Nucleic acid extraction and detection card box based on magnetic bar method and use method of nucleic acid extraction and detection card box

Abstract

The invention discloses a nucleic acid extraction and detection card box based on a magnetic bar method and a use method thereof, the card box comprises a main reaction tube, a multifunctional tube, a freeze-drying microsphere tube, an eluent tube and a plurality of washing liquid tubes which surround the main reaction tube, and a detection liquid control tube adjacent to the freeze-drying microsphere tube. The invention discloses a detection card box which adopts an integrated design and is compact in structure and convenient to manufacture and assemble. In cooperation with the use method disclosed by the invention, magnetic bar transfer is replaced by liquid transfer, so that the detection cost is low, the risk of cross contamination is avoided, and large-scale automatic nucleic acid extraction and amplification detection can be conveniently realized.

Description

Technical Field

[0001] The present invention belongs to the nucleic acid integrated extraction and detection technology branch in the fields of biological and medical detection technologies, and particularly relates to a nucleic acid extraction and detection cartridge based on a magnetic rod method and a using method thereof in this field. Background Art

[0002] At present, there are some obvious defects in nucleic acid detection-based devices on the market. For example, for nucleic acid detection methods using a microfluidic technology and an electrochemical sensing principle and adopting an overall detection cartridge design, although the specificity and sensitivity of nucleic acid detection results can be ensured, the packaging process of the overall detection cartridge and reagents is relatively complex, and the production manufacturing and single-use costs are relatively high, which limits their market application. There are also some methods that use mechanical and optoelectronic mechanisms to cooperate with a cartridge for nucleic acid extraction and detection, and rely on the movement of a magnetic rod and a magnetic rod sleeve to separate and extract nucleic acid. During the extraction and transfer process, there is an exposure risk and it is easy to cause cross-contamination, thus affecting the accuracy of the detection results. In the process of realizing automation for the external mechanical equipment they use, there are also problems such as too many mechanical actions and a large equipment volume. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a nucleic acid extraction and detection cartridge based on a magnetic rod method and a using method thereof for the problem that there is an exposure risk and it is easy to cause cross-contamination during the transfer of the magnetic rod and the magnetic rod sleeve in the current nucleic acid detection process.

[0004] The present invention adopts the following technical solutions: A nucleic acid extraction and detection card box based on a magnetic rod method, the improvement of which is that it comprises a main reaction tube and a multifunctional tube surrounding the main reaction tube, a freeze-dried microsphere tube, an eluent tube and a plurality of washing liquid tubes, and a detection liquid control tube adjacent to the freeze-dried microsphere tube, the detection liquid control tube comprising a bottom card slot and an upper tube body connected together, two transverse pipes are arranged on the card slot, one transverse pipe is located at the bottom of the card slot and communicates with the bottom of the freeze-dried microsphere tube, and the other transverse pipe is located at the upper part of the card slot and communicates with the tube body, a card-type flow detection tube is fixedly connected to the card slot, a part of its internal channel is flat, and two ports of the internal channel are respectively connected to the two transverse pipes of the above-mentioned card slot; a tube cover is installed on the tube mouth of the main reaction tube, an opening is formed on the tube cover and a plurality of air pipe joints are arranged, a magnetic rod sleeve with a closed bottom and extending to the bottom of the main reaction tube is arranged on the opening, and the bottom of the main reaction tube is respectively connected to the bottom of the multifunctional tube and the freeze-dried microsphere tube through the pipeline. The middle parts of the ball tubes are connected, and the middle parts of the main reaction tubes are connected with the middle parts of the eluent tube and each washing liquid tube through pipelines; a first piston core rod that can slide up and down along the inner wall of the multifunctional tube is inserted into the multifunctional tube, the first piston core rod comprises a core rod, a piston and a core rod head connected together, the piston seals the multifunctional tube, and a thorn-shaped protrusion is arranged at the bottom of the core rod head; a second piston core rod that can slide up and down along the inner wall of the tube is inserted into the eluent tube and each washing liquid tube, the second piston core rod comprises a core rod, a piston and a core rod head connected together, the piston seals the tube, the core rod head is a cylinder with a bottom radius the same as the inner diameter of the tube, a thorn-shaped protrusion is arranged at the bottom of the cylinder, and a vertical groove is arranged on the side of the cylinder, the groove can make the bottom of the tube communicate with the pipeline in the middle of the tube; a third piston core rod that can slide up and down along the inner wall of the tube is inserted into the freeze-dried microsphere tube and the detection liquid control tube, the third piston core rod comprises a core rod and a piston connected together, and the piston seals the tube.

