Nucleic acid real-time detection device and micro-fluidic chip

Abstract

The invention discloses a nucleic acid real-time detection device and a micro-fluidic chip, and belongs to the field of nucleic acid detection.A cracking cavity, a uniform mixing cavity and an amplification detection cavity of the micro-fluidic chip are arranged in the radial direction, and a second lifting driving part of an electrical module is in transmission connection with a spring needle to drive the spring needle to move up and down to be connected with or separated from a lower heating module; the upper heating module and the lower heating module are used for controlling the temperature of the cracking cavity, so that a sample in the cracking cavity is cracked; the rotary driving part is in transmission connection with the lower heating module, and drives the lower heating module to rotate, so that liquid in a reagent cavity and a cracking cavity in a micro-fluidic chip positioned on the lower heating module enters the uniform mixing cavity, and liquid in the uniform mixing cavity enters the amplification detection cavity; by means of the design, centrifugal force is adopted to drive liquid, an external pump and an external valve are not needed, the size of the detection device is reduced, miniaturization is facilitated, carrying is convenient, and instant detection can be conducted.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to a nucleic acid instant detection device and a microfluidic chip. Background Art

[0002] Nucleic acid testing is widely used in genomic research, genetic disease diagnosis, food and environmental safety testing, and other fields. Nucleic acid amplification technology based on polymerase chain reaction (PCR) is the gold standard in the field of molecular diagnosis. However, nucleic acid testing laboratories have strict requirements on the experimental environment, including the establishment of multiple laboratories such as the sample loading area, nucleic acid extraction area, PCR reaction area, and detection area with strict isolation and positive and negative pressure control. In addition, PCR involves multiple reaction temperatures, involves a long cycle of heating and cooling, and needs to be completed in a complex thermal cycler, all of which limit its application in point-of-care testing (POCT).

[0003] Recent advances in isothermal amplification technologies such as recombinase polymerase amplification (RPA) have made significant progress in achieving efficient and portable genetic testing solutions. Since its introduction in 2006, RPA has been widely used in the detection of viruses, bacteria, fungi, parasites, and drug-resistant genes. RPA is able to operate at a constant temperature, which can simplify the temperature control module, making it an ideal choice for point-of-care testing (POCT) without the need for the expensive thermal cycler usually required for PCR.

[0004] With the rapid development of microfluidic technology, microfluidic chips can complete the sample preparation, separation, purification, detection and other processes of traditional laboratories on a scale of a few square centimeters or even smaller. They have the advantages of small size, high integration, fast analysis speed, and low reagent consumption, and are widely used in biomedicine, environmental monitoring, analytical chemistry and other fields. The multiple analysis units of the chip can detect multiple samples at the same time, which is easy to meet the requirements of high-throughput detection, can reduce the size of the detection equipment, realize the portability of the equipment, and can be used for on-site detection.

[0005] Existing nucleic acid constant temperature amplification devices use external pumps and valves to drive liquids, which results in large size of the detection device, inability to be miniaturized, inconvenient to carry, and inability to perform instant detection. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a nucleic acid instant detection device that drives liquid through centrifugal force, has a small size, is easy to carry, and can perform instant detection.

[0007] In order to overcome the deficiencies of the prior art, the second object of the present invention is to provide a microfluidic chip that drives liquid through centrifugal force, has a small detection device, is easy to carry, and can perform instant detection.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A nucleic acid instant detection device comprises a microfluidic chip, an upper heating module and a lower heating module, wherein the microfluidic chip is provided with a reagent chamber, a lysis chamber, a mixing chamber and an amplification detection chamber, wherein the lysis chamber, the mixing chamber and the amplification detection chamber are arranged radially, and the nucleic acid instant detection device further comprises an electrical module, a rotary drive and an amplification heating module, wherein the electrical module comprises a second lifting drive and a spring needle, wherein the second lifting drive is connected to the spring needle in a transmission manner to drive the spring needle to move up and down and connect or separate from the lower heating module to control the working state of the lower heating module, wherein the upper heating module and the lower heating module control the temperature of the lysis chamber to lyse the sample in the lysis chamber; wherein the rotary drive is connected to the lower heating module in a transmission manner, wherein the rotary drive drives the lower heating module to rotate so that the reagent chamber in the microfluidic chip located on the lower heating module and the liquid in the lysis chamber enter the mixing chamber, and the liquid in the mixing chamber enters the amplification detection chamber through the rotation of the microfluidic chip, and the amplification heating module controls the temperature of the amplification detection chamber to amplify the sample.

