A compact automated microfluidic PCR instrument and methods of use thereof

Abstract

The application relates to the technical field of biology, and particularly relates to a small automatic micro-fluidic PCR instrument and a use method thereof. A temperature control fixing portion and a vortex tube fixing portion are fixed on the side of a substrate. A temperature control body is clamped in the temperature control fixing portion along the vertical direction. A temperature control reaction cavity is arranged at the axial center of the temperature control body. A heating rod and a refrigeration tube are respectively embedded in the peripheral wall of the temperature control body along the axial direction. A reaction tube is sleeved in the temperature control reaction cavity. The top surface of the reaction tube is provided with a sample inlet hole and a sample outlet hole. The vortex tube is clamped in the inner cavity of the vortex tube fixing portion along the vertical direction. The middle part of the vortex tube is a compressed air inlet. The top and bottom parts of the vortex tube are a cold air exhaust pipe and a hot air exhaust pipe respectively. The top end of the cold air exhaust pipe is sleeved with the bottom end of the refrigeration tube. A temperature sensor is sleeved in the wall of the temperature control body along the vertical direction. The output end of the temperature sensor is electrically connected with the controller input end of the compressed air pump connected with the heating rod and the compressed air inlet. The problems of slow temperature control of the PCR instrument and difficult realization of automatic sample feeding are solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a small automated microfluidic PCR instrument and its usage method. Background Technology

[0002] The basic principle of PCR (polymerase chain reaction) technology is similar to the natural replication process of DNA. Its specificity depends on oligonucleotide primers that are complementary to both ends of the target sequence. PCR basically consists of three basic reaction steps: denaturation, annealing, and extension. PCR utilizes the fact that DNA denatures into single strands at 95°C in vitro. At a low temperature (often around 60°C), primers bind to the single strands according to the principle of complementary base pairing. Then, the temperature is adjusted to the optimal reaction temperature of DNA polymerase (around 72°C), and DNA polymerase synthesizes the complementary strand along the direction from phosphate to pentose sugar.

[0003] A polymerase-based PCR instrument is essentially a temperature control device, capable of effectively controlling the denaturation, annealing, and extension temperatures. The traditional method involves fixing the sample within a temperature-variable heating device, with the device temperature fluctuating between these three temperatures as required, continuously heating and cooling the sample to achieve the reaction. This heating method requires at least several hundred microliters of sample and has a certain thermal inertia; the device itself also has thermal inertia, therefore the frequency of temperature switching cannot be too rapid. Summary of the Invention

[0004] The purpose of this invention is to provide a small, automated microfluidic PCR instrument and its usage method, which solves the problems of slow temperature control and difficulty in achieving automated sample introduction in existing PCR instruments.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a small automated microfluidic PCR instrument, wherein a temperature control fixing part and a vortex tube fixing part are fixed sequentially from top to bottom on the side of the substrate; the temperature control body is vertically clamped into the inner cavity of the temperature control fixing part, and a temperature-controlled reaction chamber is provided at the axial center of the temperature control body; a heating rod is axially embedded in the outer peripheral wall of the temperature control body; a cooling tube is axially embedded in the outer peripheral wall of the temperature control body; a reaction tube is fitted into the temperature-controlled reaction chamber, the reaction tube has a cavity structure, and its top surface has openings for inserting a sample inlet tube and a sample outlet tube, respectively. The tube has an inlet and an outlet, and the inlet and outlet tubes are connected to the solution via a micro-pump. The vortex tube is vertically fitted into the inner cavity of the vortex tube fixing part. The middle part of the vortex tube is the compressed air inlet, and the top and bottom of the vortex tube are respectively the cold air outlet pipe and the hot air outlet pipe. The top of the cold air outlet pipe is sleeved with the bottom of the refrigeration tube. The temperature sensor is vertically sleeved in the wall of the temperature control body, and the output end of the temperature sensor is electrically connected to the heating rod and the controller input end of the compressed air pump connected to the compressed air inlet.

