Germ detection kit based on microfluidics and CRISPR / Cas system and detection method thereof

Abstract

The invention discloses a pathogen detection kit based on microfluidics and a CRISPR / Cas system and a detection method thereof, and particularly relates to the technical field of biological detection.The pathogen detection kit comprises a nucleic acid releasing agent layer, an RPA reaction chamber layer and a CRISPR detection chamber layer, the nucleic acid releasing agent layer is located above the RPA reaction chamber layer, and the CRISPR detection chamber layer is located above the RPA reaction chamber layer. The RPA reaction chamber layer is positioned above the CRISPR detection chamber layer; a nucleic acid releasing chamber is formed in the bottom surface of the nucleic acid releasing agent layer in a penetrating manner. According to the present invention, the kit has the high specific recognition ability on the specific pathogen nucleic acid sequence, can accurately detect the target pathogen, can reduce the false result occurrence probability, can detect a variety of pathogens or a plurality of gene targets of the same pathogen at one time, and can rapidly amplify the nucleic acid through the optimized RPA reaction system of the RPA reaction chamber so as to substantially shorten the detection time.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically, to a pathogen detection kit based on microfluidics and CRISPR / Cas system and a detection method thereof. Background Art

[0002] In the field of modern medicine and public health, pathogen detection is a key link in disease diagnosis, epidemic prevention and control, and treatment plan formulation. Although traditional pathogen detection methods can meet detection needs to a certain extent, with the continuous development of science and technology and the increasing complexity of actual application scenarios, some limitations have gradually been exposed.

[0003] Traditional detection technology is not able to accurately distinguish pathogens with similar gene sequences, which can easily lead to false positive and false negative results, affecting the accuracy of clinical diagnosis. In the early stages of infection, it may not be able to accurately determine the type of pathogen and the infection situation, which affects the targeted treatment plan and may also cause problems such as antibiotic abuse. For complex infection cases, traditional methods are difficult to fully analyze the infection situation of pathogens at one time, and multiple tests or multiple technologies are required, which increases costs, time and error risks, and is not conducive to the formulation of comprehensive and effective treatment plans. At the same time, traditional nucleic acid extraction methods are time-consuming and cannot meet the strict requirements of detection time in emergency scenarios and epidemic outbreaks. Traditional PCR amplification technology also has the problem of long amplification time, and it is difficult to quickly screen a large number of samples at the epidemic site. This may cause the infected person to continue to spread the pathogen during the detection period, increasing the difficulty of epidemic prevention and control. Therefore, a pathogen detection kit based on microfluidics and CRISPR / Cas system and its detection method are proposed. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, the present invention provides a pathogen detection kit and a detection method thereof based on microfluidics and CRISPR / Cas system to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: A pathogen detection kit based on microfluidics and CRISPR / Cas system, comprising a nucleic acid release agent layer, an RPA reaction chamber layer and a CRISPR detection chamber layer, wherein the nucleic acid release agent layer is located above the RPA reaction chamber layer, and the RPA reaction chamber layer is located above the CRISPR detection chamber layer; A nucleic acid release chamber is provided through the bottom surface of the nucleic acid release agent layer, a first slider is fixedly connected to the bottom of the nucleic acid release agent layer, a first slide rail is provided on the top surface of the RPA reaction chamber layer, the first slider is slidably connected to the first slide rail, an RPA amplification chamber is provided through the top surface of the RPA reaction chamber layer, a second slider is fixedly connected to the bottom of the RPA reaction chamber layer, a second slide rail is provided on the top surface of the CRISPR detection chamber layer, the second slider is slidably connected to the second slide rail, and a three-electrode electrochemical detection contact is provided on the top surface of the CRISPR detection chamber layer.

[0006] Preferably, the nucleic acid releaser in the nucleic acid releaser layer is a highly efficient nucleic acid extraction reagent for quickly releasing nucleic acids in a sample; the RPA reagent in the RPA reaction chamber layer is an optimized RPA reaction system for ensuring high amplification efficiency and specificity; and the CRISPR reagent in the CRISPR detection chamber layer is a CRISPR / Cas detection system for specific pathogens, for accurately detecting target pathogens.

