layer 65 uses the insulating film used in flexible circuit boards. The base layer 65 is fixed to the housing 1 by adhesive bonding.

[0035] Optionally, such as Figure 4 As shown, multiple amplification detection chips 5 are arranged at intervals along the first direction X. The valve body 3 can control the opening and closing of the mixing chamber 11 and each amplification detection chip 5. The multiple chambers are arranged at intervals along the first direction X. Each of the multiple amplification detection chips 5 can be pre-filled with reagent microspheres of different compositions, enabling independent and parallel reactions of different amplification detection chips 5. When dispensing liquid to each amplification detection chip 5, the movement of the piston 2 is controlled according to the liquid level change in the mixing chamber 11, ensuring that the amount of liquid flowing into each amplification detection chip 5 remains consistent. In this embodiment, the amount of liquid flowing into each amplification detection chip 5 is just enough to fill the amplification detection chamber 51 of each amplification detection chip 5.

[0036] Optionally, such as Figure 4 , Figure 9 and Figure 10 As shown, the housing 1 also has a mounting cavity located below each chamber. The valve body 3 is disposed within the mounting cavity, and the operating part 31 is used to drive the valve body 3 to rotate relative to the housing 1. The bottom of each chamber is connected to the mounting cavity through a third connecting channel. The valve body 3 has a valve cavity 32 extending along the first direction X. The valve body 3 has multiple first flow holes 33, multiple second flow holes 34, and multiple third flow holes 35. The sample injection chamber 12 and each reagent chamber are each provided with a corresponding first flow hole 33. Each amplification detection chip 5 is provided with a corresponding second flow hole 34. The multiple first flow holes 33 are spaced apart along the first direction X and spaced apart circumferentially along the valve body 3. By driving the valve body 3 to rotate relative to the housing 1, the sample injection chamber 12 and each reagent chamber can be connected to the valve cavity 32 through the corresponding third connecting channel and the corresponding first flow hole 33, respectively. Multiple second flow holes 34 are spaced apart along the first direction X and circumferentially spaced along the valve body 3. By driving the valve body 3 to rotate relative to the housing 1, each amplification detection chip 5 can be connected to the valve cavity 32 through its corresponding second flow hole 34. Multiple third flow holes 35 are all located at one end of the valve body 3 along the first direction X and are spaced apart circumferentially along the valve body 3, so that when the sample injection chamber 12, reagent chamber, or amplification detection chip 5 is connected to the valve cavity 32, the mixing chamber 11 can be connected to the valve cavity 32 through its corresponding third connecting channel and a third flow hole 35. Those skilled in the art will understand that at any given time (i.e., when the valve body 3 is in a certain position), only one of the sample injection chamber 12, each reagent chamber, and each amplification detection chip 5 can be connected to the valve cavity 32. However, by rotating the valve body 3, the sample injection chamber 12, each reagent chamber, and each amplification detection chip 5 can be connected to the valve cavity 32.

[0037] For example, by rotating the valve body 3, the sample injection chamber 12 is connected to the valve cavity 32 of the valve body 3 through a first flow hole 33. At this time, the mixing chamber 11 is connected to the valve cavity 32 of the valve body 3 through a third flow hole 35. At this time, the mixing chamber 11 and the sample injection chamber 12 can achieve liquid transfer through the valve cavity 32 of the valve body 3. By rotating the valve body 3, one of the amplification detection chips 5 is connected to the valve cavity 32 of the valve body 3 through a second flow hole 34. At this time, the mixing chamber 11 is also connected to the valve cavity 32 of the valve body 3 through a third flow hole 35. At this time, the mixing chamber 11 and the amplification detection chip 5 can achieve liquid transfer through the valve cavity 32 of the valve body 3.

[0038] Those skilled in the art will understand that there is a significant difference in dielectric constant between aqueous reagents and air. By monitoring the capacitance change of the mixing chamber 11 through the capacitance detection electrode 6, the liquid level change in the mixing chamber 11 can be monitored. Furthermore, compared to the long strip continuous electrode (e.g., cylindrical electrode) used in conventional liquid level detection, the comb-shaped measurement area used in this embodiment has a relatively independent sensitive unit between each first comb tooth 623 and an adjacent second comb tooth 632. When the liquid surface in the mixing chamber 11 passes through each medium space of the capacitance detection electrode 6, a step signal is generated. By monitoring this step signal, the current liquid level in the mixing chamber 11 can be accurately determined. This method has the advantages of a small sensitive area volume and higher monitoring accuracy. Furthermore, when air enters the bottom of the mixing chamber 11 through the third connecting channel, bubbles will form at the connection between the third connecting channel and the mixing chamber 11, causing a change (decrease) in the dielectric constant of the mixing chamber 11, especially in the lower part of the mixing chamber 11. Therefore, the comb-shaped measurement area of ​​the capacitance detection electrode 6 can also detect the step signal (negative jump) generated by the bubble entering the mixing chamber 11. By monitoring this step signal, it can be determined whether air has started to enter the mixing chamber 11.

[0039] In this embodiment of the nucleic acid detection kit, the approximate volume of liquid to be transferred is known each time the piston 2 moves upward to drive the liquid from other chambers into the mixing chamber 11. Therefore, a corresponding minimum liquid level threshold can be set for each step of the piston 2 moving upward to drive the liquid from other chambers into the mixing chamber 11. When the liquid level in the mixing chamber 11 is higher than the minimum liquid level threshold corresponding to this transfer, and the capacitive detection electrode 6 detects that air begins to enter the mixing chamber 11, it is determined that this transfer is complete, and the piston 2 is controlled to stop moving upward.

