Microbial nucleic acid detection micro-fluidic chip and automatic detection box

Abstract

The utility model provides a microbe nucleic acid detection micro-fluidic chip and an automatic detection box, a first solenoid valve controls the connection and disconnection of an eluent inlet and an eluent outlet, the eluent flows out of the outlet, passes through a sample chamber of a sample introduction module, and enters a degassing flow channel through the sample inlet; the second electromagnetic valve controls connection and disconnection of an outlet of the degassing flow channel and an inlet of the lysis tank, and nucleic acid release of the sample is completed in the lysis tank; the third electromagnetic valve is used for controlling connection and disconnection between an outlet of the cracking tank and an inlet of the reaction detection tank, the reaction detection tank is provided with a plurality of reaction chambers, freeze-dried powder of an LAMP-CRISPR reaction system is preset in the reaction chambers, and nucleic acid isothermal amplification is completed in the reaction chambers. According to the utility model, the chip card box is fully integrated and has low requirements on operators; a swab is adopted for direct sample loading, and a manual extraction process is not needed; and the chip card box is convenient to install and replace due to the plugging design.

Description

TECHNICAL FIELD

[0001] The utility model belongs to the field of microorganism detection, concretely relates to a microorganism nucleic acid detection micro -fluidic chip and detection kit. BACKGROUND

[0002] The current microorganism detection process includes the collection, storage and transportation of sample and the detection steps in the laboratory. The detection in the laboratory is specifically divided into multiple links such as sample pretreatment, dilution, culture and detection, and the operation process is complex and fine, involves sample pretreatment, nucleic acid amplification and detection technology. This series of operations puts forward strict requirements to the operating environment, not only needs professional operators to ensure the accurate execution of each step, but also requires specific equipment and environmental conditions. These limiting factors hinder the wide application of microorganism detection in different scenes to some extent.

[0003] This kind of existence following insufficient: microorganism detection is restricted by operating environment and professional personnel, and there are many deficiencies. The specific performance is that the equipment and technical level are backward, it is difficult to meet the efficient and accurate detection demand;Lack of new testing method, leading to part of microorganism difficult to be effectively detected;The quality of test personnel is uneven, part of personnel professional theory knowledge is insufficient, the sense of responsibility is not strong, and the accuracy of test result is influenced;The quality of specimen is not up to standard, and it may lead to deviation of detection result;The test scheme is unreasonable, and the characteristics and detection demand of different microorganisms are not fully considered;And the communication between the test room and the clinic is insufficient, which influences the application of detection result and the treatment of patients. These problems jointly restrict the development and application of microorganism detection.

[0004] Therefore, it is urgent to automate, miniaturize and facilitate the detection, so as to realize the detection as soon as possible in different scenes. UTILITY MODEL CONTENT

[0005] In order to overcome the problems in the prior art, the utility model provides a microorganism nucleic acid detection micro -fluidic chip and automatic detection box with micro -fluidic chip as carrier and fully closed and automatic experimental process.

[0006] The utility model provides the following technical scheme: a microorganism nucleic acid detection micro -fluidic chip, including the degassing flow channel, lysis pool and reaction detection pool integrated on the micro -fluidic chip, including the degassing flow channel, lysis pool, reaction detection pool and sample module integrated on the micro -fluidic chip, the micro -fluidic chip still includes first solenoid valve, second solenoid valve and third solenoid valve, the first solenoid valve controls the on-off of eluent inlet and outlet, and eluent flows out through the sample chamber of sample module and enters the degassing flow channel through sample inlet, the liquid inlet pipe of sample chamber is connected with eluent outlet, and the liquid outlet pipe of sample chamber is connected with sample inlet;

[0007] The second electromagnetic valve controls the opening and closing of the degassing flow channel outlet and the cracking pool inlet, the cracking pool is connected with a heating assembly, and the sample is released in the cracking pool; the third electromagnetic valve controls the opening and closing of the cracking pool outlet and the reaction detection pool inlet, the reaction detection pool is provided with a plurality of reaction chambers, a freeze-dried powder of a LAMP-CRISPR reaction system is pre-placed in the reaction chamber, and a heating assembly is connected above the reaction chamber, and the nucleic acid is amplified in the reaction chamber.