[0005] Furthermore, the upper tube bodies of the main reaction tube, multifunctional tube, freeze-dried microsphere tube, eluent tube, several washing liquid tubes and detection liquid control tube are all integrally formed and embedded in a thin rectangular sheet, and the tube openings of each tube are slightly exposed on the surface of the rectangular sheet.

[0006] Furthermore, a protruding buckle is provided on the outer side of the card-type circulation detection tube, and the card-type circulation detection tube is fixedly connected to the card slot by snapping the buckle onto the card slot; the two ports of the card-type circulation detection tube protrude outside the card-type circulation detection tube, and when the card-type circulation detection tube is connected to the card slot, the two ports are inserted into the card slot and are respectively connected to the two horizontal pipes of the card slot, and a sealing ring is also provided at the connection.

[0007] Furthermore, a protrusion is provided on the outer edge of the tube opening of the main reaction tube, and a buckle corresponding to the protrusion is provided at the bottom of the edge of the tube cover; the bottom of the main reaction tube is hemispherical.

[0008] Further, the core rods of the first piston core rod, the second piston core rod, and the third piston core rod are all cross-shaped injection molded parts, with an oblate push handle at the top and an annular groove at the bottom.

[0009] Further, the core rod head of the first piston core rod is a cylinder, with an annular groove at the top of the cylinder; there is an annular groove at the top of the core rod head of the second piston core rod.

[0010] Further, the piston materials of the first piston core rod, the second piston core rod, and the third piston core rod are all made of butyl rubber. The pistons of the first piston core rod and the second piston core rod are cylinders with both ends open and the middle closed, and there is a raised ring on the inner and outer edges of both open ends. The annular groove at the bottom of the core rod of the first piston core rod and the second piston core rod is clamped in the top open end of the piston, and the annular groove at the top of the core rod head is clamped in the bottom open end of the piston; the piston of the third piston core rod is a cylinder with the top open and the rest closed, and there is a raised ring on the inner and outer edges of the top open end. The annular groove at the bottom of the core rod of the third piston core rod is clamped in the top open end of the piston.

[0011] A usage method, using the above detection cartridge, is improved in that: After putting magnetic beads and the sample to be detected into the main reaction tube and covering the tube cap, put the magnetic rod into the magnetic rod sleeve on the tube cap, and connect the gas pipe joint on the tube cap to an external air pump; put the lysis solution bag containing lysis solution into the multi-functional tube and then insert the first piston core rod; put the washing solution bag containing the same or different washing solutions into each washing solution tube and then insert the second piston core rod; put the elution solution bag containing elution solution into the elution solution tube and then insert the second piston core rod; put the freeze-dried microspheres into the freeze-dried microsphere tube and then insert the third piston core rod, and the piston of the third piston core rod seals the pipeline in the middle of the tube; insert the third piston core rod into the detection liquid control tube, and the piston of the third piston core rod seals the connection channel between the tube body and the card slot; Press down the first piston core rod of the multi-functional tube, so that the spiky protrusion on the core rod head of the first piston core rod pierces and breaks the lysis solution bag, and the flowing lysis solution is pressed into the main reaction tube. When the magnetic rod is inserted into the magnetic rod sleeve, the magnetic beads are adsorbed to the outer surface of the magnetic rod sleeve. When the magnetic rod is pulled out of the magnetic rod sleeve, the magnetic beads naturally suspend or settle in the main reaction tube. Insert and pull out the magnetic rod back and forth to make the magnetic beads fully contact with the lysis solution in the main reaction tube, enrich the nucleic acid extracted by the lysis reaction of the lysis solution and the sample to be detected on the surface of the magnetic beads. Finally, insert the magnetic rod into the magnetic rod sleeve to make the magnetic beads adsorbed to the outer surface of the magnetic rod sleeve, and pull up the first piston core rod of the multi-functional tube to draw the liquid in the main reaction tube back into the multi-functional tube; Press down the second piston core rod of a certain washing liquid tube to make the thorn-shaped protrusion on the core rod head of the second piston core rod pierce the washing liquid bag. The flowing washing liquid is pressed into the main reaction tube through the groove. Insert and pull out the magnetic bar back and forth to make the magnetic beads fully contact with the washing liquid in the main reaction tube, so as to purify the nucleic acid on the surface of the magnetic beads. Finally, insert the magnetic bar into the magnetic bar sleeve to make the magnetic beads be adsorbed on the outer surface of the magnetic bar sleeve. Pull up the first piston core rod of the multi-functional tube to suck the liquid in the main reaction tube back into the multi-functional tube. Press down the second piston core rods of the remaining washing liquid tubes in turn and repeat the process of this paragraph; Start the external air pump and introduce dry nitrogen or dry air into the main reaction tube to dry the main reaction tube; Press down the second piston core rod of the eluent tube to make the thorn-shaped protrusion on the core rod head of the second piston core rod pierce the eluent bag. The flowing eluent is pressed into the main reaction tube through the groove. Insert and pull out the magnetic bar back and forth to make the magnetic beads fully contact with the eluent in the main reaction tube, and transfer the nucleic acid on the surface of the magnetic beads to the eluent. Finally, insert the magnetic bar into the magnetic bar sleeve to make the magnetic beads be adsorbed on the outer surface of the magnetic bar sleeve; Pull up the third piston core rod of the freeze-dried microsphere tube to suck the liquid in the main reaction tube into the freeze-dried microsphere tube, so that the freeze-dried microspheres dissolve and a polymerase chain reaction occurs; Pull up the third piston core rod of the detection liquid control tube to suck the liquid in the freeze-dried microsphere tube into the card-type flow-through detection tube for subsequent detection use.