[0040] Furthermore, those skilled in the art will understand that if the piston 2 moves too slowly, the liquid transfer speed will be too slow, resulting in a decrease in nucleic acid detection efficiency. If the piston 2 moves too fast, it will be detrimental to the monitoring of capacitance changes in the mixing chamber 11 by the capacitance detection electrode 6. Therefore, the speed range of the piston 2 should be controlled between 10 mm / min and 1000 mm / min.

[0041] In this example, when piston 2 moves downward to drive the liquid in mixing chamber 11 to flow into other chambers, a moving speed of 1000 mm / min is used. When piston 2 moves upward to drive the liquid in other chambers to flow into mixing chamber 11, and when piston 2 moves downward to drive the liquid in mixing chamber 11 into amplification detection chip 5, a moving speed of 10 mm / min is used. When piston 2 moves downward to drive the liquid in mixing chamber 11 to flow into other chambers, piston 2 needs to move to the bottom of mixing chamber 11. It is not necessary to judge whether the liquid in mixing chamber 11 has been completely discharged based on the monitoring results of capacitive detection electrode 6. Therefore, piston 2 can move downward at a faster speed to save the time required for nucleic acid detection and improve detection efficiency. When piston 2 moves upward to drive the liquid in other chambers into the mixing chamber 11, it is necessary to determine whether the liquid has completely entered the mixing chamber 11 by monitoring the results of the capacitance detection electrode 6. Piston 2 moves upward at a slower speed, which can improve the control accuracy of the upward movement of piston 2. When piston 2 moves downward to drive the liquid in the mixing chamber 11 into the amplification detection chip 5, it is also necessary to determine the liquid discharge amount by monitoring the results of the capacitance detection electrode 6. Therefore, piston 2 also moves downward at a slower speed, which can improve the control accuracy of the downward movement of piston 2.

[0042] Those skilled in the art will understand that when air begins to enter the bottom of the mixing chamber 11 from the third connecting channel, the bubbles will not rise until they reach a certain volume. Because the capacitive detection electrode 6 in this embodiment has sufficient detection accuracy, and the piston 2 moves upwards slowly, it is possible to control the piston 2 to stop moving upwards before the bubbles rise. This effectively reduces the probability and amount of external gas entering the liquid within the mixing chamber 11, preventing excessive external gas from affecting the reagent mixing and cleaning effect. It also achieves precise control over liquid transfer, improving the efficiency of reagent (liquid) transfer and avoiding excessive unnecessary suction movements by the piston 2. It should be noted that during one or more instances where the piston 2 moves upwards, driving liquid from other chambers into the mixing chamber 11, a small number of bubbles may rise. Since the amount of gas entering is very small, it will not affect the nucleic acid detection process or the detection results.

[0043] Optionally, such as Figure 9 and Figure 10As shown, the distance between two adjacent first flow holes 33 along the first direction X is 10mm, and the angle between two adjacent first flow holes 33 along the first direction X along the circumference of the valve body 3 is 30°. Similarly, the distance between two adjacent second flow holes 34 along the first direction X is 10mm, and the angle between two adjacent second flow holes 34 along the first direction X along the circumference of the valve body 3 (that is, the interval angle between two adjacent second flow holes 34 along the first direction X along the circumference of the valve body 3) is 30°. The phase difference between the first first flow hole 33 and the first second flow hole 34 is 30°.

[0044] Furthermore, such as Figures 4-6 As shown, the nucleic acid detection kit provided in this embodiment also includes a sealing sleeve 8. The sealing sleeve 8 is fitted onto the valve body 3 and sandwiched between the valve body 3 and the cavity wall of the mounting cavity to ensure a sealing effect between the valve body 3 and the cavity wall of the mounting cavity, preventing liquid from entering between the valve body 3 and the cavity wall of the mounting cavity. In this embodiment, the sealing sleeve 8 is interference-fitted into the mounting cavity, and a positioning rib 81 is provided on the outer wall of the sealing sleeve 8. A positioning groove is correspondingly provided on the cavity wall of the mounting cavity, and the positioning rib 81 extends into the positioning groove so that the sealing sleeve 8 does not rotate with the valve body 3 when the valve body 3 rotates. The sealing sleeve 8 is provided with multiple fourth flow holes, and each chamber and each amplification detection chip 5 is provided with a corresponding fourth flow hole. Each chamber and each amplification detection chip 5 can communicate with the inner cavity of the sealing sleeve 8 through the corresponding fourth flow hole. The sealing sleeve 8 is made of a flexible material with good resilience.

[0045] Optionally, such as Figure 4 , Figure 9 and Figure 10 As shown, the valve body 3 is provided with a limit part 36 at one end where the operating part 31 is provided. A limit ring is threadedly connected to the opening of the mounting cavity. The limit ring limits the valve body 3 to the mounting cavity through the limit part 36, and the operating part 31 extends into the limit ring.