[0006] Preferably, the number of cooling pipes is three, the number of heating rods is two, the cooling pipes and the heating rods are circumferentially spaced and embedded in the outer peripheral wall of the temperature control body, and the three cooling pipes are connected in series.

[0007] Preferably, the outer peripheral wall of the temperature controller is uniformly provided with five arc-shaped grooves extending axially along its circumferential direction. The heating rod and the cooling pipe are respectively fitted into the arc-shaped grooves. The top surface of the temperature controller is provided with a vertical insertion hole for mounting the temperature sensor at a position outside the temperature control reaction chamber. The bottom axis of the temperature controller is provided with a tail hole communicating with the bottom end of the temperature control reaction chamber. The material of the temperature controller is one of metal, semiconductor, glass, and plastic.

[0008] Preferably, the inner wall of the reaction tube is provided with a hydrophilic layer, and the material of the reaction tube is either plastic or silicon. When the material of the reaction tube is silicon, the material of the hydrophilic layer is silicon dioxide; when the material of the reaction tube is plastic, the material of the hydrophilic layer is PVA.

[0009] Preferably, the temperature control fixing part includes a temperature control fixing seat that is directly fixed to the side of the substrate, and a temperature control fixing clip that is connected to the temperature control fixing seat by screws. The temperature control body is fitted into the cavity formed by the temperature control fixing seat and the temperature control fixing clip, and the inner wall of the cavity is provided with arc-shaped slots that correspond to and match the heating rod and the cooling pipe respectively.

[0010] Preferably, the vortex tube fixing part includes an vortex fixing seat that is directly fixed to the substrate and an vortex fixing clip that is connected to the vortex fixing seat by screws. The contact surfaces of the vortex fixing seat and the vortex fixing clip are respectively provided with semi-cylindrical grooves that are matched and engaged with the middle part of the hot gas exhaust pipe.

[0011] Preferably, it also includes an upper U-shaped tube and a lower U-shaped tube, which are used to connect the three refrigeration tubes in series.

[0012] Preferably, a heat cover is detachably fixed to the top surface of the temperature control fixing part. A through hole three is opened in the middle of the top surface of the heat cover corresponding to the position of the sample inlet and the sample outlet. A pair of through holes one are opened on the top surface of the heat cover to match the top ends of the upper U-shaped tube. A through hole two is also provided on the top surface of the heat cover to match the top end of one of the cooling tubes. A housing is fixedly connected to the side of the substrate outside the temperature control fixing part. A heat insulation layer is provided between the inner wall of the housing and the outer wall of the temperature control fixing part.

[0013] A method for using a small, automated microfluidic PCR instrument includes the following steps:

[0014] S1. Pretreatment of the reaction tube;

[0015] First, when the reaction tube is used for the first time, 1 mL of pure water is first flowed into the sample inlet, left to stand for 10 minutes, and then the pure water is discharged from the sample outlet by the action of the micro pump. The process of pure water flowing in and out is repeated twice.

[0016] Secondly, the pure water in the flow channel is evacuated by reverse pumping through the inlet port;

[0017] Next, blow air through the injection port for 5 minutes to dry the reaction tube, then set it aside for later use.

[0018] S2. Inject PCR solution into the reaction tube;

[0019] 1 μL of template, 2 μL of upstream and downstream mixed primer solution, and 47 μL of enzyme raw material MIX solution are sequentially injected into the inner lumen of the reaction tube through the injection port using a micropump.

[0020] S3. Heat the hot cover to 105°C and then maintain the temperature.

[0021] S4. Preheat the reaction tube;

[0022] The heating rod heats the temperature control body, raising the temperature of the temperature control reaction chamber to 98°C for 2 minutes.

[0023] S5. Perform the first reaction cycle;

[0024] Compressed air is input into the vortex tube through the compressed air inlet, and cold air enters the refrigeration tube through the cold air outlet pipe, causing the temperature-controlled reaction chamber to cool down rapidly. When the temperature drops to 60°C, it is maintained for 10 seconds. Then, the heating rod heats the temperature-controlled reaction chamber, raising the temperature to 72°C and maintaining it for 20 seconds to complete the first reaction cycle.