[0010] Furthermore, the upper heating module includes a first lifting drive component, a fixed plate, a first circuit board and a heating block. The first lifting drive component is connected to the fixed plate to drive the fixed plate to lift and lower. The first circuit board is fixed to the fixed plate, and the heating block is fixed to the first circuit board.

[0011] Furthermore, the upper heating module also includes a pressure column, which is fixed to the fixed plate. The microfluidic chip also includes a second valve body, which is located between the lysis chamber and the mixing chamber. When the first lifting drive drives the fixed plate to descend, the heating block heats the lysis chamber, and the pressure column presses the second valve body to prevent the liquid in the lysis chamber from overflowing during heating.

[0012] Furthermore, the microfluidic chip includes multiple chip modules, each of the chip modules is fan-shaped, and the multiple chip modules are arranged in a circular shape. The multiple chip modules are all provided with the reagent chamber, the lysis chamber, the mixing chamber and the amplification detection chamber.

[0013] Furthermore, each of the chip modules is provided with a plurality of amplification detection cavities, the plurality of amplification detection cavities are located on an arc line, and the amplification detection cavities of the plurality of chip modules are located on a circumference.

[0014] Furthermore, the amplification heating module is ring-shaped, and the lower heating module is located inside the amplification heating module and forms a gap with the amplification heating module.

[0015] Furthermore, the amplification heating module includes an annular radiation element and a heating element, the heating element is annular, and the heating element is fixed to the bottom of the annular radiation element.

[0016] Furthermore, the instant nucleic acid detection device also includes a fiber optic probe, and the amplification and heating module is provided with an avoidance hole, and the fiber optic probe detects the liquid in the amplification detection cavity through the avoidance hole.

[0017] The second object of the present invention is achieved by adopting the following technical solution:

[0018] A microfluidic chip, the microfluidic chip comprising a reagent chamber, a lysis chamber, a mixing chamber and an amplification detection chamber, wherein the lysis chamber, the mixing chamber and the amplification detection chamber are arranged radially, and when the microfluidic chip rotates, centrifugal force causes the liquid in the reagent chamber and the lysis chamber to enter the mixing chamber, and the liquid in the mixing chamber enters the amplification detection chamber.

[0019] Furthermore, the microfluidic chip includes multiple chip modules, each of the chip modules is fan-shaped, and multiple chip modules are arranged in a circular shape. Multiple chip modules are provided with the reagent chamber, the lysis chamber, the mixing chamber and the amplification detection chamber. Each chip module is provided with multiple amplification detection chambers, and multiple amplification detection chambers are located on an arc line. The amplification detection chambers of multiple chip modules are located on a circumference.

[0020] Compared with the prior art, the lysis chamber, mixing chamber and amplification detection chamber of the microfluidic chip of the nucleic acid instant detection device of the present invention are arranged radially, and the nucleic acid instant detection device also includes an electrical module, a rotating drive member and an amplification heating module. The electrical module includes a second lifting drive member and a spring needle. The second lifting drive member is connected to the spring needle in a transmission manner to drive the spring needle to move up and down and connect or separate from the lower heating module to control the working state of the lower heating module. The upper heating module and the lower heating module control the temperature of the lysis chamber to lyse the sample in the lysis chamber; the rotating drive member is connected to the lower heating module in a transmission manner, and the rotating drive member drives the lower heating module to rotate so that the reagent chamber in the microfluidic chip located on the lower heating module and the liquid in the lysis chamber enter the mixing chamber, and the liquid in the mixing chamber enters the amplification detection chamber through the rotation of the microfluidic chip. The amplification heating module controls the temperature of the amplification detection chamber to amplify the sample. Through the above design, centrifugal force is used to drive the liquid, and no external pump or valve is required, which reduces the volume of the detection device, facilitates miniaturization, is easy to carry, can perform instant detection, and can realize automated multiple detection of multiple steps of lysis, mixing, amplification and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A three-dimensional diagram of the nucleic acid instant detection device of the present invention;

[0022] Figure 2 for Figure 1 A three-dimensional diagram of a microfluidic chip of a nucleic acid instant detection device;