[0046] Optionally, such as Figures 1-6 As shown, all amplification detection chips 5 are connected to the housing 1. Each amplification detection chip 5 is provided with an amplification detection chamber 51, a reagent inlet channel 52, and an exhaust channel 53. The reagent inlet channel 52 connects the amplification detection chamber 51 to the mounting cavity, and the exhaust channel 53 connects the amplification detection chamber 51 to the outside of the amplification detection chip 5. The depth of the amplification detection chamber 51 (in this embodiment, the depth direction of the amplification detection chamber 51 is also the width direction of the housing 1) can be designed to be 100μm-1000μm according to the volume of the reaction system. Preferably, in this example, the depth of the amplification detection chamber 51 is set to 100μm to improve the speed of the PCR reaction.

[0047] Furthermore, such as Figures 4-6As shown, the reagent inlet channel 52 includes a first channel section 521 and a second channel section 522 connected in sequence. One end of the first channel section 521 is connected to the mounting cavity, and the other end is connected to one end of the second channel section 522. The other end of the second channel section 522 is connected to the amplification detection cavity 51. The cross-sectional dimension of the second channel section 522 is smaller than that of the first channel section 521, and the reagent microspheres are disposed within the first channel section 521. The size of the reagent microspheres is smaller than the cross-sectional dimension of the first channel section 521 but larger than that of the second channel section 522. When the test liquid flows through the reagent microspheres, the reagent microspheres dissolve rapidly. Compared to directly placing the reagent microspheres in the amplification detection cavity 51, this method of placing the reagent microspheres within the first channel section 521 can improve the dissolution rate of the reagent microspheres and facilitate the uniform mixing of the components in the test liquid.

[0048] In this embodiment, as Figures 4-6 As shown, the cross-section of the first flow channel section 521 is circular, and the cross-section of the second flow channel section 522 is slit-shaped. The cross-sectional size of the second flow channel section 522 is much smaller than that of the first flow channel section 521. The slit-shaped second flow channel section 522 can fully dissolve the reagent microspheres in the first flow channel section 521, ensuring that the components in the test liquid are mixed evenly.

[0049] Furthermore, such as Figure 5 and Figure 11 As shown, the nucleic acid detection kit provided in this embodiment also includes a chip holder 9, which includes a base 91 and clips 92. The amplification detection chips 5 are all connected to the bottom end of the base 91. That is, in this embodiment, the amplification detection chips 5 are all connected to the kit body 1 through the chip holder 9. The top of the base 91 is provided with multiple spaced clips 92, and the bottom surface of the kit body 1 is provided with slots 17 corresponding to the clips 92. The clips 92 are inserted into the slots 17 to achieve the connection between the chip holder 9 and the kit body 1.

[0050] Furthermore, such as Figures 4-6 As shown, the base 91 is provided with a plurality of first connecting channels 911, and the first connecting channels 911 are configured one-to-one with the reagent inlet channels 52. One end of the first connecting channel 911 is used to communicate with the mounting cavity, and the other end is connected to the first channel section 521 of the reagent inlet channel 52.

[0051] Furthermore, such as Figures 4-6 As shown, the box body 1 is also provided with a plurality of second connecting channels 18, and the second connecting channels 18 are provided in a one-to-one correspondence with the first connecting channels 911. The first connecting channels 911 are all connected to the mounting cavity of the box body 1 through the corresponding second connecting channels 18.

[0052] Furthermore, such as Figure 4 , Figure 5 , Figure 6 and Figure 11 As shown, the chip socket 9 provided in this embodiment also includes a sealing ring 93. The sealing ring 93 is provided in a one-to-one correspondence with the second connecting flow channel 18. The bottom surface of the housing 1 is provided with receiving grooves that correspond to the second connecting flow channels 18. The sealing ring 93 is disposed in the corresponding receiving groove and surrounds the corresponding second connecting flow channel 18. The sealing ring 93 can be manufactured using soft materials such as silicone, rubber, and TPU. In this example, silicone is preferably used.

[0053] Optionally, such as Figure 2 , Figure 3 and Figure 5 As shown, each amplification detection chip 5 includes a chip body 54 and a top cover 55. The chip body 54 is fixedly connected to the bottom end of the base 91 of the chip holder 9. The top cover 55 covers the chip body 54 and is sealed to it. The chip body 54 and the top cover 55 form an amplification detection chamber 51 and a reagent inlet channel 52. An exhaust channel 53 is provided on the chip body 54. The top cover 55 is made of transparent material to facilitate fluorescence collection. In this embodiment, the top cover 55 is connected to the chip body 54 by hot pressing or adhesive bonding.

[0054] Optionally, the amplification detection chip 5 also includes a waterproof and breathable plug. A waterproof and breathable plug is provided in the exhaust channel 53. The plug is breathable but not liquid-permeable, thus achieving pressure balance in the amplification detection chamber 51 to ensure the test liquid can smoothly enter the chamber while preventing liquid leakage and avoiding overflow of amplification products from the exhaust channel 53, which could cause laboratory contamination.

[0055] Furthermore, the valve body 3 has a sealed working position. When the valve body 3 is in the sealed working position, the valve cavity 32 of the valve body 3 is not connected to any chamber or any amplification detection chip 5. This allows the reagent inlet channel 52 of the amplification detection chip 5 to be sealed during amplification and fluorescence acquisition, preventing amplification products (aerosols) from overflowing from the reagent inlet channel 52 and causing laboratory contamination. It should be noted that the valve body 3 is in the sealed working position when the nucleic acid detection kit is not in use.