[0007] Preferably, the number of nucleic acid release chambers in the nucleic acid release agent layer is multiple to meet the demand of processing multiple samples simultaneously, and the nucleic acid release agent layer, RPA reaction chamber layer and CRISPR detection chamber layer are all made of transparent materials to facilitate observation of the reaction process.

[0008] Preferably, the nucleic acid release chamber is used to place the sample, and a nucleic acid release agent is added thereto to achieve rapid release of the nucleic acid in the sample. The RPA amplification chamber is used to perform a recombinase polymerase amplification reaction, receive the nucleic acid transferred from the nucleic acid release chamber, and amplify the nucleic acid under suitable conditions to provide sufficient nucleic acid for subsequent detection.

[0009] Preferably, the CRISPR detection chamber is used to receive the amplification product from the RPA amplification chamber and perform a CRISPR shearing reaction therein.

[0010] The present invention also provides a detection method, which uses a pathogen detection kit based on microfluidics and CRISPR / Cas system for detection, comprising the following steps: S1: Preparation before testing, check all components of the test kit, including the nucleic acid release agent layer, the RPA reaction chamber layer and the CRISPR detection chamber layer, and check that the first slider and the second slider between the layers are connected normally with the first and second slide rails, and the three-electrode electrochemical detection contacts function normally. Check whether the nucleic acid release agent, RPA reagent and CRISPR reagent in the kit are sufficient and not expired, and that each chamber is well sealed and ready for use; S2: Sample collection and processing: collect samples, place the samples in a nucleic acid release chamber, add an appropriate amount of nucleic acid release agent, immerse the samples in the nucleic acid release agent, seal the nucleic acid release chamber containing the samples and the nucleic acid release agent, and place it in an instrument for nucleic acid release; S3: Nucleic acid amplification. After the nucleic acid is released, the nucleic acid release agent layer is rotated slowly and steadily, so that the first slider slides uniformly from one end to the other end along the first slide rail, so that the nucleic acid release agent layer is accurately connected with the RPA reaction chamber layer, and the RPA amplification chamber is connected with the nucleic acid release chamber. The entire detection kit is instantaneously centrifuged using a centrifuge, and the centrifugation parameters are adjusted so that the nucleic acid is transferred from the nucleic acid release chamber into the RPA amplification chamber under the action of centrifugal force. The device is placed in a constant temperature device, and the RPA reagent in the RPA amplification chamber performs an amplification reaction on the transferred nucleic acid; S4: CRISPR shearing reaction. After the amplification reaction is completed, the nucleic acid release agent layer and the RPA reaction chamber layer are rotated. At this time, the nucleic acid release agent layer and the RPA reaction chamber layer are regarded as a whole, and the second slider is smoothly slid from one end to the other end along the second slide rail, so that the RPA amplification chamber is accurately connected with the CRISPR detection chamber. In the CRISPR detection chamber, the CRISPR reagent performs a CRISPR shearing reaction on the amplification product entering; S5: Detection and result analysis. After the CRISPR reaction is completed, use an electrochemical detection device to connect the three-electrode electrochemical detection contacts on the top surface of the CRISPR detection chamber layer for electrochemical signal detection. Before detection, ensure that the detection equipment has been calibrated and that the connection between the electrodes and the detection contacts is firm and in good contact. Record the changes in the electrochemical signal, including the change curves of the current and potential parameters. Based on the electrochemical signal data obtained from the test and combined with the standard curve or reference value provided by the kit, analyze and determine whether the target pathogen exists in the sample and the content information of the pathogen. Record the test results to complete the test.

[0011] Preferably, the sample collected in S2 is a swab or saliva. For respiratory pathogen detection, a throat swab or nasal swab collection method can be used. For saliva samples, the subject should be required to rinse the mouth thoroughly before collection, and then naturally spit out an appropriate amount of saliva into a special collection container.

[0012] Preferably, the first sliding block slides along the first sliding rail from one end to the other end at a constant speed to ensure that the connection between the layers remains stable during the rotation process without dislocation or separation.

[0013] Preferably, the centrifugation parameters in S3 and S4 are that the centrifugation speed is set at 2500-3500 rpm and the centrifugation time is 1-2 minutes. The amplification reaction time in S3 is adjusted according to the requirements of the nucleic acid amount and amplification efficiency, specifically 15-30 minutes.