[0008] Further, the sample chamber is arranged in the sample injection module, and the sample chamber is connected with the sample injection module through a sample injection port, the sample injection port is opened and closed through a sealing screw, the sample chamber inlet is connected with a liquid inlet pipe, the liquid inlet pipe is connected with an eluent outlet of the microorganism nucleic acid detection microfluidic chip, and the sample chamber outlet is connected with a liquid outlet pipe.

[0009] Further, an eluent storage bag is arranged on the microorganism nucleic acid detection microfluidic chip, and the eluent storage bag is connected with the eluent inlet through a peristaltic pump.

[0010] Further, a waterproof and breathable pad is arranged on the degassing flow channel, and a pressing plate is arranged on the waterproof and breathable pad, the degassing flow channel is sealed by the pressing plate, and the width of the degassing flow channel is 1-3 mm.

[0011] Further, the cracking pool is a spindle-shaped pool body, and the volume of the cracking pool is greater than the total volume of the reaction chambers.

[0012] Further, the reaction detection pool inlet is connected with the reaction chambers through a plurality of liquid inlet branch pipes, the volumes of the reaction chambers are the same, the reaction chambers are connected with liquid outlet branch pipes, the outlets of the liquid outlet branch pipes are connected with an air outlet, a waterproof and breathable pad is arranged on the air outlet, the air outlet is sealed by an air outlet pressing plate, and a light transmission window is arranged below the reaction chambers.

[0013] Further, the reaction detection pool inlet is connected with the liquid inlet branch pipes through a liquid inlet main pipe, a bypass liquid flow channel is further connected in parallel on one side of the liquid inlet branch pipe, the diameter of the bypass liquid flow channel is wider than that of the liquid inlet branch pipe, the outlet of the bypass liquid flow channel is connected with the air outlet, and the volumes of the liquid outlet branch pipes are the same.

[0014] Further, the heating assembly is sequentially provided with a heat conduction plate, a heating plate and an insulation plate from bottom to top, a temperature sensor is arranged in the heat conduction plate, and the heat conduction plate is sealed and fixed on the corresponding position of the microfluidic chip through a sealing ring.

[0015] Further, an oval liquid level detection pool is arranged in the liquid inlet pipeline of the cracking pool, metal electrodes are arranged at both ends of the liquid level detection pool, one end of the electrode is in the detection pool, and the other end is connected with a lead wire outside the chip.

[0016] Furthermore, the pyrolysis tank inlet pipe is equipped with a serpentine pipe.

[0017] Furthermore, the connection between the sample inlet and the degassing channel is through the water-proof and breathable pad, which seals the passageway.

[0018] An automated detection kit includes a microfluidic chip for microbial nucleic acid detection. The automated detection kit is detachably connected to the top of a detection device housing by a chip housing. The microbial nucleic acid detection microfluidic chip is fixed inside the chip housing, and the sample injection module is fixed inside the chip housing. The sample dispensing port abuts against the surface of the chip housing, and the sealing screw protrudes outside the chip housing. A fluorescence detector is disposed inside the detection device housing, and the fluorescence probe of the fluorescence detector corresponds to the position of the reaction chamber. A main control board is also provided in the detection device housing for electrical control of various electrical components.

[0019] Furthermore, the fluorescence detector is connected to the detection equipment housing via a connecting rod. The connecting rod is equipped with a rotating wheel and a belt. A stepper motor drives the rotating wheel to rotate the belt, thereby enabling the fluorescence detector to move laterally below each reaction chamber. The range of lateral movement is limited by a photoelectric switch. The fluorescence detector has FAM, VIC, ROX, and CY5 channels.

[0020] Furthermore, the main control board controls the heating element to heat up and collects the temperature of the temperature sensor inside the heat-conducting plate, controls the opening and closing of the peristaltic pump and solenoid valve, reads the liquid detection signal from the liquid level sensor, controls the rotation of the stepper motor and the collection of the fluorescence signal from the fluorescence detector, and reads the position signal from the photoelectric switch.

[0021] A method for detecting Staphylococcus aureus nucleic acid using an automated detection kit includes the following steps:

[0022] Step 1) Pre-contain lyophilized powder of LAMP amplification primers for Staphylococcus aureus nuc gene, sgRNA targeting Staphylococcus aureus nuc gene, AapCas12b protease, fluorescent probe, Bst3.0 polymerase, dNTPs reagent and reaction buffer in the reaction chamber; one reaction chamber is set as a blank control.