[0025] S6. By repeatedly raising and lowering the temperature of the refrigeration assembly consisting of the heating rod, the vortex tube, and the cooling tube, PCR cycling is achieved to enable the large-scale amplification of nucleic acids.

[0026] S7. After PCR amplification is completed, the micropump runs and the PCR solution flows to the next module for nucleic acid detection through the sample outlet.

[0027] S8. Rinse the reaction tube;

[0028] First, the heating rod heats the temperature control body, and the temperature is controlled at 65°C;

[0029] Next, 1 mL of pH 8 TE buffer solution was injected into the reaction tube through the inlet using a micropump. After standing for 10 minutes, the TE buffer solution was discharged from the outlet using the micropump. This process was repeated twice.

[0030] Next, the micropump injects 1 mL of pure water into the reaction tube through the inlet port, lets it stand for 10 minutes, and then the pure water is discharged from the outlet port under the control of the micropump. This process is repeated twice.

[0031] Finally, the micropump back-evacuates the pure water in the flow channel through the sample inlet, and then blows air through the sample inlet for 5 minutes to dry the reaction tube in order to prepare for the next reaction cycle.

[0032] Preferably, when storing the reaction tube, the reaction tube is first rinsed and dried according to step S8, and then placed at room temperature.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] 1. The present invention relates to a small automated microfluidic PCR instrument that uses a vortex tube to generate a vortex in a high-speed airflow to separate cold and hot airflows. The cold airflow is used to achieve a cooling method, which can quickly cool the airflow. The heating rod embedded in the peripheral wall of the temperature controller can also achieve rapid heating, thereby further reducing the amplification time. On the other hand, the device has a simple structure, is easy to operate, and can easily achieve automated sample loading and unloading.

[0035] 2. The method of using a small automated microfluidic PCR instrument, as disclosed in this invention, can fully utilize the advantages of rapid heating and rapid cooling of the microfluidic PCR instrument, ensuring high efficiency and precision of the reaction. Attached Figure Description

[0036] Figure 1 This is a three-dimensional structural diagram of the entire invention.

[0037] Figure 2 For the present invention Figure 1 Enlarged structural diagram at point A;

[0038] Figure 3 This is a three-dimensional structural diagram of the temperature control fixing part of the present invention;

[0039] Figure 4 This is a three-dimensional structural diagram of the vortex tube fixing part of the present invention;

[0040] Figure 5 This is a top view of the temperature control body of the present invention;

[0041] Figure 6 For the present invention Figure 5 Schematic diagram of the cross-sectional structure of the middle BB.

[0042] In the figure: 1-substrate; 11-temperature control fixing part; 111-temperature control fixing seat; 112-temperature control fixing clip; 113-arc-surface slot; 12-vortex tube fixing part; 121-vortex tube fixing seat; 122-vortex tube fixing clip; 123-semi-cylindrical groove; 13-insulation layer; 14-shell

[0043] 2-Temperature controller; 21-Temperature-controlled reaction chamber; 22-Arc-shaped groove; 23-Insertion hole; 24-Tail hole;

[0044] 3-Heating rod;

[0045] 4-Refrigeration pipe;

[0046] 5-Reaction tube; 51-Inlet port; 52-Outlet port;

[0047] 6-Vortex tube; 61-Compressed air inlet; 62-Cold air exhaust pipe; 63-Hot air exhaust pipe;

[0048] 7-Temperature sensor;

[0049] 8-Upper U-shaped tube;

[0050] 9-Lower U-shaped tube;