[0014] Preferably, the shear reaction temperature in S4 is between 30-37°C for 10-20 minutes, and the temperature in S2 nucleic acid release process is incubated at 37°C for 15-30 minutes to promote the full release of nucleic acid of pathogens in the sample into the solution.

[0015] Technical effects and advantages of the present invention: By utilizing the highly specific recognition ability of the CRISPR / Cas system for specific pathogen nucleic acid sequences, the target pathogen can be accurately detected, greatly reducing the probability of false positive and false negative results. For some pathogens with similar gene sequences, the CRISPR / Cas system can accurately distinguish their subtle differences and accurately lock the target pathogen, providing a reliable basis for clinical diagnosis and epidemic prevention and control. It can detect multiple pathogens or multiple gene targets of the same pathogen at one time, which is particularly important in complex infection cases. It can comprehensively analyze the infection status of pathogens and provide richer information for accurate diagnosis of diseases and formulation of treatment plans. The kit can complete the test at one time, greatly improving the detection efficiency and accuracy, and helping to take targeted treatment measures in a timely manner and reduce the occurrence of complications. The nucleic acid in the sample can be quickly released by the efficient nucleic acid extraction reagent in the nucleic acid releaser layer, and the nucleic acid extraction process can be completed in a relatively short time. Generally, the nucleic acid can be fully released within 15-30 minutes. Compared with the traditional nucleic acid extraction method, the sample processing time is greatly shortened. The optimized RPA reaction system in the RPA reaction chamber has the characteristics of rapid amplification, and can amplify a large amount of nucleic acid within 15-30 minutes to provide sufficient nucleic acid for subsequent detection. This rapid amplification method enables the entire detection process to be completed in a relatively short time. Compared with the traditional PCR amplification technology, a lot of time is saved. In emergency scenarios or rapid screening at the epidemic site, the test results can be quickly obtained, which is of great significance for timely isolation of infected persons, control of epidemic spread, and guidance of clinical treatment. It helps to improve medical efficiency and the ability to respond to public health emergencies. The detection kit can be applied to a variety of sample types, including throat swabs, nasal swabs and saliva. For different pathogen infection routes and infection sites, the corresponding appropriate sample type can be selected for detection, which improves the flexibility and applicability of the detection; In summary, through the mutual influence of the above-mentioned multiple effects, the pathogen detection kit based on microfluidics and CRISPR / Cas system and its detection method have highly specific recognition ability for specific pathogen nucleic acid sequences, can accurately detect target pathogens, reduce the probability of false positive and false negative results, and can also detect multiple pathogens or multiple gene targets of the same pathogen at one time, providing rich information for accurate disease diagnosis and treatment plan formulation. Secondly, the efficient nucleic acid extraction reagent in the nucleic acid releaser layer can quickly release the nucleic acid in the sample, and the optimized RPA reaction chamber RPA reaction system can quickly amplify nucleic acid, greatly shorten the detection time, and is suitable for emergency scenarios and rapid screening at the epidemic site, improving medical efficiency and the ability to respond to public health emergencies. The detection kit is suitable for a variety of sample types, such as swabs and saliva, which improves the flexibility and applicability of the test. In summary, the kit is of great significance in clinical diagnosis, epidemic prevention and control, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the overall structure of the present invention.

[0017] Figure 2 It is a schematic diagram of the three-dimensional structure of the CRISPR detection chamber layer of the present invention.

[0018] Figure 3 It is a schematic diagram of the three-dimensional structure of the RPA reaction chamber layer of the present invention.

[0019] Figure 4 It is a schematic diagram of the bottom structure of the RPA reaction chamber layer of the present invention.

[0020] Figure 5 It is a schematic diagram of the three-dimensional structure of the nucleic acid releasing agent layer of the present invention.

[0021] Figure 6 It is a schematic diagram of the bottom structure of the nucleic acid release agent layer of the present invention.

[0022] Figure 7 It is a perspective structural schematic diagram of the present invention.

[0023] Figure 8 It is a schematic diagram of the detection method process structure of the present invention.