[0012] Further, after putting magnetic beads into the main reaction tube, cover the tube cap. Put the sample to be detected into the multi-functional tube. Press down the first piston core rod of the multi-functional tube to make the thorn-shaped protrusion on the core rod head of the first piston core rod pierce the lysis solution bag, and press the flowing lysis solution into the main reaction tube. Pull up and press down the first piston core rod of the multi-functional tube repeatedly to suck the lysis solution back and press it out of the multi-functional tube. During this process, the lysis solution is fully contacted and mixed with the sample to be detected, and finally the lysis solution is pressed into the main reaction tube again; or after putting magnetic beads, lysis solution and the sample to be detected into the main reaction tube, cover the tube cap. Do not put the lysis solution bag into the multi-functional tube, and only insert the first piston core rod to the bottom of the multi-functional tube.

[0013] Further, a heating and temperature control unit is arranged at the bottom of the main reaction tube; a temperature control device is installed on the side of the card-type flow-through detection tube.

[0014] The beneficial effects of the present invention are: In view of the problems existing in the field of nucleic acid extraction and detection, such as the inability of current equipment to fully automate all steps of nucleic acid extraction and detection, the complex structure of some integrated nucleic acid extraction and amplification detection equipment, the high difficulty in manufacturing and assembly, and the high risk in the detection process, the present invention discloses a detection cartridge, which adopts an integrated design, has a compact structure, and is convenient for manufacturing and assembly. In combination with the usage method disclosed in the present invention, liquid transfer is used to replace magnetic rod transfer, with low detection cost, no risk of cross-contamination, and facilitating large-scale automated nucleic acid extraction and amplification detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 FIG. 1 is a schematic structural diagram I of the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 2 FIG. 2 is a schematic structural diagram II of the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 3 FIG. 3 is a schematic structural diagram III of the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 4 FIG. 4 is a sectional view of the distribution of the pipeline 4 on the inner wall of the main reaction tube in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 5 FIG. 5 is a schematic structural diagram of the card-type flow-through detection tube in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 6 FIG. 6 is a schematic structural diagram of the tube cap in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 7 FIG. 7 is a connection relationship diagram of the main reaction tube with the multi-functional tube and the freeze-dried microsphere tube in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 8 FIG. 8 is a connection relationship diagram of the main reaction tube with the washing liquid tube and the elution liquid tube in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 9 FIG. 9 is a connection relationship diagram of the main reaction tube with the washing liquid tube in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 10 FIG. 10 is a schematic structural diagram of the first piston core rod in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 11 FIG. 11 is a schematic structural diagram of the second piston core rod in the detection cartridge disclosed in Embodiment 1 of the present invention; Figure 12 FIG. 12 is a schematic structural diagram of the third piston core rod in the detection cartridge disclosed in Embodiment 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] Example 1 discloses a nucleic acid extraction and detection cartridge based on the magnetic rod method. As shown in FIG. Figure 1 -4, it includes a main reaction tube and a multi-functional tube, a freeze-dried microsphere tube, an eluent tube, and three washing solution tubes surrounding the main reaction tube. There is also a detection solution control tube adjacent to the freeze-dried microsphere tube. The detection solution control tube includes a bottom card slot and an upper tube body connected together. Two horizontal pipelines are arranged on the card slot. One horizontal pipeline is located at the bottom of the card slot and communicates with the bottom of the freeze-dried microsphere tube. The other horizontal pipeline is located at the upper part of the card slot and communicates with the center of the bottom of the tube body through a vertical hole. The card-type flow-through detection tube is fixedly connected to the card slot. A part of its internal channel is flat, and the two ports of the internal channel are respectively connected to the two horizontal pipelines of the above-mentioned card slot. As shown in FIG. Figure 5 As shown, the card-type flow-through detection tube is a thin cuboid, and the flat internal channel 1 facilitates optical detection.