[0051] 10-Heat cover; 1001-Through hole one; 1002-Through hole two; 1003-Through hole three. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Please see Figure 1-6This invention provides a technical solution: a small automated microfluidic PCR instrument. A temperature control fixing part 11 and a vortex tube fixing part 12 are sequentially fixed from top to bottom on the side of a substrate 1. A temperature control body 2 is vertically fitted into the inner cavity of the temperature control fixing part 11, and a temperature-controlled reaction chamber 21 is located at the axial center of the temperature control body 2. A heating rod 3 is axially embedded in the outer peripheral wall of the temperature control body 2, with a heating power of 5-40 watts, and its external power source is a battery. A cooling tube 4 is axially embedded in the outer peripheral wall of the temperature control body 2. A reaction tube 5 is fitted into the temperature-controlled reaction chamber 21; the reaction tube 5 has a cavity structure, and its top surface has openings for... The sample inlet 51 and sample outlet 52 of the sample inlet and outlet tubes are inserted, and the sample inlet and outlet tubes realize the entry and exit of the solution through the micro pump; the vortex tube 6 is vertically clamped in the inner cavity of the vortex tube fixing part 12, the middle part of the vortex tube 6 is the compressed air inlet 61, the top and bottom of the vortex tube 6 are the cold air outlet pipe 62 and the hot air outlet pipe 63 respectively, and the top of the cold air outlet pipe 62 is sleeved with the bottom of the cooling pipe 4; the temperature sensor 7 is vertically sleeved in the wall of the temperature controller 2, and the output end of the temperature sensor 7 is electrically connected to the heating rod 3 and the controller input end of the compressed air pump connected to the compressed air inlet 61 respectively.

[0054] In this embodiment, there are three cooling pipes 4 and two heating rods 3. The cooling pipes 4 and heating rods 3 are embedded in the outer peripheral wall of the temperature control body 2 at intervals along the circumference, and the three cooling pipes 4 are connected in series.

[0055] In this embodiment, the outer peripheral wall of the temperature controller 2 is uniformly provided with 5 arc-shaped grooves 22 extending axially along its circumferential direction. The heating rod 3 and the cooling pipe 4 are respectively fitted into the arc-shaped grooves 22. The top surface of the temperature controller 2 is provided with a vertical insertion hole 23 for mounting the temperature sensor 7 at the position outside the temperature control reaction chamber 21. The bottom axis of the temperature controller 2 is provided with a tail hole 24 communicating with the bottom end of the temperature control reaction chamber 21. The material of the temperature controller 2 is one of metal, semiconductor, glass and plastic.

[0056] In this embodiment, the inner wall of the reaction tube 5 is provided with a hydrophilic layer. The material of the reaction tube 5 is either plastic or silicon. When the material of the reaction tube 5 is silicon, the material of the hydrophilic layer is silicon dioxide; when the material of the reaction tube 5 is plastic, the material of the hydrophilic layer is PVA.

[0057] In this embodiment, the temperature control fixing part 11 includes a temperature control fixing seat 111 that is directly fixed to the side of the substrate 1, and a temperature control fixing clip 112 that is connected to the temperature control fixing seat 111 by screws. The temperature control body 2 is fitted into the cavity formed by the temperature control fixing seat 111 and the temperature control fixing clip 112, and the inner wall of the cavity is provided with arc-shaped slots 113 that correspond to and match the heating rod 3 and the cooling pipe 4 respectively.

[0058] In this embodiment, the vortex tube fixing part 12 includes an vortex fixing seat 121 that is directly fixed to the substrate 1 and an vortex fixing clip 122 that is connected to the vortex fixing seat 121 by screws. The contact surfaces of the vortex fixing seat 121 and the vortex fixing clip 122 are respectively provided with semi-cylindrical grooves 123 that are matched and engaged with the middle part of the hot gas exhaust pipe 63.

[0059] In this embodiment, an upper U-shaped tube 8 and a lower U-shaped tube 9 are also included, which are used to connect three refrigeration tubes 4 in series.

[0060] In this embodiment, to prevent the reaction solution from evaporating during the temperature control cycle, a heat cover 10 is detachably fixed to the top surface of the temperature control fixing part 11. A through hole 1003 is provided in the middle of the top surface of the heat cover 10 corresponding to the position of the sample inlet hole 51 and the sample outlet hole 52. A pair of through holes 1001 are provided on the top surface of the heat cover 10 to be fitted and matched with the top ends of the upper U-shaped tube 8. A through hole 2 1002 is also provided on the top surface of the heat cover 10 to be fitted and matched with the top end of one of the cooling tubes 4. A housing 14 is fixedly connected to the side of the substrate 1 around the temperature control fixing part 11. A heat insulation layer 13 is provided between the inner wall of the housing 14 and the outer wall of the temperature control fixing part 11.