[0056] Furthermore, the amplification detection chip 5 also includes a sealing membrane that covers the exhaust port of the exhaust channel 53. When the nucleic acid detection box is not in use, the presence of the sealing membrane can prevent external gas from entering the amplification detection chip 5, thereby protecting the reagent microspheres and enabling the nucleic acid detection box to be stored for a long time. When dispensing liquid into the amplification detection chip 5, the pressure generated by the piston 2 pressing down in the mixing chamber 11 can break open the sealing membrane on the amplification detection chip 5.

[0057] Optionally, such as Figures 1-5As shown, the nucleic acid testing kit provided in this embodiment also includes a cover 7, which is detachably connected to the kit body 1 to cover the opening of the sample injection chamber 12. Before use, the cover 7 prevents external debris from entering the sample injection chamber 12. When it is necessary to inject the sample into the sample injection chamber 12, the cover 7 is removed using an external device. After the sample is injected into the sample injection chamber 12, the cover 7 is put back on to prevent liquid in the nucleic acid testing kit from splashing out through the opening of the sample injection chamber 12 and to prevent external debris from entering the sample injection chamber 12. When the cover 7 is placed on the opening of the sample injection chamber 12, the sample injection chamber 12 can still communicate with the outside atmosphere through its opening. That is to say, the cover 7 does not airtighten the opening of the sample injection chamber 12 to ensure that liquid can be smoothly transferred between the mixing chamber 11 and the sample injection chamber 12 during the use of the nucleic acid testing kit.

[0058] This embodiment also provides a nucleic acid detection method for non-disease diagnosis purposes, using the above-described nucleic acid detection kit, including: S1. Inject the sample to be tested into the sample injection chamber 12 through the chamber seal 4, then place the entire nucleic acid detection box into the compatible instrument, clamp the nucleic acid detection box with the external device, insert the external operating structure into the mixing chamber 11 through the chamber seal 4, and connect it to the piston 2 through the connecting part 21, puncture the chamber seal 4 at the opening of each reagent chamber. In this embodiment, the amount of sample to be tested injected into the sample injection chamber 12 is 200μL. When the external operating structure is connected to the piston 2, the external operating structure presses down, punctures the chamber seal 4, and the external operating structure continues to press down and extends into the connecting part 21 of the piston 2. Rotate the external operating structure so that the external operating structure is threadedly connected to the piston 2 through the connecting part 21, and drive the piston 2 to move down to the bottom of the mixing chamber 11. The current position of the piston 2 is used as the calibration position of the piston 2. In this embodiment, the external operating structure is a piston control rod. S2 drives the valve body 3 and piston 2, and monitors the liquid transfer process through the capacitance detection electrode 6. The sample to be tested in the sample injection chamber 12 is mixed with the lysis buffer and magnetic bead mixture in the mixing chamber 11 and the lysis buffer chamber 13 to form a first mixture. The first mixture is contained in the lysis buffer chamber 13. Then, the first mixture in the lysis buffer chamber 13 is ultrasonically lysed using an ultrasonic device so that the magnetic beads in the first mixture adsorb nucleic acid molecules. The lysis frequency can be set to 20-25 kHz depending on the sample. In this example, the lysis frequency is set to 20 kHz, the ultrasonic power is set to 50 W, and it is maintained for 1 min. Specifically, when the sample to be tested is mixed with the lysis buffer and magnetic bead mixture in the mixing chamber 11 to form the first mixture, the mixture is transferred back and forth between the mixing chamber 11 and the lysis buffer chamber 13 multiple times to form the final first mixture to improve the mixing uniformity. In this embodiment, the mixture is transferred back and forth between the mixing chamber 11 and the lysis buffer chamber 13 5 times. S3. Use a magnetic device to attract the magnetic beads in the lysis chamber 13 to the wall of the lysis chamber 13, drive the valve body 3 and piston 2, and monitor the liquid transfer process through the capacitance detection electrode 6, so that the liquid in the lysis chamber 13 enters the sample injection chamber 12 through the mixing chamber 11. S4 drives the valve body 3 and piston 2, and monitors the liquid transfer process through the capacitive detection electrode 6, so that the cleaning liquid in the cleaning liquid chamber 14 is mixed with the magnetic beads in the lysis liquid chamber 13 to clean the magnetic beads, and drives the waste liquid generated by cleaning into the sample injection chamber 12. The cleaned magnetic beads are contained in the lysis liquid chamber 13. Preferably, multiple cleaning liquid chambers 14 are provided, and the cleaning liquid in multiple cleaning liquid chambers 14 cleans the magnetic beads respectively to improve the cleaning effect. In this embodiment, two cleaning liquid chambers 14 are provided. S5 drives the valve body 3 and piston 2, and monitors the liquid transfer process through the capacitance detection electrode 6, so that the eluent in the elution chamber 15 is mixed with the magnetic beads in the lysis chamber 13 to elute the nucleic acid molecules on the magnetic beads. The magnetic beads are adsorbed onto the cavity wall of the lysis chamber 13 using a magnetic device, and the eluent containing nucleic acid molecules is driven to mix with the PCR reaction solution in the PCR reaction chamber 16 through the mixing chamber 11 to form a second mixture, and the second mixture is contained in the mixing chamber 11. Specifically, in this embodiment, after the eluent in the elution chamber 15 is mixed with the magnetic beads and transferred to the lysis chamber 13, the lysis chamber 13 is accelerated to elute by an ultrasonic device. Preferably, the ultrasonic application frequency is 10 kHz and the ultrasonic application time is 1 min. S6 drives the valve body 3 and piston 2 to allow the second mixture to enter the amplification detection chip 5 to dissolve the reagent microspheres and form a third mixture. The third mixture in the amplification detection chip 5 is then amplified and fluorescence is collected.