[0018] Specifically, a protruding buckle 2 is arranged on the outside of the card-type flow-through detection tube. By clamping the buckle on the card slot, the card-type flow-through detection tube is fixedly connected to the card slot. The two ports of the card-type flow-through detection tube protrude outside the card-type flow-through detection tube, and the inner diameters of the two ports are the same as the inner diameters of the two horizontal pipelines in the card slot. When the card-type flow-through detection tube is connected to the card slot, these two ports are inserted into the card slot and are respectively connected to the two horizontal pipelines of the card slot, and a sealing ring is arranged at the connection.

[0019] This embodiment consists of an integrated box body composed of a main reaction tube and a multi-functional tube, a freeze-dried microsphere tube, an eluent tube, three washing solution tubes, and a detection solution control tube adjacent to the freeze-dried microsphere tube, with a total of 8 vertically arranged tubular structures. Specifically, the upper tube bodies of the main reaction tube, multi-functional tube, freeze-dried microsphere tube, eluent tube, three washing solution tubes, and detection solution control tube are integrally formed and embedded in a thin cuboid sheet, and the tube orifices of each tube slightly protrude from the surface of the cuboid sheet.

[0020] The multi-functional tube is named tube No. 0. The multi-functional tube is a flat-bottomed cylindrical tube with an upward orifice, and it has the largest volume.

[0021] The main reaction tube is named tube No. 1. The main reaction tube is a cylindrical tube with an open top and a hemispherical bottom. A tube cap is installed on the orifice of the main reaction tube. As shown in FIG. Figure 6As shown in the figure, the tube cap is a circular pressing cap that matches the size of the opening of the main reaction tube. There is an opening in the center of the tube cap, and two gas connectors 3 are provided. On the opening, there is a slender magnetic rod sleeve 4 with a closed bottom that extends to the bottom of the main reaction tube. The magnetic rod can be repeatedly inserted into and pulled out of the magnetic rod sleeve. A protrusion is provided on the outer edge of the opening of the main reaction tube, and a buckle corresponding to the above-mentioned protrusion is provided at the bottom edge of the tube cap; by slightly applying external force to press, the buckle of the tube cap can be closed with the protrusion of the tube opening, so that the tube cap seals the opening of the main reaction tube.

[0022] As Figure 7 shown, the bottom of the main reaction tube is respectively communicated with the bottom of the multi-functional tube and the middle part of the freeze-dried microsphere tube through pipelines. As Figure 8 , as shown in Fig. 9, the middle part of the main reaction tube is respectively communicated with the middle parts of the eluent tube and each washing liquid tube through horizontal pipelines. Here, the middle part refers to about 1 / 2 - 2 / 3 of the tube body from the bottom of the tube.

[0023] The three washing liquid tubes are respectively named as No. 2 - 4 tubes. The structures of the three washing liquid tubes are the same, and they are all flat-bottomed cylindrical tubes with the openings facing upward.

[0024] The eluent tube is named as No. 5 tube. The eluent tube is a flat-bottomed cylindrical tube with the opening facing upward. The volumes of No. 2 - 5 tubes are the same.

[0025] The freeze-dried microsphere tube is named as No. 6 tube. The freeze-dried microsphere tube is a flat-bottomed cylindrical tube with the opening facing upward. The volume of No. 6 tube is larger than that of No. 2 - 5 tubes.

[0026] The detection liquid control tube is named as No. 7 tube. The upper tube body of the detection liquid control tube is a flat-bottomed cylindrical tube with the opening facing upward.

[0027] Insert a first piston core rod that can slide up and down along the inner wall of the multi-functional tube into the multi-functional tube. As Figure 10 shown, the first piston core rod includes a core rod 51, a piston 52, and a core rod head 53 that are connected together. The piston seals the multi-functional tube, and a spiky protrusion 54 is provided at the bottom of the core rod head; insert a second piston core rod that can slide up and down along the inner wall of the tube into the eluent tube and each washing liquid tube. As Figure 11 shown, the second piston core rod includes a core rod 61, a piston 62, and a core rod head 63 that are connected together. The piston seals the tube. The core rod head is a cylinder with the bottom surface radius the same as the inner diameter of the tube. A spiky protrusion 64 is provided at the bottom of the cylinder. These spiky protrusions (needle tips) are distributed radially with the center of the cylinder as the center; a vertical groove 65 is provided on the side surface of the cylinder. The groove and the inner wall of the tube form a channel for liquid flow. Therefore, this groove can make the pipeline at the bottom of the tube communicate with the pipeline in the middle of the tube; insert a third piston core rod that can slide up and down along the inner wall of the tube into the freeze-dried microsphere tube and the detection liquid control tube. As Figure 12As shown, the third piston core rod includes a core rod 71 and a piston 72 connected together, and the piston seals the tube.