[0023] Figure 3 for Figure 2 An exploded view of the chip module of the microfluidic chip;

[0024] Figure 4 for Figure 3 Schematic diagram of the internal structure of the chip module;

[0025] Figure 5 for Figure 1 A three-dimensional diagram of an upper heating module of a nucleic acid instant detection device;

[0026] Figure 6 for Figure 5 Exploded view of the upper heating module;

[0027] Figure 7 for Figure 1 A three-dimensional diagram of a lower heating module of a nucleic acid instant detection device;

[0028] Figure 8 for Figure 7 Another stereoscopic view of the lower heating module;

[0029] Fig. 9 for Figure 1 An exploded view of an amplification heating module of a nucleic acid instant detection device;

[0030] Fig.10 for Figure 1 A three-dimensional diagram of the electrical module of the nucleic acid instant detection device.

[0031] In the figure: 10, bracket; 11, bottom plate; 12, support column; 13, panel; 20, rotating drive member; 30, microfluidic chip; 31, chip module; 310, top layer; 311, first adhesive layer; 312, middle layer; 313, second adhesive layer; 314, bottom layer; 315, pores; 316, sample loading hole; 317, reagent chamber; 318, lysis chamber; 319, mixing chamber; 320, first valve body; 321, second valve body; 322, hollow part; 323, third valve body; 327, detection flow path; 324, metering chamber; 325, capillary valve; 326, amplification detection chamber; 328, waste liquid chamber; 329, positioning hole; 40 , upper heating module; 41, first lifting drive member; 42, fixing plate; 43, first circuit board; 44, heating block; 45, pressure column; 46, baffle; 47, column; 48, position detection sensor; 50, lower heating module; 51, mounting plate; 510, groove; 52, second circuit board; 53, positioning pin; 54, connecting part; 60, amplified heating module; 61, annular radiation member; 610, first avoidance hole; 62, heating member; 620, second avoidance hole; 70, electrical module; 71, mounting plate; 72, spring pin; 73, connecting line; 74, adapter plate; 75, second lifting drive member; 76, slide plate; 80, optical fiber probe. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] It should be noted that when a component is referred to as being "fixed to" another component, it may be directly on the other component or there may be another intermediate component through which it is fixed. When a component is considered to be "connected to" another component, it may be directly connected to the other component or there may be another intermediate component at the same time. When a component is considered to be "set on" another component, it may be directly set on the other component or there may be another intermediate component at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0035] See also Figure 1 The nucleic acid instant detection device includes a bracket 10, a rotating driving member 20, a microfluidic chip 30, an upper heating module 40, a lower heating module 50, an amplification heating module 60, an electrical module 70 and an optical fiber probe 80.

[0036] The bracket 10 includes a bottom plate 11, a support column 12 and a panel 13. One end of the support column 12 is fixed to the bottom plate 11, and the other end is fixed to the panel 13, so that a space for installing the rotating drive member 20 is formed between the bottom plate 11 and the panel 13. Specifically, the bottom plate 11 is parallel to the panel 13.

[0037] The rotary drive member 20 is fixed to the bottom of the panel 13, and the rotary drive member 20 is connected to the lower heating module 50 by transmission, driving the lower heating module 50 to rotate relative to the panel 13, so that the microfluidic chip 30 on the lower heating module 50 rotates, generating centrifugal force, so that the liquid in the microfluidic chip 30 flows with the centrifugal force, thereby driving the liquid. Specifically, the rotary drive member 20 is a motor. The rotary drive member 20 generates forces of different directions and magnitudes by adjusting parameters such as acceleration, speed, and deceleration to manipulate the fluid in the chip.

[0038] See also Figures 2 to 4 The microfluidic chip 30 is used for sample lysis, mixing, amplification and detection. The microfluidic chip 30 is circular in shape and includes a plurality of chip modules 31 , each of which is fan-shaped.

[0039] The chip module 31 is manufactured by injection molding, laser cutting or CNC engraving machine processing.