[0059] Furthermore, the liquid transfer process is monitored by the capacitance detection electrode 6, including: when the piston 2 moves upward to allow liquid from other chambers to enter the mixing chamber 11, the capacitance detection electrode 6 monitors the capacitance change in the mixing chamber 11, and determines whether the liquid transfer is complete based on the capacitance change in the mixing chamber 11. If the liquid transfer is determined to be complete, the piston 2 is controlled to stop moving upward.

[0060] Specifically, in this embodiment, determining whether the current pipetting is completed based on the capacitance change in the mixing chamber 11 includes: determining the current liquid level in the mixing chamber 11 based on the capacitance change in the mixing chamber 11, and whether air has entered the mixing chamber 11. If the current liquid level in the mixing chamber 11 is higher than the minimum liquid level threshold corresponding to this pipetting, and air has entered the mixing chamber 11, then the pipetting is determined to be completed.

[0061] It should be noted that since the nucleic acid test kit requires multiple pipetting operations during use, and a small number of tiny air bubbles may remain in the liquid during the pipetting process, if no minimum liquid level threshold is set for each pipetting operation, and the control system only monitors whether air bubbles have entered the mixing chamber 11 to determine whether the pipetting is complete, the control system may misjudge that the pipetting is complete when a small number of air bubbles in the liquid have entered the mixing chamber 11. Furthermore, since the pre-filling volume of each reagent in the nucleic acid test kit may have errors, and the sample volume injected into the sample injection chamber 12 may also have errors, if only a minimum liquid level threshold is set to determine whether the pipetting is complete, the liquid level in the mixing chamber 11 may reach the minimum liquid level threshold before some liquid has entered the mixing chamber 11, resulting in incomplete pipetting. Therefore, this embodiment uses both the minimum liquid level threshold corresponding to the main body of the kit and the presence of air in the mixing chamber 11 as two conditions to determine whether the pipetting is complete, making the pipetting judgment more accurate.

[0062] Furthermore, during the process of driving the valve body 3 and piston 2 to allow the second mixture to enter the amplification detection chip 5 and dissolve the reagent microspheres to form the third mixture, the liquid level change in the mixing chamber 11 is monitored by the capacitive detection electrode 6, and the movement of piston 2 is controlled according to the liquid level in the mixing chamber 11 to ensure that the amount of liquid flowing into each amplification detection chip 5 remains consistent. Preferably, the amount of liquid flowing into each amplification detection chip 5 is just enough to fill the amplification detection chamber 51 of each amplification detection chip 5.

[0063] Optionally, in step S4, the cleaning solution in the cleaning solution chamber 14 is mixed with the magnetic beads in the lysis solution chamber 13 to clean the magnetic beads, and the waste liquid generated during cleaning is driven into the sample injection chamber 12. The cleaned magnetic beads are then contained in the lysis solution chamber 13. Specifically, this includes: driving the cleaning solution in the cleaning solution chamber 14 through the mixing chamber 11 into the lysis solution chamber 13 to form a first intermediate mixture containing the magnetic beads; then driving the first intermediate mixture to move back and forth between the lysis solution chamber 13 and the mixing chamber 11; and determining whether the cleaning solution and magnetic beads have been completely cleaned. If the cleaning solution and magnetic beads are determined to be mixed, the first intermediate mixture contained in the mixing chamber 11 is driven into the lysis chamber 13. Then, the magnetic device adsorbs the magnetic beads in the lysis chamber 13 onto the chamber wall of the lysis chamber 13 and drives the liquid in the lysis chamber 13 (that is, the waste liquid generated in this cleaning) into the sample injection chamber 12 through the mixing chamber 11. If it is determined that the cleaning solution and magnetic beads are not mixed, the first intermediate mixture continues to move back and forth between the lysis chamber 13 and the mixing chamber 11. It should be noted that, since the magnetic beads are adsorbed onto the side wall of the pyrolysis chamber 13 using an external magnetic device in step S3, although the external magnetic device no longer adsorbs the magnetic beads when the cleaning liquid in the cleaning liquid chamber 14 is mixed with the magnetic beads in the pyrolysis chamber 13, some or even most of the magnetic beads may still be in an adherent and agglomerated state. Therefore, in this cleaning step, it is necessary to mix the cleaning liquid and the magnetic beads to achieve resuspension of the magnetic beads. The uniformity of the mixing of the cleaning liquid and the magnetic beads directly affects the cleaning effect. Therefore, in this cleaning step of this embodiment, the first intermediate mixture is driven to move back and forth between the pyrolysis chamber 13 and the mixing chamber 11 to improve the uniformity of the mixing of the magnetic beads and the cleaning liquid.

[0064] Furthermore, determining whether the cleaning fluid and magnetic beads have been completely mixed includes: determining whether the number of times the first intermediate mixture has been contained in the mixing chamber 11 has reached a first preset number; if the number of times the first intermediate mixture has been contained in the mixing chamber 11 has reached the first preset number, then it is determined that the cleaning fluid and magnetic beads have been completely mixed.