[0028] The core rods of the first piston core rod, the second piston core rod, and the third piston core rod are all cross-shaped injection-molded parts, with an oblate push handle at the top and an annular groove at the bottom. The entire core rod is integrally injection-molded from a polymer material such as polypropylene.

[0029] The core rod head of the first piston core rod is a cylinder, and an annular groove is provided at the top of the cylinder; an annular groove is provided at the top of the core rod head of the second piston core rod.

[0030] The pistons of the first piston core rod, the second piston core rod, and the third piston core rod are all integrally injection-molded from a butyl rubber-like material. The pistons of the first piston core rod and the second piston core rod are cylinders with open ends at both ends and a closed middle, and a raised ring is provided on the inner and outer edges of the open ends at both ends. The inner raised ring is used to clamp and fix the annular groove, and the outer raised ring is used to enhance the seal between the piston and the tube wall to ensure the isolation of the liquid medicine in the tube by the piston. The annular groove at the bottom of the core rod of the first piston core rod and the second piston core rod is clamped in the open end at the top of the piston, and the annular groove at the top of the core rod head is clamped in the open end at the bottom of the piston; the piston of the third piston core rod is a cylinder with an open top and the rest closed, and a raised ring is provided on the inner and outer edges of the open top. The inner raised ring is used to clamp and fix the annular groove, and the outer raised ring is used to enhance the seal between the piston and the tube wall to ensure the isolation of the liquid medicine in the tube by the piston. The annular groove at the bottom of the core rod of the third piston core rod is clamped in the open end at the top of the piston.

[0031] Before use, the integrated box body, each piston core rod, and the card-type flow detection tube are respectively placed in a clean sealed bag. When in use, each piston core rod is inserted into the corresponding tube, two sealing rings are respectively placed at the connection points between the two horizontal pipes in the card slot and the two ends of the card-type flow detection tube, and then the card-type flow detection tube is fixedly connected to the card slot.

[0032] This embodiment also discloses a usage method, using the above detection card box: After putting magnetic beads and the sample to be detected into the main reaction tube and covering the tube cap, put the magnetic rod into the magnetic rod sleeve on the tube cap, and connect the air pipe joint on the tube cap to an external air pump; after putting the lysis solution bag containing the lysis solution into the multi-functional tube, insert the first piston core rod; after putting the washing solution bag containing the same or different washing solutions into each washing solution tube, insert the second piston core rod; after putting the elution solution bag containing the elution solution into the elution solution tube, insert the second piston core rod; after putting a few freeze-dried microspheres into the freeze-dried microsphere tube, insert the third piston core rod, and the piston of the third piston core rod seals the pipe in the middle of the tube; insert the third piston core rod into the detection liquid control tube, and the piston of the third piston core rod seals the connection channel between the tube body and the card slot; The above-mentioned sample to be detected is a homogenized dissolved sample.

[0033] The above-mentioned liquid sac uses natural or synthetic polymer materials as the capsule membrane to wrap the liquid reagent. After being punctured by a sharp object, the liquid in the liquid sac flows out; the appearance of the liquid sac can be processed into different shapes such as spherical or olive-shaped according to needs.

[0034] The above-mentioned freeze-dried microspheres are microspheres made from a mixed reagent of a freeze-drying protectant, buffer solution, enzyme, primer, probe, etc. required for the amplification reaction through freeze-drying technology. The freeze-dried microspheres dissolve when they encounter a purified nucleic acid liquid sample and undergo a polymerase chain reaction.