[0061] A method for using a small, automated microfluidic PCR instrument includes the following steps:

[0062] S1, Pretreatment of reaction tube 5;

[0063] First, when using reaction tube 5 for the first time, 1 mL of pure water is first introduced into the inlet port 51, and after standing for 10 minutes, the pure water is discharged from the outlet port 52 by the action of the micro pump. The process of pure water inflow and outflow is repeated twice.

[0064] Secondly, pure water in the flow channel is evacuated through the injection port 51;

[0065] Next, blow air through the injection port 51 for 5 minutes to dry the reaction tube 5, and then set it aside for later use;

[0066] S2. Inject PCR solution into reaction tube 5;

[0067] 1 μL of template, 2 μL of upstream and downstream mixed primer solution, and 47 μL of enzyme raw material MIX solution are sequentially injected into the inner cavity of reaction tube 5 through sample inlet 51 using a micropump.

[0068] S3. Heat the hot cover 10 to 105°C and then keep it at a constant temperature.

[0069] S4. Preheat reaction tube 5;

[0070] Heating rod 3 heats temperature control body 2, causing temperature control reaction chamber 21 to rise to 98°C and remain at that temperature for 2 minutes;

[0071] S5. Perform the first reaction cycle;

[0072] Compressed air is input into the vortex tube 6 through the compressed air inlet 61, and cold air enters the refrigeration tube 4 through the cold air outlet 62, which causes the temperature-controlled reaction chamber 21 to cool down rapidly. When the temperature drops to 60°C, it is maintained for 10 seconds. Then the heating rod 3 heats the temperature-controlled reaction chamber 21, raising the temperature to 72°C and maintaining it for 20 seconds to complete the first reaction cycle.

[0073] S6. Through multiple temperature increases and decreases of the cooling assembly consisting of heating rod 3, vortex tube 6, and cooling tube 4, PCR cycling is achieved to enable large-scale amplification of nucleic acids.

[0074] S7. After PCR amplification is completed, the micropump runs and the PCR solution flows to the next module for nucleic acid detection through sample outlet 52.

[0075] S8. Rinse reaction tube 5.

[0076] First, the heating rod 3 heats the temperature control body 2, and the temperature is controlled at 65°C;

[0077] Next, 1 mL of pH 8 TE buffer solution was injected into the reaction tube 5 through the sample inlet 51 using a micropump. After standing for 10 min, the TE buffer solution was discharged from the sample outlet 52 under the control of the micropump. This process was repeated twice.

[0078] Next, the micropump injects 1 mL of pure water into the reaction tube 5 through the sample inlet 51, lets it stand for 10 min, and then the pure water is discharged from the sample outlet 52 under the control of the micropump. This process is repeated twice.

[0079] Finally, the micropump back-evacuates the pure water in the flow channel through the injection port 51, and then blows air through the injection port 51 for 5 minutes to dry the reaction tube 5, so as to facilitate the next reaction cycle.

[0080] In this embodiment, when storing the reaction tube 5, the reaction tube 5 is first rinsed and dried according to step S8, and then placed at room temperature.

[0081] Taking the amplification of Navicula nucleic acid as an example, the template is the Navicula genome nucleic acid, the upstream and downstream primers are a specific nucleic acid chain of the V3 region of Navicula, and the enzyme and raw material mixture is the Gold Medal MIX Green mixed solution.

[0082] The temperature controller 2 is preferably silicon-based, utilizing the high thermal conductivity of silicon to achieve rapid temperature rise and fall during PCR testing. Its heating rate can reach 35℃ / s, and its cooling rate can reach 9℃ / s, significantly shortening the detection reaction time and improving detection efficiency. Furthermore, the temperature sensor 7, through the nonlinearity between resistance and temperature, after calibration, can accurately determine the real-time temperature of the temperature-controlled reaction chamber 21, thus ensuring precise control of the reaction temperature within the chamber.