[0065] Furthermore, determining whether the cleaning solution and magnetic beads have been completely mixed also includes: when the first intermediate mixture is contained in the mixing chamber 11, calculating the standard deviation of the monitoring signal of the capacitance detection electrode 6 in real time; if the standard deviation of the monitoring signal of the capacitance detection electrode 6 is within a preset threshold range within a first preset time period, then it is determined that the cleaning solution and magnetic beads have been completely mixed. That is to say, before the number of times the first intermediate mixture is contained in the mixing chamber 11 reaches a first preset number, the first intermediate mixture needs to remain in the mixing chamber 11 for at least a first preset time each time it is completely introduced, so that the control system can determine whether the cleaning solution and magnetic beads have been completely mixed.

[0066] The standard deviation of the monitoring signal of the capacitive detection electrode 6 can reflect the uniformity of the mixing of the cleaning solution and the magnetic beads in the first intermediate mixture in the mixing chamber 11. Before the number of times the first intermediate mixture is contained in the mixing chamber 11 reaches the first preset number, by judging the uniformity of the mixing of the cleaning solution and the magnetic beads, the reciprocating movement of the first intermediate mixture between the lysis chamber 13 and the mixing chamber 11 can be interrupted in time when the uniformity of the mixing of the cleaning solution and the magnetic beads meets the requirements, thereby saving the time required for nucleic acid detection and improving the detection efficiency.

[0067] It should be noted that the capacitive detection electrode 6 can monitor the distribution of magnetic beads in the first intermediate mixture within the mixing chamber 11 by capturing the influence of the magnetic bead distribution on the equivalent dielectric constant. When a large number of magnetic beads are in an adherent and agglomerated state in the mixing chamber 11, the monitoring signal of the capacitive detection electrode 6 exhibits irregular, low-frequency, and violent fluctuations due to the influence of these agglomerated magnetic bead clusters on the dielectric constant within the mixing chamber 11. When the magnetic beads in the mixing chamber 11 are resuspended (i.e., when the uniformity of the mixing of the cleaning liquid and the magnetic beads meets the requirements), the monitoring signal of the capacitive detection electrode 6 transforms into a stable high-frequency micro-vibration. The control system can autonomously determine the resuspending effect of the magnetic beads by calculating the standard deviation fluctuation of the monitoring signal in real time, thereby adjusting the number of mixing steps (i.e., the number of times the first intermediate mixture is contained in the mixing chamber 11 during this cleaning process).

[0068] Optionally, in step S5, the eluent in the elution chamber 15 is mixed with the magnetic beads in the lysis chamber 13 to elute nucleic acid molecules on the magnetic beads. Specifically, this includes: driving the eluent in the elution chamber 15 through the mixing chamber 11 into the lysis chamber 13 to form a second intermediate mixture containing magnetic beads; then driving the second intermediate mixture to move back and forth between the lysis chamber 13 and the mixing chamber 11; and determining whether the eluent and the magnetic beads have been completely mixed. If it is determined that the eluent and the magnetic beads have been completely mixed, the second intermediate mixture contained in the mixing chamber 11 is driven into the lysis chamber 13. If it is determined that the eluent and the magnetic beads have not been completely mixed, the second intermediate mixture is continued to move back and forth between the lysis chamber 13 and the mixing chamber 11.

[0069] Further, determining whether the eluent and magnetic beads have been completely mixed includes: determining whether the number of times the second intermediate mixture has been contained in the mixing chamber 11 has reached a second preset number; if the number of times the second intermediate mixture has been contained in the mixing chamber 11 has reached the second preset number, then it is determined that the eluent and magnetic beads have been completely mixed.

[0070] Furthermore, determining whether the eluent and magnetic beads are completely mixed also includes: when the second intermediate mixture is contained in the mixing chamber 11, calculating the standard deviation of the monitoring signal of the capacitive detection electrode 6 in real time; if the standard deviation of the monitoring signal of the capacitive detection electrode 6 is within a preset threshold range within a second preset time period, then it is determined that the eluent and magnetic beads have been completely mixed. That is to say, before the second intermediate mixture is contained in the mixing chamber 11 a second preset number of times, the second intermediate mixture needs to remain in the mixing chamber 11 for at least a second preset time each time it is fully introduced, so that the control system can determine whether the eluent and magnetic beads are completely mixed.

[0071] Before the second intermediate mixture has been contained in the mixing chamber 11 for the second preset number of times, by judging the degree of uniformity of the mixing of the elution buffer and the magnetic beads, the reciprocating movement of the second intermediate mixture between the lysis buffer chamber 13 and the mixing chamber 11 can be interrupted in time when the uniformity of the mixing of the elution buffer and the magnetic beads meets the requirements, thereby saving the time required for nucleic acid detection and improving detection efficiency.

[0072] In this embodiment, both the first preset number of times and the second preset number of times are 5 times.

[0073] Optionally, amplification and fluorescence acquisition of the third mixture within the amplification detection chip 5 include: rotating the valve body 3 to a sealed working position, so that the valve chamber 32 of the valve body 3 is not connected to any chamber or any amplification detection chip 5; then, using an external temperature control device closely attached to each amplification detection chip 5, performing multiple heating and cooling cycles on the liquid in each amplification detection chamber 51 (one heating plus one cooling constitutes one heating and cooling cycle); and acquiring fluorescence from each amplification detection chip 5 after each heating and cooling cycle to complete the PCR amplification reaction process. In this embodiment, the external temperature control device is a semiconductor cooling chip.