[0035] Press down the first piston core rod of the multi-functional tube to make the spiky protrusion on the core rod head of the first piston core rod puncture the lysis liquid sac, and press the flowing lysis liquid into the main reaction tube. When the magnetic rod is inserted into the magnetic rod sleeve, the magnetic beads are adsorbed to the outer surface of the magnetic rod sleeve. When the magnetic rod is pulled out of the magnetic rod sleeve, the magnetic beads naturally suspend or settle in the main reaction tube. Insert and pull out the magnetic rod back and forth to make the magnetic beads repeatedly aggregate and disperse, so as to fully contact with the lysis liquid in the main reaction tube, and enrich the nucleic acid extracted by the lysis reaction of the lysis liquid and the sample to be detected on the surface of the magnetic beads. In order to improve the extraction efficiency of the lysis reaction, a heating and temperature control unit can be set at the bottom of the main reaction tube to heat the main reaction tube; finally, insert the magnetic rod into the magnetic rod sleeve to make the magnetic beads adsorbed to the outer surface of the magnetic rod sleeve, and pull up the first piston core rod of the multi-functional tube to draw the liquid in the main reaction tube back into the multi-functional tube; Press down the second piston core rod of a certain washing liquid tube to make the spiky protrusion on the core rod head of the second piston core rod puncture the washing liquid sac, and the flowing washing liquid is pressed into the main reaction tube through the groove. Insert and pull out the magnetic rod back and forth to make the magnetic beads repeatedly aggregate and disperse, so as to fully contact with the washing liquid in the main reaction tube, and purify the nucleic acid on the surface of the magnetic beads. Finally, insert the magnetic rod into the magnetic rod sleeve to make the magnetic beads adsorbed to the outer surface of the magnetic rod sleeve, and pull up the first piston core rod of the multi-functional tube to draw the liquid in the main reaction tube back into the multi-functional tube. Press down the second piston core rods of the other two washing liquid tubes in turn and repeat the process of this paragraph; Start the external air pump to introduce dry nitrogen or dry air into the main reaction tube to dry the main reaction tube; in the case where ventilation drying treatment is not required or not satisfied, one of the two gas pipe joints on the tube cap of the main reaction tube can be closed, and the other is connected to an external membrane filter, which only allows air to pass through, and liquids or aerosols cannot pass through.

[0036] Press down the second piston core rod of the elution liquid tube to make the spiky protrusion on the core rod head of the second piston core rod puncture the elution liquid sac, and the flowing elution liquid is pressed into the main reaction tube through the groove. Insert and pull out the magnetic rod back and forth to make the magnetic beads repeatedly aggregate and disperse, so as to fully contact with the elution liquid in the main reaction tube, and transfer the nucleic acid on the surface of the magnetic beads to the elution liquid. Finally, insert the magnetic rod into the magnetic rod sleeve to make the magnetic beads adsorbed to the outer surface of the magnetic rod sleeve; Pull up the third piston core rod of the freeze-dried microsphere tube to draw the liquid in the main reaction tube into the freeze-dried microsphere tube, dissolve the freeze-dried microspheres and initiate a polymerase chain reaction. To improve the efficiency of the polymerase chain reaction, a temperature control device can be installed on the side of the card-type flow-through detection tube to ensure that the temperature is well controlled between the denaturation temperature (about 95°C), the annealing temperature (about 60°C), and the extension temperature (about 72°C).

[0037] Pull up the third piston core rod of the detection liquid control tube to draw the liquid in the freeze-dried microsphere tube into the card-type flow-through detection tube, control the denaturation temperature, annealing temperature, and extension temperature, and use the movement of the external fluorescence detection module to perform real-time detection of the fluorescence before and after the amplification reaction in the flat channel, thus completing the entire nucleic acid extraction and detection process.

[0038] Example 2. The difference in the usage method between this example and Example 1 is as follows: After placing magnetic beads in the main reaction tube, cover the tube cap. Place the sample to be detected in the multi-functional tube, press down the first piston core rod of the multi-functional tube, so that the spiky protrusion at the head of the first piston core rod pierces and ruptures the lysis solution capsule. The flowing-out lysis solution is mixed with the sample to be detected and then pressed into the main reaction tube. Repeatedly pull up and press down the first piston core rod of the multi-functional tube to continuously draw back and press out the lysis solution from the multi-functional tube. During this process, the lysis solution comes into full contact and mixing with the soluble substances in the sample to be detected. Finally, press the lysis solution into the main reaction tube again. The subsequent process is the same as that in Example 1 and will not be elaborated here.

[0039] The number of washing times in Example 1 and Example 2 can be flexibly selected according to the actual situation of the specific sample to be detected.

[0040] Example 3. The difference in the usage method between this example and Example 1 is as follows: Do not use the liquid capsule. Pour the liquid reagent directly into the corresponding tube. After placing magnetic beads, lysis solution, and the sample to be detected in the main reaction tube, cover the tube cap. Do not pour lysis solution into the multi-functional tube, and only insert the first piston core rod to the bottom of the multi-functional tube. With the cooperation of the magnetic beads and the magnetic rod, the RNA or DNA nucleic acid in the main reaction tube is detached from the sample by the action of the lysis solution and enriched on the surface of the magnetic beads. The subsequent process is the same as that in Example 1 and will not be elaborated here.