[0023] Step 2) Open the sealing screw, and put the swab carrying the sample into the sample chamber through the sample inlet. Break off the swab rod and leave the swab head in the sample chamber. Tighten the sealing screw. The main control board controls the peristaltic pump to pump the eluent in the storage bag into the eluent inlet. Open the first solenoid valve. The eluent flows into the sample chamber of the sample injection module for elution. Then it enters the degassing channel from the sample inlet for degassing of the eluent.

[0024] Step 3) The main control board controls the peristaltic pump to continue pumping liquid. After the electrode detects that the eluent has flowed into the liquid level detection pool, the timer is started. The peristaltic pump is controlled by the time to deliver the eluent to fill the lysis pool. Then the peristaltic pump, the first solenoid valve and the second solenoid valve are closed, and the heating component connected above the lysis pool is controlled to heat to 90°C. The lysis is carried out for 5-15 minutes to complete the nucleic acid extraction.

[0025] Step 4) The main control board controls the peristaltic pump and the third solenoid valve to deliver the lysis buffer into the reaction chamber. Then, the third solenoid valve is closed, and the heating component connected above the reaction chamber is controlled to heat to 65°C. The reaction is carried out for 60 minutes, and the nucleic acid isothermal amplification is performed for 60 cycles, with each cycle lasting 1 minute.

[0026] Step 5) The main control board controls the stepper motor to drive the fluorescence detector to pass under each reaction chamber in turn. The fluorescence probe opens the FAM channel for fixed-point fluorescence detection. The main control board controls the fluorescence probe to perform fluorescence signal detection once in each cycle.

[0027] By adopting the above technical solution, this utility model has the following beneficial effects:

[0028] 1. This utility model features a fully integrated chip card holder, requiring minimal operator skill; it uses swabs for direct sample loading, eliminating the need for manual extraction; and its plug-and-play chip card holder design makes installation and replacement convenient.

[0029] 2. This utility model uses a one-step thermal pyrolysis method for detection, with controllable pyrolysis time and effect; multiple detection wells can be combined to perform different detection items; and LAMP-CRISPR detection technology is used, resulting in higher detection sensitivity. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the structure of the microfluidic chip and sample introduction module for microbial nucleic acid detection of this utility model;

[0031] Figure 2 This is an exploded view of the structure of the microfluidic chip for microbial nucleic acid detection of this utility model;

[0032] Figure 3 This is a schematic diagram of the sample introduction module of this utility model;

[0033] Figure 4 This is a schematic diagram of the liquid channel structure of the microfluidic chip for microbial nucleic acid detection of this utility model;

[0034] Figure 5 This is a schematic diagram of another liquid channel structure of the microfluidic chip for microbial nucleic acid detection of this utility model;

[0035] Figure 6 This is a schematic diagram of the connection of the microfluidic chip for microbial nucleic acid detection in the automatic detection kit of this utility model;

[0036] Figure 7 This is a cross-sectional view of the internal components of the automatic detection box of this utility model;

[0037] Figure 8 This is a perspective view of the automatic detection box of this utility model.

[0038] Figure 9 This is a fluorescence detection result diagram in Embodiment 3 of this utility model.

[0039] Among them: 1. Microfluidic chip for microbial nucleic acid detection, 101. Degassing channel, 102. Lysis cell, 103. Reaction chamber, 104. First solenoid valve, 105. Second solenoid valve, 106. Third solenoid valve, 107. Sample inlet, 108. Degassing channel outlet, 109. Lysis cell inlet, 1010. Lysis cell outlet, 1011. Reaction detection cell inlet, 1012. Elution solution outlet, 1013. Elution solution inlet, 1014. Storage bag, 1015. Peristaltic pump, 1016. Gas outlet, 1017. Heat-conducting plate, 1018. Heating plate, 1019. Heat insulation plate, 1020. Sealing ring, 1021. Bypass liquid channel, 1022. Liquid level detection cell;

[0040] 2. Sample injection module; 201. Sample chamber; 202. Sample inlet; 203. Sealing screw; 204. Sample chamber inlet; 205. Sample chamber outlet; 3. Chip housing; 4. Detection equipment housing; 401. Fluorescence detector; 402. Main control board; 403. Stepper motor; 404. Photoelectric switch Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this utility model clearer, the present utility model will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only for explaining the present utility model and are not intended to limit the present utility model.