[0074] Those skilled in the art will understand that after amplification and fluorescence acquisition are completed, the acquisition results still need to be analyzed.

[0075] In summary, the embodiments of the present invention provide a nucleic acid detection kit and a nucleic acid detection method for non-disease diagnosis purposes, which have a high degree of automation and integration; can effectively reduce the risk of sample contamination during the detection process; have high fluid control precision; have good cleaning effect of the cleaning solution on the magnetic beads and good elution effect of the elution solution on the magnetic beads; can simultaneously achieve parallel detection of multiple targets and low sample consumption in a single device, which is beneficial for applications in primary healthcare or point-of-care testing scenarios; and the reagents in the pre-loaded nucleic acid detection kit can be stably stored for a long time.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid detection kit, characterized in that, It includes a housing (1), a piston (2), a valve body (3), a chamber seal (4), an amplification detection chip (5), and a capacitance detection electrode (6). The box body (1) is provided with multiple chambers with openings. The multiple chambers include a mixing chamber (11), a sample injection chamber (12), and multiple reagent chambers. The opening of the mixing chamber (11) and the opening of the reagent chambers are provided with chamber seals (4). The multiple reagent chambers include a lysis buffer chamber (13), a washing buffer chamber (14), an elution buffer chamber (15), and a PCR reaction chamber (16). The lysis buffer chamber (13) contains a mixture of lysis buffer and magnetic beads, the washing buffer chamber (14) contains washing solution, the elution buffer chamber (15) contains elution solution, and the PCR reaction chamber (16) contains PCR reaction solution. The amplification detection chip (5) is equipped with reagent microspheres. The piston (2) is provided in the mixing chamber (11), and a connecting part (21) is provided at the top of the piston (2). The connecting part (21) is used to connect with an external operating structure to drive the piston (2). The valve body (3) is disposed on the housing (1), and the valve body (3) is provided with an operating part (31) exposed on the housing (1). The operating part (31) is used to drive the valve body (3) to control the connection and disconnection between the mixing chamber (11) and the sample injection chamber (12), between the mixing chamber (11) and each of the reagent chambers, and between the mixing chamber (11) and the amplification detection chip (5). The capacitance detection electrode (6) is disposed on the housing (1) and is used to monitor the capacitance change in the mixing chamber (11). It is also used to monitor whether the magnetic beads and the cleaning solution are completely mixed when the cleaning solution cleans the magnetic beads, and to monitor whether the magnetic beads and the elution solution are completely mixed when the elution solution elutes the nucleic acid molecules on the magnetic beads. The capacitance detection electrode (6) has a comb-shaped measurement area and a top reference area. The top reference area is located above the preset highest liquid level of the mixing chamber (11), and the comb-shaped measurement area is located below the top reference area. The comb-shaped measurement area adopts a staggered comb-shaped electrode structure.

2. The nucleic acid detection kit according to claim 1, characterized in that, The capacitance detection electrode (6) is provided with a connecting contact point (61), which is exposed outside the housing (1).

3. The nucleic acid detection kit according to claim 1, characterized in that, The amplification detection chip (5) is provided in multiple ways, and the multiple amplification detection chips (5) are spaced apart along the first direction (X). The valve body (3) can control the connection and disconnection between the mixing chamber (11) and each of the amplification detection chips (5). The multiple chambers are spaced apart along the first direction (X), and the first direction (X) is the length direction of the valve body (3).

4. The nucleic acid detection kit according to claim 3, characterized in that, The box body (1) is also provided with an installation cavity, the valve body (3) is disposed in the installation cavity, and the operating part (31) is used to drive the valve body (3) to rotate relative to the box body (1); Each of the chambers is connected to the mounting cavity. The valve body (3) is provided with a valve cavity (32) extending along the first direction (X). The valve body (3) is provided with a plurality of first flow holes (33), a plurality of second flow holes (34) and a plurality of third flow holes (35). Multiple first flow holes (33) are spaced apart along the first direction (X) and spaced apart along the circumferential direction of the valve body (3), driving the valve body (3) to rotate relative to the box body (1), so that the sample injection chamber (12) and each of the reagent chambers are connected to the valve chamber (32) through the first flow holes (33); Multiple second flow holes (34) are spaced apart along the first direction (X) and spaced apart along the circumferential direction of the valve body (3), driving the valve body (3) to rotate relative to the housing (1), so that each amplification detection chip (5) can be connected to the valve cavity (32) through the second flow holes (34); Multiple third flow holes (35) are disposed at one end of the valve body (3) along the first direction (X) and are spaced apart circumferentially along the valve body (3) so that when the sample injection chamber (12), the reagent chamber or the amplification detection chip (5) is connected to the valve chamber (32), the mixing chamber (11) can be connected to the valve chamber (32) through the third flow holes (35).

5. The nucleic acid detection kit according to claim 4, characterized in that, The amplification detection chips (5) are all connected to the housing (1). The amplification detection chip (5) is provided with an amplification detection chamber (51), a reagent inlet channel (52) and an exhaust channel (53). The reagent inlet channel (52) is used to connect the amplification detection chamber (51) with the mounting cavity. The exhaust channel (53) connects the amplification detection chamber (51) with the outside of the amplification detection chip (5).