Claims

1. A nucleic acid extraction and detection cartridge based on a magnetic rod method, characterized in that: The invention comprises a main reaction tube and a multifunctional tube, a freeze-dried microsphere tube, an eluent tube and a plurality of washing liquid tubes surrounding the main reaction tube, and a detection liquid control tube adjacent to the freeze-dried microsphere tube, wherein the detection liquid control tube comprises a bottom card slot and an upper tube body connected together, two transverse pipes are arranged on the card slot, one transverse pipe is located at the bottom of the card slot and communicates with the bottom of the freeze-dried microsphere tube, and the other transverse pipe is located at the upper part of the card slot and communicates with the tube body, and the card-type flow detection tube is fixedly connected to the card slot, a part of the internal channel thereof is flat, and the two ports of the internal channel are respectively connected to the two transverse pipes of the above-mentioned card slot. The main reaction tube is connected with the pipeline; a tube cover is installed on the tube mouth of the main reaction tube, an opening is formed on the tube cover and a plurality of air pipe joints are arranged, a magnetic rod sleeve with a closed bottom and extending to the bottom of the main reaction tube is arranged on the opening, the bottom of the main reaction tube is communicated with the bottom of the multifunctional tube and the middle of the freeze-dried microsphere tube through the pipeline, and the middle of the main reaction tube is communicated with the eluent tube and the middle of each washing liquid tube through the pipeline; a first piston core rod that can slide up and down along the inner wall of the multifunctional tube is inserted into the multifunctional tube, the first piston core rod comprises a core rod, a piston and a core rod head connected together, the piston seals the multifunctional tube, and a thorn-shaped protrusion is arranged at the bottom of the core rod head; A second piston core rod capable of sliding up and down along the inner wall of the tube is inserted into the eluent tube and each washing liquid tube. The second piston core rod comprises a core rod, a piston and a core rod head connected together. The piston seals the tube. The core rod head is a cylinder with a bottom radius the same as the inner diameter of the tube. A thorn-shaped protrusion is arranged at the bottom of the cylinder. A vertical groove is arranged on the side of the cylinder. The groove enables the bottom of the tube to communicate with the pipeline in the middle of the tube. A third piston core rod capable of sliding up and down along the inner wall of the tube is inserted into the freeze-dried microsphere tube and the detection liquid control tube. The third piston core rod comprises a core rod and a piston connected together. The piston seals the tube.

2. The nucleic acid extraction and detection cartridge based on the magnetic rod method according to claim 1, characterized in that: The upper tube bodies of the main reaction tube, multifunctional tube, freeze-dried microsphere tube, eluent tube, several washing liquid tubes and detection liquid control tube are all integrally formed and embedded in a thin rectangular sheet, and the tube openings of each tube are slightly exposed on the surface of the rectangular sheet.

3. The nucleic acid extraction and detection cartridge based on the magnetic rod method according to claim 1, characterized in that: A protruding buckle is arranged on the outer side of the card-type circulation detection tube, and the card-type circulation detection tube is fixedly connected to the card slot by snapping the buckle onto the card slot; the two ports of the card-type circulation detection tube protrude from the card-type circulation detection tube, and when the card-type circulation detection tube is connected to the card slot, the two ports are inserted into the card slot and are respectively connected to the two horizontal pipes of the card slot, and a sealing ring is also arranged at the connection.

4. The nucleic acid extraction and detection cartridge based on the magnetic rod method according to claim 1, characterized in that: A protrusion is arranged on the outer edge of the tube opening of the main reaction tube, and a buckle corresponding to the protrusion is arranged on the bottom of the edge of the tube cover; the bottom of the main reaction tube is hemispherical.

5. The nucleic acid extraction and detection cartridge based on the magnetic rod method according to claim 1, characterized in that: The core rods of the first piston core rod, the second piston core rod and the third piston core rod are all cross-shaped injection molded parts, with a flat circular push handle on the top and an annular groove on the bottom.

6. The nucleic acid extraction and detection cartridge based on the magnetic rod method according to claim 5, characterized in that: The core rod head of the first piston core rod is a cylinder, and an annular groove is arranged on the top of the cylinder; an annular groove is arranged on the top of the core rod head of the second piston core rod.

7. The nucleic acid extraction and detection cartridge based on the magnetic rod method according to claim 6, characterized in that: The piston materials of the first piston core rod, the second piston core rod and the third piston core rod are all butyl rubber. The pistons of the first piston core rod and the second piston core rod are cylinders that are open at both ends and closed in the middle, and a circle of ridges are arranged on the inner and outer edges of the openings at both ends. The annular grooves at the bottom of the core rods of the first piston core rod and the second piston core rod are clamped in the top opening of the piston, and the annular grooves at the top of the core rod head are clamped in the bottom opening of the piston; the piston of the third piston core rod is a cylinder that is open at the top and closed at the rest, and a circle of ridges are arranged on the inner and outer edges of the top opening, and the annular grooves at the bottom of the core rod of the third piston core rod are clamped in the top opening of the piston.