[0042] Example 1

[0043] like Figures 1-4 As shown, this utility model provides a microfluidic chip 1 for microbial nucleic acid detection, including a degassing channel 101, a lysis cell 102, a reaction detection cell, and a sample injection module 2 integrated on the microfluidic chip. The microfluidic chip also includes a first solenoid valve 104, a second solenoid valve 105, and a third solenoid valve 106. The first solenoid valve controls the opening and closing of the eluent inlet and outlet. The eluent flows out from the outlet through the sample chamber 201 of the sample injection module 2 and enters the degassing channel through the sample inlet 107. The inlet pipe of the sample chamber is connected to the eluent outlet 1012, and the outlet pipe of the sample chamber is connected to the sample inlet 107.

[0044] The sample chamber is located within the sample injection module, which is connected to the sample injection module via a sample dispensing port 202. The sample dispensing port is switched on and off by a sealing screw 203. The sample chamber inlet 204 is connected to a liquid inlet tube, which is connected to the elution outlet 1012 of the microbial nucleic acid detection microfluidic chip. The sample chamber outlet 205 is connected to an outlet tube, which is connected to the sample inlet 107 of the microbial nucleic acid detection microfluidic chip. The microbial nucleic acid detection microfluidic chip has an elution reservoir bag 1014, which is connected to the elution inlet 1013 via a peristaltic pump 1015.

[0045] The second solenoid valve controls the opening and closing of the degassing channel outlet 108 and the lysis cell inlet 109. A heating component is connected above the lysis cell, and the sample completes nucleic acid release in the lysis cell. The third solenoid valve controls the opening and closing of the lysis cell outlet 1010 and the reaction detection cell inlet 1011. The reaction detection cell is equipped with several reaction chambers 103. The lyophilized powder of the LAMP-CRISPR reaction system is pre-placed in the reaction chamber. A heating component is connected above the reaction chamber, and the nucleic acid isothermal amplification is completed in the reaction chamber.

[0046] A peristaltic pump controls the eluent in the storage bag to be pumped into the eluent inlet 1013. A first solenoid valve connects the eluent inlet to the eluent outlet 1012, allowing the eluent to enter the sample chamber. The eluent flowing out of the sample chamber outlet 205 reaches the sample inlet 107 and enters the degassing channel for degassing. A second solenoid valve connects the degassing channel outlet 108 to the lysis cell inlet 109, allowing the eluent to enter the lysis cell for nucleic acid lysis. Preferably, the lysis cell is spindle-shaped, and its volume is larger than the total volume of all the reaction chambers. A third solenoid valve connects the lysis cell outlet 1010 to the reaction detection cell inlet 1011, allowing the lysate to enter each reaction chamber 103 for isothermal nucleic acid amplification.

[0047] A water-proof and breathable pad is installed on the degassing channel, and a pressure plate is installed on the water-proof and breathable pad. The pressure plate presses and seals the degassing channel, and the width of the degassing channel is 1-3mm. The appropriate channel width can ensure the efficiency of bubble removal, while ensuring that the liquid does not stagnate in the channel.

[0048] The reaction detection cell inlet 1011 is connected to the reaction chamber via several liquid inlet branches. All reaction chambers have the same volume. Each reaction chamber is connected to an outlet branch, and the outlet of the outlet branch is connected to a gas outlet 1016. A water-proof and breathable pad is installed on the gas outlet, which is sealed by a pressure plate. A light-transmitting window is located at the bottom of the reaction chamber. The function of the water-proof and breathable pad is to seal the liquid while allowing gas to pass through. During the process of the pyrolysis solution filling the reaction chamber, the gas in the chamber can be discharged through the water-proof and breathable pad, while the liquid is trapped in the outlet branch.

[0049] The heating assembly consists of a heat-conducting plate 1017, a heating plate 1018, and a heat insulation plate 1019 from bottom to top. A temperature sensor is installed inside the heat-conducting plate and is sealed and fixed to the corresponding position on the microfluidic chip by a sealing ring 10120.

[0050] Example 2

[0051] like Figure 5 As shown, the inlet of the reaction detection cell is connected to each inlet branch pipe via a main inlet pipe. A bypass liquid channel 1021 is also connected in parallel to one side of each inlet branch pipe. The diameter of the bypass liquid channel is wider than that of the inlet branch pipe, and the outlet of the bypass liquid channel is connected to an outlet. The volume of the outlet branch pipe is the same. Liquid first flows from the main inlet pipe into the bypass liquid channel, and only after the bypass liquid channel is filled does it flow into the inlet branch pipe and be distributed into each reaction detection cell. This design prevents eluent exceeding the lysis cell from entering the reaction detection cell, avoiding unlysolved samples occupying space in the reaction detection cell, which could lead to decreased detection sensitivity or even detection failure.