[0024] In the figure: 1. Nucleic acid release agent layer; 2. RPA reaction chamber layer; 3. CRISPR detection chamber layer; 4. Nucleic acid release chamber; 5. First slider; 6. First slide rail; 7. RPA amplification chamber; 8. Second slider; 9. Second slide rail; 10. CRISPR detection chamber; 11. Three-electrode electrochemical detection contacts. DETAILED DESCRIPTION

[0025] 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.

[0026] like Figure 1-7 The pathogen detection kit based on microfluidics and CRISPR / Cas system shown includes a nucleic acid release agent layer 1, an RPA reaction chamber layer 2 and a CRISPR detection chamber layer 3, wherein the nucleic acid release agent layer 1 is located above the RPA reaction chamber layer 2, and the RPA reaction chamber layer 2 is located above the CRISPR detection chamber layer 3; A nucleic acid release chamber 4 is provided through the bottom surface of the nucleic acid release agent layer 1, a first slider 5 is fixedly connected to the bottom of the nucleic acid release agent layer 1, a first slide rail 6 is provided on the top surface of the RPA reaction chamber layer 2, the first slider 5 is slidably connected to the first slide rail 6, an RPA amplification chamber 7 is provided through the top surface of the RPA reaction chamber layer 2, a second slider 8 is fixedly connected to the bottom of the RPA reaction chamber layer 2, a second slide rail 9 is provided on the top surface of the CRISPR detection chamber layer 3, the second slider 8 is slidably connected to the second slide rail 9, and a three-electrode electrochemical detection contact 11 is provided on the top surface of the CRISPR detection chamber layer 3.

[0027] As attached Figure 1-7 As shown, the nucleic acid releaser in the nucleic acid releaser layer 1 is a highly efficient nucleic acid extraction reagent, which is used to quickly release the nucleic acid in the sample. The RPA reagent in the RPA reaction chamber layer 2 is an optimized RPA reaction system, which is used to ensure high amplification efficiency and specificity. The CRISPR reagent in the CRISPR detection chamber layer 3 is a CRISPR / Cas detection system for specific pathogens, which is used to accurately detect the target pathogens.

[0028] As attached Figure 1-7 As shown, there are multiple nucleic acid release chambers 4 in the nucleic acid release agent layer 1 to meet the needs of processing multiple samples at the same time. The nucleic acid release agent layer 1, the RPA reaction chamber layer 2 and the CRISPR detection chamber layer 3 are all made of transparent materials to facilitate observation of the reaction process.

[0029] As attached Figure 1 , 3 As shown in Figures 4, 5, 6 and 7, the nucleic acid release chamber 4 is used to place the sample and add a nucleic acid release agent therein to achieve rapid release of the nucleic acid in the sample. The RPA amplification chamber 7 is used to perform a recombinase polymerase amplification reaction, receive the nucleic acid transferred from the nucleic acid release chamber, amplify the nucleic acid under suitable conditions, and provide sufficient nucleic acid for subsequent detection.

[0030] As attached Figure 2 , 7 As shown, the CRISPR detection chamber 10 is used to receive the amplification product from the RPA amplification chamber and perform a CRISPR shearing reaction therein.