[0083] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A small, automated microfluidic PCR instrument, characterized in that, include: A substrate (1) has a temperature control fixing part (11) and a vortex tube fixing part (12) fixed on its side from top to bottom. Temperature control body (2), the temperature control body (2) is vertically mounted in the inner cavity of the temperature control fixing part (11), and a temperature control reaction chamber (21) is provided at the axial position of the temperature control body (2); Heating rod (3), the heating rod (3) is embedded axially in the outer peripheral wall of the temperature control body (2); A refrigeration pipe (4) is axially embedded in the outer peripheral wall of the temperature control body (2); The reaction tube (5) is fitted inside the temperature-controlled reaction chamber (21). The reaction tube (5) has a cavity structure and its top surface is provided with an inlet hole (51) and an outlet hole (52) for inserting the inlet tube and the outlet tube, respectively. The inlet tube and the outlet tube realize the entry and exit of the solution through a micro pump. Vortex tube (6), the vortex tube (6) is vertically fitted into the inner cavity of the vortex tube fixing part (12), the middle part of the vortex tube (6) is the compressed air inlet (61), the top and bottom of the vortex tube (6) are respectively the cold air exhaust pipe (62) and the hot air exhaust pipe (63), the top end of the cold air exhaust pipe (62) is connected to the bottom end of the refrigeration pipe (4); Temperature sensor (7) is vertically fitted into the wall of the temperature control body (2). The output end of the temperature sensor (7) is electrically connected to the controller input end of the heating rod (3) and the compressed air pump connected to the compressed air inlet (61).

2. The small automated microfluidic PCR instrument according to claim 1, characterized in that: The number of the refrigeration tubes (4) is three, and the number of the heating rods (3) is two. The refrigeration tubes (4) and the heating rods (3) are circumferentially spaced and embedded in the outer peripheral wall of the temperature control body (2). The three refrigeration tubes (4) are connected in series.

3. The small automated microfluidic PCR instrument according to claim 2, characterized in that: The outer peripheral wall of the temperature control body (2) is uniformly provided with five arc-shaped grooves (22) extending along its axial direction. The heating rod (3) and the cooling tube (4) are respectively fitted into the arc-shaped grooves (22). The top surface of the temperature control body (2) is provided with a vertical insertion hole (23) for mounting the temperature sensor (7) at the position outside the temperature control reaction chamber (21). The bottom axis of the temperature control body (2) is provided with a tail hole (24) communicating with the bottom end of the temperature control reaction chamber (21). The material of the temperature control body (2) is one of metal, semiconductor, glass and plastic.

4. The small automated microfluidic PCR instrument according to claim 1, characterized in that: The inner wall of the reaction tube (5) is provided with a hydrophilic layer. The material of the reaction tube (5) is either plastic or silicon, and the material of the hydrophilic layer is either PVA or silicon dioxide.

5. A small automated microfluidic PCR instrument according to claim 3, characterized in that: The temperature control fixing part (11) includes a temperature control fixing seat (111) that is directly fixed to the side of the substrate (1) and a temperature control fixing clip (112) that is connected to the temperature control fixing seat (111) by screws. The temperature control body (2) is fitted into the cavity formed by the temperature control fixing seat (111) and the temperature control fixing clip (112), and the inner wall of the cavity is provided with arc-shaped slots (113) that correspond to and match the heating rod (3) and the cooling pipe (4).

6. A small automated microfluidic PCR instrument according to claim 1, characterized in that: The vortex tube fixing part (12) includes a vortex fixing seat (121) directly fixed to the base plate (1) and a vortex fixing clip (122) connected to the vortex fixing seat (121) by screws. The contact surfaces of the vortex fixing seat (121) and the vortex fixing clip (122) are respectively provided with semi-cylindrical grooves (123) that are matched and engaged with the middle part of the hot gas exhaust pipe (63).