[0052] An elliptical liquid level detection cell 1022 is installed in the liquid inlet pipe of the pyrolysis cell. Metal electrodes are located at both ends of the liquid level detection cell, with one end of the electrode inside the detection cell and the other end protruding outside the chip and connected to the wires. The part of the metal electrode in contact with the chip is kept sealed. This design ensures that there is enough liquid in the liquid level detection cell to contact the two electrodes, while the wider detection cell reduces the resistance between the electrodes, ensuring detection sensitivity.

[0053] Preferably, the inlet pipe of the pyrolysis tank can be equipped with a serpentine pipe. The serpentine pipe can increase the mixing effect of the liquid during the flow process.

[0054] The sample inlet 107 can also be designed next to the gas outlet, so that the connecting pipe between the sample inlet 107 and the degassing channel is sealed by a water-proof and breathable pad. This design allows the sample degassing and the gas outlet of the reaction detection cell to share a single water-proof and breathable pad, reducing the difficulty of chip fabrication.

[0055] Example 3

[0056] like Figures 6-8 As shown, this utility model provides an automated detection box including a microfluidic chip for microbial nucleic acid detection. The automated detection box is detachably connected to the top of the detection device box body 4 by a chip box body 3. The microfluidic chip 1 for microbial nucleic acid detection is fixed inside the chip box body, the sample injection module 2 is fixed inside the chip box body, the sample dispensing port abuts against the surface of the chip box body, and the sealing screw protrudes outside the chip box body. The sample is added by opening the sealing screw.

[0057] The fluorescence detector 401 is installed inside the detection equipment box. The fluorescence probe of the fluorescence detector corresponds to the position of the reaction chamber. The detection equipment box is also equipped with a main control board 402, which is used to electrically control various electrical components.

[0058] The fluorescence detector is connected to the detection equipment housing via a connecting rod. The connecting rod is equipped with a rotating wheel and a belt. The rotating wheel is driven by a stepper motor 403, which in turn drives the belt to rotate, thereby enabling the fluorescence detector to move laterally below each reaction chamber. The range of lateral movement is limited by a photoelectric switch 404. The fluorescence detector has FAM, VIC, ROX, and CY5 channels.

[0059] The main control board controls the heating element to heat up and collects the temperature from the temperature sensor inside the heat-conducting plate. It also controls the opening and closing of the peristaltic pump and solenoid valve, reads the liquid detection signal from the liquid level sensor, controls the rotation of the stepper motor and the collection of the fluorescence signal from the fluorescence detector, and reads the position signal from the photoelectric switch.

[0060] Detection of Staphylococcus aureus nucleic acid using an automated detection kit

[0061] Step 1) Pre-contain the LAMP amplification primers for the Staphylococcus aureus nuc gene, sgRNA targeting the Staphylococcus aureus nuc gene, AapCas12b protease, fluorescent probe, Bst3.0 polymerase, dNTPs reagent, and lyophilized reaction buffer in the reaction chamber; one reaction chamber is set as a blank control.

[0062] The nucleotide sequences of LAMP amplification primers F3, B3, FIP, BIP, LF and LB and the nucleotide sequence of sgRNA are shown in Table 1. Based on the LAMP-CRISPR / Cas12b method, combined with microfluidic chips and devices, one-step automated detection of microorganisms can be realized, which has the advantages of high sensitivity and short detection time.

[0063] Table 1

[0064]

[0065]

[0066] Step 2) Open the sealing screw, and put the swab carrying the sample into the sample chamber through the sample inlet. Break off the swab rod and leave the swab head in the sample chamber. Tighten the sealing screw. The main control board controls the peristaltic pump to pump the eluent in the storage bag into the eluent inlet. Open the first solenoid valve. The eluent flows into the sample chamber of the sample injection module for elution. Then it enters the degassing channel from the sample inlet for degassing of the eluent.

[0067] Step 3) The main control board controls the peristaltic pump to continue pumping liquid. After the electrode detects that the eluent has flowed into the liquid level detection pool, the timer is started. The peristaltic pump is controlled by the time to deliver the eluent to fill the lysis pool. Then the peristaltic pump, the first solenoid valve and the second solenoid valve are closed, and the heating component connected above the lysis pool is controlled to heat to 90°C. The lysis is carried out for 5-15 minutes to complete the nucleic acid extraction.