[0040] Each chip module 31 includes a top layer 310, a first adhesive layer 311, a middle layer 312, a second adhesive layer 313 and a bottom layer 314 arranged in sequence. The top layer 310, the middle layer 312 and the bottom layer 314 are made of plastic material, and the plastic material is a thermoplastic material, which is any one of PMMA, PC, and COC. The thickness of the top layer 310, the middle layer 312 and the bottom layer 314 are 0.6-1mm, 0.6-1mm and 0.4-0.8mm respectively. Preferably, the thickness of the top layer 310, the middle layer 312 and the bottom layer 314 are 0.8mm, 0.8mm and 0.6mm respectively. The first adhesive layer 311 and the second adhesive layer 313 are double-sided pressure-sensitive adhesives, and the thickness of the first adhesive layer 311 and the second adhesive layer 313 are both 130-170μm. Preferably, the thickness of the first adhesive layer 311 and the second adhesive layer 313 are both 150μm.

[0041] The top layer 310 is provided with an air hole 315 and two sample addition holes 316 . The air hole 315 is used to exhaust the waste liquid chamber 328 , and the two sample addition holes 316 are used to add reagents and samples respectively.

[0042] The first adhesive layer 311, the middle layer 312, and the second adhesive layer 313 are all provided with through grooves of the same shape, which, together with the top layer 310 and the bottom layer 314, form a reagent chamber 317, a lysis chamber 318, a mixing chamber 319, a third valve body 323, a detection flow path 327, and a waste liquid chamber 328. The reagent chamber 317, the lysis chamber 318, the mixing chamber 319, the third valve body 323, the detection flow path 327, and the waste liquid chamber 328 have a depth of 0.9-1.3 mm, preferably, a depth of 1.1 mm.

[0043] The reagent chamber 317 is connected to one sample adding hole 316, the lysis chamber 318 is connected to another sample adding hole 316, the mixing chamber 319 is connected to the reagent chamber 317 and the lysis chamber 318, the mixing chamber 319 is connected to the detection flow path 327, the end of the detection flow path 327 is connected to the waste liquid chamber 328, and the end of the waste liquid chamber 328 is connected to the air hole 315. Reagents are added to the reagent chamber 317 through the sample adding hole 316, and samples are added to the lysis chamber 318 through another sample adding hole 316.

[0044] A first valve body 320 is provided between the reagent chamber 317 and the mixing chamber 319. The first valve body 320 is a steam trap, which can prevent the liquid in the reagent chamber 317 from entering the mixing chamber 319 in advance. The steam trap has a depth of 0.8 mm and a cross-section of 1.5×2.2 mm.

[0045] A second valve body 321 is provided between the lysis chamber 318 and the mixing chamber 319. The second valve body 321 is a silicone valve with a diameter of 2 mm and a depth of 0.8 mm. When the silicone valve is impacted, it can prevent liquid from overflowing during the heating process.

[0046] The edge of the mixing chamber 319 is provided with a protrusion to form a third valve body 323. The third valve body 323 stops the microfluidic chip 30 from rotating. When the microfluidic chip 30 stops suddenly, the liquid will flow through the third valve body 323 due to inertia.

[0047] There are multiple detection flow paths 327, and the multiple detection flow paths 327 are arranged along the circumferential direction. Each detection flow path 327 includes a metering chamber 324, a capillary valve 325, and an amplification detection chamber 326. The metering chamber 324 is connected to the mixing chamber 319, and the metering chamber 324 is connected to the amplification detection chamber 326 through the capillary valve 325. The capillary valve 325 has a depth of 0.8 mm and a cross-section of 0.1×1.4 mm.

[0048] Each chip module 31 is also provided with a positioning hole 329 and a hollow portion 322, and the positioning hole 329 and the hollow portion 322 both penetrate the top layer 310, the first adhesive layer 311, the middle layer 312, the second adhesive layer 313 and the bottom layer 314. The positioning hole 329 is used for positioning the chip module 31 during installation, and the hollow portion 322 surrounds the cracking cavity 318, and the hollow portion 322 is used for heat dissipation to reduce chip deformation caused by thermal stress on the material.

[0049] In order to remove the residues caused by flying chips in and around the chamber and the flow channel during the processing, the microfluidic chip 30 was first ultrasonically cleaned with 0.1% Micro-90, then ultrasonically cleaned with deionized water, and then its surface was blown dry with nitrogen. The side wall of the middle layer 312 was treated with 1% Teflon and placed in a 70°C oven for drying.