6. The nucleic acid detection kit according to claim 5, characterized in that, The reagent entry channel (52) includes a first channel section (521) and a second channel section (522) connected in sequence. One end of the first flow channel segment (521) is connected to the mounting cavity, and the other end is connected to one end of the second flow channel segment (522). The other end of the second flow channel segment (522) is connected to the amplification detection cavity (51). The cross-sectional size of the second flow channel segment (522) is smaller than that of the first flow channel segment (521). The reagent microspheres are disposed in the first flow channel segment (521).

7. The nucleic acid detection kit according to claim 6, characterized in that, The amplification detection chip (5) also includes a waterproof and breathable plug, and the waterproof and breathable plug is provided in the exhaust channel (53).

8. The nucleic acid detection kit according to claim 1, characterized in that, It also includes a cover (7) which is detachably connected to the box body (1) to cover the opening of the sample injection cavity (12).

9. A nucleic acid detection method for non-disease diagnostic purposes, characterized in that, Using the nucleic acid detection kit according to any one of claims 1-8 includes the following steps: S1. The sample to be tested is injected into the sample injection chamber (12) through the chamber seal (4), and the external operating structure is inserted into the mixing chamber (11) through the chamber seal (4) and connected to the piston (2) through the connecting part (21) to puncture the chamber seal (4) at the opening of each reagent chamber. S2. Drive the valve body (3) and the piston (2), and monitor the liquid transfer process through the capacitance detection electrode (6), so that the sample to be tested in the sample injection chamber (12) is mixed with the lysis solution and magnetic bead mixture in the mixing chamber (11) and the lysis solution chamber (13) to form a first mixture, and the first mixture is contained in the lysis solution chamber (13). Use an ultrasonic device to perform ultrasonic lysis on the first mixture in the lysis solution chamber (13) so that the magnetic beads in the first mixture adsorb nucleic acid molecules. S3. Use a magnetic device to attract the magnetic beads in the lysis chamber (13) to the wall of the lysis chamber (13), drive the valve body (3) and the piston (2), and monitor the liquid transfer process through the capacitance detection electrode (6) so that the liquid in the lysis chamber (13) enters the sample injection chamber (12) through the mixing chamber (11). S4. Drive the valve body (3) and the piston (2), and monitor the liquid transfer process through the capacitor detection electrode (6). Drive the cleaning liquid in the cleaning liquid chamber (14) through the mixing chamber (11) into the lysis liquid chamber (13) to form a first intermediate mixture containing magnetic beads. Then drive the first intermediate mixture to move back and forth between the lysis liquid chamber (13) and the mixing chamber (11), and determine whether the cleaning liquid and the magnetic beads have been mixed. If it is determined that the cleaning liquid and the magnetic beads have been mixed, drive the first intermediate mixture contained in the mixing chamber (11) into the lysis liquid chamber (13). Then the magnetic device adsorbs the magnetic beads in the lysis liquid chamber (13) onto the cavity wall of the lysis liquid chamber (13), and drive the liquid in the lysis liquid chamber (13) through the mixing chamber (11) into the sample injection chamber (12). S5. Drive the valve body (3) and the piston (2), and monitor the liquid transfer process through the capacitor detection electrode (6). Drive the elution liquid in the elution chamber (15) through the mixing chamber (11) into the lysis chamber (13) to form a second intermediate mixture containing magnetic beads. Then drive the second intermediate mixture to move back and forth between the lysis chamber (13) and the mixing chamber (11), and determine whether the elution liquid and the magnetic beads have been mixed. If it is determined that the elution liquid and the magnetic beads have been mixed, drive the second intermediate mixture contained in the mixing chamber (11) into the lysis chamber (13). Use a magnetic device to adsorb the magnetic beads onto the wall of the lysis chamber (13), and drive the elution liquid containing nucleic acid molecules through the mixing chamber (11) to mix with the PCR reaction liquid in the PCR reaction chamber (16) to form a second mixture. S6. Drive the valve body (3) and the piston (2) to allow the second mixture to enter the amplification detection chip (5) to dissolve the reagent microspheres and form a third mixture. Amplify and collect fluorescence in the third mixture in the amplification detection chip (5). Determining whether the cleaning solution and the magnetic beads have been completely mixed includes: when the first intermediate mixture is contained in the mixing chamber (11), the standard deviation of the monitoring signal of the capacitance detection electrode (6) is calculated in real time. If the standard deviation of the monitoring signal of the capacitance detection electrode (6) is within the preset threshold range within the first preset time period, it is determined that the cleaning solution and the magnetic beads have been completely mixed. Determining whether the eluent and magnetic beads have been completely mixed includes: when the second intermediate mixture is contained in the mixing chamber (11), the standard deviation of the monitoring signal of the capacitance detection electrode (6) is calculated in real time. If the standard deviation of the monitoring signal of the capacitance detection electrode (6) is within a preset threshold range within a second preset time period, it is determined that the eluent and magnetic beads have been completely mixed.

10. The nucleic acid detection method for non-disease diagnostic purposes according to claim 9, characterized in that, The monitoring of the liquid transfer process via the capacitance detection electrode (6) includes: When the piston (2) moves upward to allow liquid from other chambers to enter the mixing chamber (11), the capacitance change in the mixing chamber (11) is monitored by the capacitance detection electrode (6), and the liquid transfer is judged to be completed based on the capacitance change in the mixing chamber (11). If the liquid transfer is judged to be completed, the piston (2) is controlled to stop moving upward.

Images