[0031] As attached Figure 1-8 As shown, the present invention also provides a detection method, which uses a pathogen detection kit based on microfluidics and CRISPR / Cas system for detection, comprising the following steps: S1: Preparation before testing, check all components of the test kit, including the nucleic acid release agent layer, the RPA reaction chamber layer and the CRISPR detection chamber layer, and check that the first slider and the second slider between the layers are connected normally with the first and second slide rails, and the three-electrode electrochemical detection contacts function normally. Check whether the nucleic acid release agent, RPA reagent and CRISPR reagent in the kit are sufficient and not expired, and that each chamber is well sealed and ready for use; S2: Sample collection and processing: collect samples, place the samples in a nucleic acid release chamber, add an appropriate amount of nucleic acid release agent, immerse the samples in the nucleic acid release agent, seal the nucleic acid release chamber containing the samples and the nucleic acid release agent, and place it in an instrument for nucleic acid release; The samples collected in S2 are swabs or saliva. For respiratory pathogen detection, throat swabs or nasal swabs can be used for collection. For saliva samples, the examinee should be required to rinse the mouth thoroughly before collection, and then naturally spit out an appropriate amount of saliva into a special collection container; During the S2 nucleic acid release process, the temperature is kept constant at 37°C for 15-30 minutes to promote the full release of nucleic acids of pathogens in the sample into the solution; S3: Nucleic acid amplification. After the nucleic acid is released, the nucleic acid release agent layer is rotated slowly and steadily, so that the first slider slides uniformly from one end to the other end along the first slide rail, so that the nucleic acid release agent layer is accurately connected with the RPA reaction chamber layer, and the RPA amplification chamber is connected with the nucleic acid release chamber. The entire detection kit is instantaneously centrifuged using a centrifuge, and the centrifugation parameters are adjusted so that the nucleic acid is transferred from the nucleic acid release chamber into the RPA amplification chamber under the action of centrifugal force. The device is placed in a constant temperature device, and the RPA reagent in the RPA amplification chamber performs an amplification reaction on the transferred nucleic acid; The first sliding block slides along the first sliding rail from one end to the other end at a uniform speed to ensure that the connection between the layers remains stable during the rotation process without dislocation or separation; S4: CRISPR shearing reaction. After the amplification reaction is completed, the nucleic acid release agent layer and the RPA reaction chamber layer are rotated. At this time, the nucleic acid release agent layer and the RPA reaction chamber layer are regarded as a whole, and the second slider is smoothly slid from one end to the other end along the second slide rail, so that the RPA amplification chamber is accurately connected with the CRISPR detection chamber. In the CRISPR detection chamber, the CRISPR reagent performs a CRISPR shearing reaction on the amplification product entering; In S4, the shear reaction temperature is between 30-37°C and the time is 10-20 minutes; The centrifugation parameters in S3 and S4 are that the centrifugation speed is set at 2500-3500 rpm and the centrifugation time is 1-2 minutes. The amplification reaction time in S3 is adjusted according to the requirements of nucleic acid amount and amplification efficiency, specifically 15-30 minutes; S5: Detection and result analysis. After the CRISPR reaction is completed, use an electrochemical detection device to connect the three-electrode electrochemical detection contacts on the top surface of the CRISPR detection chamber layer for electrochemical signal detection. Before detection, ensure that the detection equipment has been calibrated and that the connection between the electrodes and the detection contacts is firm and in good contact. Record the changes in the electrochemical signal, including the change curves of the current and potential parameters. Based on the electrochemical signal data obtained from the test and combined with the standard curve or reference value provided by the kit, analyze and determine whether the target pathogen exists in the sample and the content information of the pathogen. Record the test results to complete the test.