[0050] After the remaining layers are assembled except the bottom layer 314, each amplification detection chamber 326 is coated with 0.5 mg / mL BSA solution at 37°C for 1 hour, and all primers and probes are spotted in the center of each amplification detection chamber 326. The freeze-dried powder of the reaction components containing the recombinase, auxiliary protein and single-stranded binding protein is loaded into the mixing chamber 319. The entire microfluidic chip 30 is pressed by a hydraulic press for 120 seconds. After 46 μL of RPA premix is ​​added to the reagent chamber 317 through the sample injection hole 316, the sample injection hole is sealed with a PCR film, and the chip with the pre-embedded reagent is sealed in an aluminum foil bag and kept dry and stored at 4°C until use.

[0051] Please continue reading Figure 5 as well as Figure 6The upper heating module 40 includes a first lifting drive member 41, a fixed plate 42, a first circuit board 43, a heating block 44, a pressure column 45, a baffle 46, a column 47 and a position detection sensor 48. The first lifting drive member 41 is fixed to the bottom of the panel 13, specifically, the first lifting drive member 41 is a screw motor. The fixed plate 42 is transmission-connected to the first lifting drive member 41, and the fixed plate 42 is a metal plate. The first circuit board 43 is fixed to the fixed plate 42, and the heating block 44 is installed on the first circuit board 43. Specifically, the heating block 44 is a copper block, which is installed on the first circuit board 43 and coated with thermal grease. The copper traces on the first circuit board 43 serve as a heat source to control the temperature of the copper block; the four pressure columns 45 provide pressure for the chip silicone active valve. The baffle 46 is fixed to the fixed plate 42, the column 47 is fixed to the panel 13, and the position detection sensor 48 is fixed to the top of the column 47. The baffle 46 moves up and down with the fixed plate 42. When the baffle 46 moves to the position detection sensor 48, it can trigger the position detection sensor 48 to detect the position of the fixed plate 42.

[0052] Please continue reading Figure 7 as well as Figure 8 , the lower heating module 50 includes a mounting plate 51, a second circuit board 52, a positioning pin 53 and a connecting portion 54. The mounting plate 51 is a metal plate, and a groove 510 is provided on the mounting plate 51 to facilitate temperature control. The mounting plate 51 is used to install the microfluidic chip 30, the second circuit board 52, the positioning pin 53 and the connecting portion 54. The second circuit board 52 is provided with four copper blocks, and the temperature of the four copper blocks is controlled by the second circuit board 52 in the same manner as the upper heating module 40. The lower heating module 50 is transmission-connected to the rotating drive member 20 through the connecting portion 54. Since the lower heating module 50 can rotate, it cannot be connected to the external temperature controller by a wire. Therefore, it is necessary to establish an electrical connection for it through the electrical module 70 when the motor is stationary; the positioning pin 53 is used to cooperate with the photoelectric sensor to calibrate the motor position.

[0053] Please continue reading Fig. 9 , the amplification heating module 60 includes an annular radiation member 61 and a heating member 62, and the heating member 62 is located at the bottom of the annular radiation member 61. The amplification heating module 60 is located at the bottom of the microfluidic chip 30, and the annular radiation member 61 is an annular metal block. The heat radiated by the annular metal block provides the necessary temperature for the RPA reaction. The heating member 62 is an annular heating film pasted under the annular radiation member 61. The gap between the microfluidic chip and the annular radiation member 61 allows the chip to rotate during the amplification process to detect fluorescent signals from different detection units. A first avoidance hole 610 is provided on the annular radiation member 61, and a second avoidance hole 620 is provided on the heating member 62. The first avoidance hole 610 and the second avoidance hole 620 enable the optical fiber probe 80 to detect fluorescent signals from different detection units.

[0054] Please continue reading Fig.10The electrical module 70 includes a mounting plate 71, a plurality of spring pins 72, a connecting line 73, an adapter plate 74, a second lifting drive member 75 and a slide plate 76. The second lifting drive member 75 is fixed to the panel 13, and the mounting plate 71 is transmission-connected to the second lifting drive member 75, and the second lifting drive member 75 drives the mounting plate 71 to move in the vertical direction. The spring pins 72 are floatingly mounted on the mounting plate 71, and each spring pin 72 is connected to the adapter plate 74 through a connecting line 73 to ensure that there is no interference during the rotation of the chip. The slide plate 76 is fixed to the panel 13, and the mounting plate 71 is slidably mounted on the slide plate 76. The second lifting drive member 75 is a screw motor, which controls the spring pins 72 to move up and down through the screw motor to connect or disconnect the electrical connection of the lower heating module 50.