[0068] Step 4) The main control board controls the peristaltic pump and the third solenoid valve to deliver the lysis buffer into the reaction chamber. Then, the third solenoid valve is closed, and the heating component connected above the reaction chamber is controlled to heat to 65°C. The reaction is carried out for 60 minutes, and the nucleic acid is amplified at an isothermal temperature for 60 cycles, with each cycle lasting 1 minute.

[0069] LAMP reaction: The reaction system is 25 μL, containing 2 μL template solution, 8 U Bst 3.0 polymerase, 6 mM MgSO4, 1.4 mM dNTPs, 1x reaction buffer, 0.2 μM F3, 0.2 μM B3, 0.4 μM LF, 0.4 μM LB, 1.6 μM FIP, and 1.6 μM M IP. The nucleic acid amplification process is 40 cycles at 65℃, with each cycle lasting 1 minute.

[0070] LAMP-CRISPR / Cas12b reaction system (20 μL): contains 5 μL LAMP reaction mixture, 250 nM fluorescent probe ( / 5'-FAM-XXXX-BHQ1-3' / ), 250 mM AapCas12b and 250 mM sgRNA, amplified at 65 °C for 60 cycles, each cycle lasting 1 minute.

[0071] The *Staphylococcus aureus* nuc gene was selected as the target region through specific sequence analysis and comparison. LAMP primers were designed, and the recognition RNA (sgRNA) sequence of AapCas12b was designed using the complementary sequence of the F2-B2 amplification region of the LAMP primers. LAMP amplification produced a large amount of DNA amplification products containing the nuc gene. These products can serve as target DNA for the CRISPR / Cas12b reaction, activating the trans-cleavage activity of Cas12b, which cleaves the fluorescent probe labeled with a single-stranded fluorescent molecule-quencher, generating a fluorescent signal.

[0072] Step 5) The main control board controls the stepper motor to drive the fluorescence detector to pass under each reaction chamber in turn. The fluorescence probe opens the FAM channel for fixed-point fluorescence detection. The main control board controls the fluorescence probe to perform fluorescence signal detection once in each cycle.

[0073] The feasibility of using the LAMP-CRISPR / Cas12b system for detection was evaluated, and the results are as follows: Figure 9As shown, DEPC water was added to the negative control group, while a target solution containing the nuc gene was added to the experimental group. After 20 minutes of reaction, the fluorescence signal intensity of each group was compared. The experimental group was significantly higher than that of the negative control group, proving that the automatic detection kit of this invention can quickly and accurately identify the nuc gene of Staphylococcus aureus for microbial nucleic acid detection.

[0074] The embodiments described above are merely illustrative of the implementation of this utility model, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this utility model patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this utility model, and these all fall within the protection scope of this utility model. Therefore, the protection scope of this utility model patent should be determined by the appended claims.

Claims

1. A microfluidic chip for microbial nucleic acid detection, comprising a degassing channel, a lysis cell, a reaction detection cell, and a sample introduction module integrated on the microfluidic chip, characterized in that, The microfluidic chip also includes a first solenoid valve, a second solenoid valve, and a third solenoid valve. The first solenoid valve controls the opening and closing of the eluent inlet and outlet. The eluent flows out from the outlet through the sample chamber of the sample injection module and enters the desorption channel through the sample inlet. The inlet pipe of the sample chamber is connected to the eluent outlet, and the outlet pipe of the sample chamber is connected to the sample inlet. The second solenoid valve controls the opening and closing of the degassing channel outlet and the lysis cell inlet. A heating component is connected above the lysis cell, and the sample completes nucleic acid release in the lysis cell. The third solenoid valve controls the opening and closing of the lysis cell outlet and the reaction detection cell inlet. The reaction detection cell is equipped with several reaction chambers, in which lyophilized powder of the LAMP-CRISPR reaction system is pre-placed. A heating component is connected above the reaction chamber, and nucleic acid isothermal amplification is completed in the reaction chamber.

2. The microfluidic chip for microbial nucleic acid detection according to claim 1, characterized in that, The sample chamber is located within the sample injection module. The sample injection module is connected to the sample chamber via a sample dispensing port, which is switched by a sealing screw. The sample chamber inlet is connected to a liquid inlet tube, which is connected to the elution outlet of the microbial nucleic acid detection microfluidic chip. The sample chamber outlet is connected to an outlet tube, which is connected to the sample inlet of the microbial nucleic acid detection microfluidic chip.