7. A small automated microfluidic PCR instrument according to claim 1, characterized in that, It also includes an upper U-shaped tube (8) and a lower U-shaped tube (9), which are used to connect the three refrigeration tubes (4) in series.

8. A small automated microfluidic PCR instrument according to claim 7, characterized in that: The top surface of the temperature control fixing part (11) is detachably fixed with a heat cover (10). The top surface of the heat cover (10) has a through hole three (1003) at the position corresponding to the sample inlet hole (51) and the sample outlet hole (52). The top surface of the heat cover (10) has a pair of through holes one (1001) that are fitted and matched with the top two ends of the upper U-shaped tube (8). The top surface of the heat cover (10) also has a through hole two (1002) that is fitted and matched with the top end of one of the cooling tubes (4). The side of the substrate (1) is fixedly connected to a shell (14) around the temperature control fixing part (11). The inner wall of the shell (14) and the outer wall of the temperature control fixing part (11) are provided with a heat insulation layer (13).

9. The method of using a small automated microfluidic PCR instrument according to claim 8, characterized in that, Includes the following steps: S1. Pretreatment of the reaction tube (5); First, when the reaction tube (5) is used for the first time, 1 mL of pure water is first flowed into the sample inlet (51), left to stand for 10 minutes, and then the pure water is discharged from the sample outlet (52) by the action of the micro pump. The process of pure water flowing in and out is repeated twice. Secondly, pure water in the flow channel is evacuated by reverse pumping through the injection port (51); Next, blow air through the injection port (51) for 5 minutes to dry the reaction tube (5) for later use; S2. Inject PCR solution into the reaction tube (5); 1 μL of template, 2 μL of upstream and downstream mixed primer solution, and 47 μL of enzyme raw material MIX solution are sequentially injected into the inner cavity of the reaction tube (5) through the injection port (51) using a micropump. S3. Heat the hot cover (10) to 105°C and then keep it at a constant temperature; S4. Preheat the reaction tube (5); The heating rod (3) heats the temperature control body (2), causing the temperature control reaction chamber (21) to rise to 98°C for 2 minutes; S5. Perform the first reaction cycle; Compressed air is input into the vortex tube (6) through the compressed air inlet (61), and cold air enters the refrigeration tube (4) through the cold air outlet (62), causing the temperature-controlled reaction chamber (21) to cool down rapidly. When the temperature drops to 60°C, it is maintained for 10 seconds. Then the heating rod (3) heats the temperature-controlled reaction chamber (21), raising the temperature to 72°C and maintaining it for 20 seconds to complete the first reaction cycle. S6. Through multiple temperature increases and decreases of the refrigeration assembly consisting of the heating rod (3), the vortex tube (6), and the cooling tube (4), PCR cycling is achieved to enable the large-scale amplification of nucleic acids. S7. After PCR amplification is completed, the micropump runs and the PCR solution flows to the next module for nucleic acid detection through the sample outlet (52); S8. Rinse the reaction tube (5); First, the heating rod (3) heats the temperature control body (2) and controls the temperature at 65°C; Next, 1 mL of TE buffer solution with pH 8 is injected into the reaction tube (5) through the sample inlet (51) using a micropump. After standing for 10 min, the TE buffer solution is discharged from the sample outlet (52) under the control of the micropump. This process is repeated twice. Next, the micropump injects 1 mL of pure water into the reaction tube (5) through the sample inlet (51), lets it stand for 10 min, and then the pure water is discharged from the sample outlet (52) under the control of the micropump. This process is repeated twice. Finally, the micropump back-evacuates the pure water in the flow channel through the sample inlet (51), and then blows air through the sample inlet (51) for 5 minutes to dry the reaction tube (5) in order to facilitate the next reaction cycle.

10. The method of using a small automated microfluidic PCR instrument according to claim 9, characterized in that: When storing the reaction tube (5), first rinse and dry the reaction tube (5) according to step S8, and then place it at room temperature.

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