[0055] There are two optical fiber probes 80 , which are connected to an external fluorescence detection optical path to detect the signal values ​​of the two fluorescent probes, FAM and TAMRA, respectively.

[0056] When using the nucleic acid instant detection device, the following steps are included:

[0057] (1) Sample processing: Use a disposable throat swab to scrape oral epithelial cells, immerse it in nuclease-free water, shake it several times to wash off the oral epithelial cells on the swab, and press the swab to the edge of the tube to squeeze out as much liquid as possible. Use a 50μL precision pipette to transfer a drop of cell suspension to the lysis solution containing prepGEM thermophilic protease and buffer. Use a 5μL precision pipette to drop a drop of the above liquid from the sample loading hole 316 of the chip into the sample lysis chamber 318, and then seal the sample loading hole 316 with a PCR membrane. Load the chip into the instrument through the positioning hole 329. At this time, the first valve body 320 can prevent the liquid in the reagent chamber 317 from entering the mixing chamber 319 prematurely.

[0058] (2) Cracking: The rotating drive member 20 is calibrated and then rotated to the cracking position; the second lifting drive member 75 of the electrical module 70 drives the spring pin 75 to move upward to establish an electrical connection with the lower heating module 50; the first lifting drive member 41 rotates to move the upper heating module 40 downward, and the four copper blocks fit into the four cracking chambers 318 of the chip. The four pressure columns 45 press the second valve body 321 on the surface of the chip to prevent liquid from overflowing during the heating process; then the eight copper blocks are heated and stabilized at 75°C for 5 minutes; after the cracking is completed, the upper heating module 40 and the electrical module 70 are removed.

[0059] (3) Liquid mixing: high-speed centrifugation, the liquids in the reagent chamber 317 and the lysis chamber 318 break through the first valve body 320 and the second valve body 321 respectively, and are transferred to the mixing chamber 319; the two liquids are mixed by alternating rotation.

[0060] (4) Preparation for amplification: Heat the amplification heating module 60 to the required temperature; transfer the liquid in the mixing chamber 319 to the metering chamber 324 and the waste liquid chamber 328 by repeating the following operations: rotate the chip counterclockwise and accelerate steadily, then stop suddenly. Due to inertia, the liquid will break through the third valve body 328; finally, through high-speed centrifugation, the liquid in the metering chamber 324 breaks through the capillary valve 325 and is transferred to the amplification detection chamber 326, and then alternately rotate to mix the liquid and the dry powder.

[0061] (5) Amplification: The reagents in the amplification detection chamber 326 undergo RPA reaction in a constant temperature environment; the chip is moved so that each amplification detection chamber 326 is aligned with the optical fiber probe 80, and the change of fluorescence value is continuously detected.

[0062] The repeatability of the system was verified using four chips, with three samples (genotype HLA-B*58:01, HLA-B*38:02, and HLA-B*38:02 oral swab spiked with HLA-B*58:01) and a negative control sample. The target HLA-B*58:01 and the housekeeping gene β-globin were detected simultaneously through three amplification detection chambers 326 on the chip, and the mean and standard deviation of Tp were calculated. The detection differences of the HLA-B*58:01 allele in the three repeated detection chambers were minimal. Based on the Tp value, it can be determined whether the oral swab carries the HLA-B*58:01 allele.

[0063] The microfluidic chip 30 of the present application can realize the integrated functions of orderly transport, mixing and detection of trace samples and reagents by designing micro chambers, micro channels, capillary valves 325 and silicone valves, and pre-installing reagents on the chip. A portable, fully automatic nucleic acid detection system is designed around the microfluidic chip 30. Through the upper heating module 40, the lower heating module 50 and the two-color fluorescence light path design, rapid sample lysis and multi-target detection can be achieved, and oral swab HLA-B*58:01 alleles and housekeeping genes are detected simultaneously on the system. The whole process only requires sample addition, and the processes of sample lysis, reagent re-dissolution, mixing, amplification and fluorescence detection are all completed on the chip. The liquid is driven by centrifugal force, and no external pump or valve is required, which reduces the volume of the detection device, facilitates miniaturization, is easy to carry, can be used for instant detection, and can realize automated multiple detection of multiple steps of lysis, mixing, amplification and detection. Multiple identical unit structures on the chip can complete the parallel detection of multiple samples or multiple indicators, and the detection of 4 different samples and 18 targets can be completed in about 50 minutes. The equipment is portable, easy to operate, requires little sample and has high detection efficiency.