3. The microfluidic chip for microbial nucleic acid detection according to claim 2, characterized in that, The microfluidic chip for microbial nucleic acid detection is equipped with a storage bag for elution solution, which is connected to the elution solution inlet via a peristaltic pump.

4. The microfluidic chip for microbial nucleic acid detection according to claim 3, characterized in that, A water-proof and breathable pad is provided on the degassing channel, and a pressure plate is provided on the water-proof and breathable pad. The pressure plate presses and seals the degassing channel, and the width of the degassing channel is 1 to 3 mm.

5. The microfluidic chip for microbial nucleic acid detection according to claim 3, characterized in that, The pyrolysis cell is a spindle-shaped cell, and its volume is larger than the total volume of all the reaction chambers.

6. The microfluidic chip for microbial nucleic acid detection according to claim 5, characterized in that, The inlet of the reaction detection pool is connected to the reaction chamber through several liquid inlet branch pipes. Each reaction chamber has the same volume. The reaction chamber is connected to the liquid outlet branch pipe. The outlet of the liquid outlet branch pipe is connected to the gas outlet. A water-proof and breathable pad is provided on the gas outlet. The gas outlet is sealed by pressing with a gas outlet pressure plate. A light-transmitting window is provided at the bottom of the reaction chamber.

7. The microfluidic chip for microbial nucleic acid detection according to claim 6, characterized in that, The inlet of the reaction detection cell is connected to each of the inlet branch pipes via the main inlet pipe. A bypass liquid flow channel is also connected in parallel on one side of the inlet branch pipe. The diameter of the bypass liquid flow channel is wider than that of the inlet branch pipe. The outlet of the bypass liquid flow channel is connected to the gas outlet. The volume of the outlet branch pipe is the same.

8. The microfluidic chip for microbial nucleic acid detection according to claim 1, characterized in that, The heating assembly consists of a heat-conducting plate, a heating plate, and a heat-insulating plate from bottom to top. A temperature sensor is installed inside the heat-conducting plate and is sealed and fixed to the corresponding position on the microfluidic chip by a sealing ring.

9. The microfluidic chip for microbial nucleic acid detection according to claim 1, characterized in that, An elliptical liquid level detection pool is provided in the liquid inlet pipe of the pyrolysis cell. Metal electrodes are provided at both ends of the liquid level detection pool. One end of the electrode is inside the detection pool, and the other end protrudes outside the chip and is connected to the wire.

10. The microfluidic chip for microbial nucleic acid detection according to claim 9, characterized in that, The pyrolysis tank inlet pipe is equipped with a serpentine pipe.

11. The microfluidic chip for microbial nucleic acid detection according to claim 6, characterized in that, The connection between the sample inlet and the degassing channel is through the water-proof and breathable pad, which seals the passageway.

12. An automated detection kit comprising the microbial nucleic acid detection microfluidic chip according to any one of claims 1-11, characterized in that, The automatic detection box is detachably connected to the top of the detection device box by a chip box. The microbial nucleic acid detection microfluidic chip is fixed inside the chip box, the sample injection module is fixed inside the chip box, the sample dispensing port abuts against the surface of the chip box, and the sealing screw protrudes outside the chip box. The fluorescence detector is set inside the detection device box, and the fluorescence probe of the fluorescence detector corresponds to the position of the reaction chamber. The detection device box also has a main control board, which is used to electrically control the various electrical components.

13. The automatic detection box according to claim 12, characterized in that, The fluorescence detector is connected to the detection equipment housing via a connecting rod. The connecting rod is equipped with a rotating wheel and a belt. A stepper motor drives the rotating wheel to rotate the belt, thereby enabling the fluorescence detector to move laterally below each reaction chamber. The range of lateral movement is limited by a photoelectric switch. The fluorescence detector has FAM, VIC, ROX, and CY5 channels.

14. The automatic detection box according to claim 13, characterized in that, The main control board controls the heating element to heat up and collects the temperature of the temperature sensor inside the heat-conducting plate, controls the opening and closing of the peristaltic pump and solenoid valve, reads the liquid detection signal from the liquid level sensor, controls the rotation of the stepper motor and the collection of the fluorescence signal from the fluorescence detector, and reads the position signal from the photoelectric switch.

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