8. A method of use, using the detection cartridge according to claim 1, characterized in that: After placing magnetic beads and samples to be tested in the main reaction tube, the tube cover is closed, a magnetic rod is placed in the magnetic rod sleeve on the tube cover, and the air pipe joint on the tube cover is connected to an external air pump; after placing a lysis liquid capsule containing lysis solution in the multifunctional tube, a first piston core rod is inserted; after placing a washing liquid capsule containing the same or different washing solutions in each washing liquid tube, a second piston core rod is inserted; after placing an elution liquid capsule containing elution solution in the eluent tube, a second piston core rod is inserted; after placing freeze-dried microspheres in the freeze-dried microsphere tube, a third piston core rod is inserted, and the piston of the third piston core rod seals the pipeline in the middle of the tube; after inserting a third piston core rod in the detection liquid control tube, the piston of the third piston core rod seals the connection channel between the tube body and the card slot; The first piston core rod of the multifunctional tube is pressed down, so that the thorn-like protrusion on the core rod head of the first piston core rod punctures the lysis liquid capsule, and the outflowing lysis liquid is pumped into the main reaction tube. When the magnetic rod is inserted into the magnetic rod sleeve, the magnetic beads are adsorbed to the outer surface of the magnetic rod sleeve. When the magnetic rod is pulled out of the magnetic rod sleeve, the magnetic beads are naturally suspended or settled in the main reaction tube. The magnetic rod is inserted and pulled out back and forth to make the magnetic beads fully contact with the lysis liquid in the main reaction tube, and the nucleic acid extracted by the lysis reaction of the lysis liquid and the sample to be detected is enriched on the surface of the magnetic beads. Finally, the magnetic rod is inserted into the magnetic rod sleeve so that the magnetic beads are adsorbed to the outer surface of the magnetic rod sleeve, and the first piston core rod of the multifunctional tube is pulled up to draw the liquid in the main reaction tube back to the multifunctional tube; The second piston core rod of a washing liquid tube is pressed down, so that the thorn-like protrusion on the core rod head of the second piston core rod pierces the washing liquid capsule, and the outflowing washing liquid is pressed into the main reaction tube through the groove, and the magnetic rod is inserted and pulled back and forth to make the magnetic beads fully contact with the washing liquid in the main reaction tube, so that the nucleic acid on the surface of the magnetic beads is purified, and finally the magnetic rod is inserted into the magnetic rod sleeve, so that the magnetic beads are adsorbed to the outer surface of the magnetic rod sleeve, and the first piston core rod of the multifunctional tube is pulled up to draw the liquid in the main reaction tube back to the multifunctional tube, and the second piston core rods of the remaining washing liquid tubes are pressed down in turn and this process is repeated; Start the external air pump to introduce dry nitrogen or dry air into the main reaction tube to dry the main reaction tube; The second piston core rod of the eluent tube is pressed down, so that the thorn-like protrusion on the core rod head of the second piston core rod pierces the eluent bag, and the outflowing eluent is pressed into the main reaction tube through the groove, and the magnetic rod is inserted and pulled back and forth to make the magnetic beads fully contact with the eluent in the main reaction tube, and the nucleic acid on the surface of the magnetic beads is transferred to the eluent, and finally the magnetic rod is inserted into the magnetic rod sleeve, so that the magnetic beads are adsorbed to the outer surface of the magnetic rod sleeve; Pull up the third piston core rod of the freeze-dried microsphere tube to draw the liquid in the main reaction tube into the freeze-dried microsphere tube, so that the freeze-dried microspheres are dissolved and polymerase chain reaction occurs; Pull up the third piston core rod of the test liquid control tube to draw the liquid in the freeze-dried microsphere tube into the card-type flow test tube for subsequent testing.

9. The method of use according to claim 8, characterized in that: After placing magnetic beads in the main reaction tube, the tube cap is closed, and the sample to be tested is placed in the multifunctional tube. The first piston core rod of the multifunctional tube is pressed down, so that the thorn-like protrusions on the core rod head of the first piston core rod puncture the lysis liquid capsule, and the outflowing lysis liquid is pressed into the main reaction tube. The first piston core rod of the multifunctional tube is repeatedly pulled up and pressed down to continuously draw back and press the lysis liquid out of the multifunctional tube. During this process, the lysis liquid and the sample to be tested are fully contacted and mixed, and finally the lysis liquid is pressed into the main reaction tube; or after placing magnetic beads, lysis liquid and the sample to be tested in the main reaction tube, the tube cap is closed, and no lysis liquid capsule is placed in the multifunctional tube, and only the first piston core rod is inserted to the bottom of the multifunctional tube.

10. The method of use according to claim 8, characterized in that: A heating and temperature control unit is arranged at the bottom of the main reaction tube; and a temperature control device is installed on the side of the card-type flow detection tube.

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