[0064] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which are equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of them belong to the protection scope of the present invention.

Claims

1. A nucleic acid instant detection device, comprising a microfluidic chip, an upper heating module and a lower heating module, wherein the microfluidic chip is provided with a reagent chamber, a lysis chamber, a mixing chamber and an amplification detection chamber, characterized in that: The lysis chamber, the mixing chamber and the amplification detection chamber are arranged radially. The nucleic acid instant detection device also includes an electrical module, a rotating drive and an amplification heating module. The electrical module includes a second lifting drive and a spring needle. The second lifting drive is connected to the spring needle to drive the spring needle to move up and down and connect or separate from the lower heating module to control the working state of the lower heating module. The upper heating module and the lower heating module control the temperature of the lysis chamber to lyse the sample in the lysis chamber; the rotating drive is connected to the lower heating module, and the rotating drive drives the lower heating module to rotate so that the reagent chamber in the microfluidic chip located on the lower heating module and the liquid in the lysis chamber enter the mixing chamber, and the liquid in the mixing chamber enters the amplification detection chamber through the rotation of the microfluidic chip. The amplification heating module controls the temperature of the amplification detection chamber to amplify the sample.

2. The nucleic acid instant detection device according to claim 1, characterized in that: The upper heating module includes a first lifting drive member, a fixed plate, a first circuit board and a heating block. The first lifting drive member is connected to the fixed plate to drive the fixed plate to lift and lower. The first circuit board is fixed to the fixed plate, and the heating block is fixed to the first circuit board.

3. The nucleic acid instant detection device according to claim 2, characterized in that: The upper heating module also includes a pressure column, which is fixed to the fixed plate. The microfluidic chip also includes a second valve body, which is located between the lysis chamber and the mixing chamber. When the first lifting drive drives the fixed plate to descend, the heating block heats the lysis chamber, and the pressure column presses the second valve body to prevent the liquid in the lysis chamber from overflowing during heating.

4. The nucleic acid instant detection device according to claim 2, characterized in that: The microfluidic chip comprises a plurality of chip modules, each of which is fan-shaped, and a plurality of the chip modules are arranged in a circular shape. The plurality of chip modules are provided with the reagent chamber, the lysis chamber, the mixing chamber and the amplification detection chamber.

5. The nucleic acid instant detection device according to claim 4, characterized in that: Each of the chip modules is provided with a plurality of amplification detection cavities, the plurality of amplification detection cavities are located on an arc line, and the amplification detection cavities of the plurality of chip modules are located on a circumference.

6. The nucleic acid instant detection device according to claim 1, characterized in that: The amplification heating module is ring-shaped, and the lower heating module is located inside the amplification heating module and forms a gap with the amplification heating module.

7. The nucleic acid instant detection device according to claim 6, characterized in that: The amplification heating module includes an annular radiation element and a heating element. The heating element is annular and is fixed to the bottom of the annular radiation element.

8. The nucleic acid instant detection device according to claim 1, characterized in that: The instant nucleic acid detection device also includes an optical fiber probe, and the amplification and heating module is provided with an avoidance hole. The optical fiber probe detects the fluorescent signal of the liquid in the amplification detection cavity through the avoidance hole.

9. A microfluidic chip, comprising a reagent chamber, a lysis chamber, a mixing chamber, and an amplification detection chamber, characterized in that: The lysis chamber, the mixing chamber and the amplification detection chamber are arranged radially. When the microfluidic chip rotates, the centrifugal force causes the liquid in the reagent chamber and the lysis chamber to enter the mixing chamber, and the liquid in the mixing chamber enters the amplification detection chamber.

10. The microfluidic chip according to claim 9, characterized in that: The microfluidic chip includes multiple chip modules, each of which is fan-shaped, and multiple chip modules are arranged in a circular shape. Multiple chip modules are provided with the reagent chamber, the lysis chamber, the mixing chamber and the amplification detection chamber. Each chip module is provided with multiple amplification detection chambers, and multiple amplification detection chambers are located on an arc line. The amplification detection chambers of multiple chip modules are located on a circumference.

Images