[0032] Working principle of the present invention: after collecting the sample swab or saliva, place it in the nucleic acid release chamber 4. The nucleic acid release chamber 4 in the nucleic acid release agent layer 1 is the starting point of sample processing, which provides space for the contact between the sample and the nucleic acid release agent. After adding the nucleic acid release agent, incubate at a constant temperature of 37°C for 15-30 minutes, and the nucleic acid release agent begins to work. It can quickly destroy the cell structure of the pathogen in the sample, release the nucleic acid from the pathogen cells, and fully dissolve it in the solution to prepare for subsequent nucleic acid detection. After the nucleic acid release is completed, by rotating the nucleic acid release agent layer 1, the first slider 5 slides uniformly from one end to the other end along the first slide rail 6. This operation enables the nucleic acid release agent layer 1 to be accurately connected with the RPA reaction chamber layer 2. At this time, the RPA amplification chamber 7 is connected with the nucleic acid release chamber 4. This sliding connection design ensures that the layers can be accurately docked when they need to be connected, and at the same time maintain their independence when they are not connected, ensuring the separate storage and sealing of the reagents; A centrifuge is used to perform instantaneous centrifugation operation, and the centrifugal speed is set at 2500-3500rpm, and the centrifugation time is 1-2 minutes. Under the action of centrifugal force, the nucleic acid is smoothly transferred from the nucleic acid release chamber 4 into the RPA amplification chamber 7. The RPA reagent in the RPA reaction chamber layer 2 is an optimized RPA reaction system. In a constant temperature device, usually at a temperature of 37-42°C, the transferred nucleic acid is amplified. The reaction time is adjusted according to the requirements of the nucleic acid amount and amplification efficiency, generally 15-30 minutes. The RPA amplification reaction can amplify a large amount of nucleic acid at an extremely fast speed, providing sufficient nucleic acid for subsequent detection. Its high amplification efficiency and specificity ensure the accuracy and sensitivity of the detection. After the amplification reaction is completed, the nucleic acid release agent layer 1 and the RPA reaction chamber layer 2 are rotated. At this time, the two layers are regarded as a whole, and the first The second slider 8 slides smoothly from one end to the other end along the second slide rail 9, so that the RPA amplification chamber 7 is accurately connected to the CRISPR detection chamber 10. This layered sliding connection structure design enables the corresponding chambers to be connected in order at different reaction stages, realizing the automation and accuracy of the detection process. In the CRISPR detection chamber 10, the CRISPR reagent performs a CRISPR shearing reaction on the amplified product entering. The CRISPR reagent in the CRISPR detection chamber layer 3 is a CRISPR / Cas detection system for specific pathogens, which can specifically identify the nucleic acid sequence of the target pathogen. At a temperature of 30-37°C, the reaction takes 10-20 minutes, and the target nucleic acid sequence in the amplified product is identified and sheared by the CRISPR / Cas system to generate a detectable signal change; After the CRISPR reaction is completed, an electrochemical detection device is used to connect the three-electrode electrochemical detection contacts 11 on the top surface of the CRISPR detection chamber layer 3 for electrochemical signal detection. Before the detection, ensure that the detection device has been calibrated, the connection between the electrode and the detection contact is firm and the contact is good, and record the changes in the electrochemical signal, including the change curves of the current and potential parameters. According to the electrochemical signal data obtained by the detection, combined with the standard curve or reference value provided by the kit, analyze and judge whether the target pathogens are present in the sample and the content information of the pathogens. Through the accurate detection and analysis of the electrochemical signals, the qualitative and quantitative detection of the pathogens is achieved, the entire detection process is completed and reliable detection results are obtained. The entire detection kit is based on the precise control of microfluidic technology and the high specificity detection capability of the CRISPR / Cas system. Through the synergistic effect of each layer structure and a reasonable operation process, efficient and accurate detection of pathogens is achieved.

[0033] Finally, a few points should be explained: First, in the description of this application, it should be noted that, unless otherwise specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, which may refer to mechanical connection or electrical connection, or internal communication between two components, or direct connection. "upper", "lower", "left", "right", etc. are only used to indicate relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may change; Secondly: In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other; Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A pathogen detection kit based on microfluidics and CRISPR / Cas system, comprising a nucleic acid release agent layer (1), an RPA reaction chamber layer (2) and a CRISPR detection chamber layer (3), characterized in that: The nucleic acid release agent layer (1) is located above the RPA reaction chamber layer (2), and the RPA reaction chamber layer (2) is located above the CRISPR detection chamber layer (3); a nucleic acid release chamber (4) is provided through the bottom surface of the nucleic acid release agent layer (1), a first slider (5) is fixedly connected to the bottom of the nucleic acid release agent layer (1), a first slide rail (6) is provided on the top surface of the RPA reaction chamber layer (2), and the first slider (5) and the first slide rail (6) are slidably connected, an RPA amplification chamber (7) is provided through the top surface of the RPA reaction chamber layer (2), a second slider (8) is fixedly connected to the bottom of the RPA reaction chamber layer (2), a second slide rail (9) is provided on the top surface of the CRISPR detection chamber layer (3), and the second slider (8) and the second slide rail (9) are slidably connected, and a three-electrode electrochemical detection contact (11) is provided on the top surface of the CRISPR detection chamber layer (3).

2. The pathogen detection kit based on microfluidics and CRISPR / Cas system according to claim 1, characterized in that: The nucleic acid releaser in the nucleic acid releaser layer (1) is a highly efficient nucleic acid extraction reagent, which is used to quickly release the nucleic acid in the sample; the RPA reagent in the RPA reaction chamber layer (2) is an optimized RPA reaction system, which is used to ensure high amplification efficiency and specificity; and the CRISPR reagent in the CRISPR detection chamber layer (3) is a CRISPR / Cas detection system for specific pathogens, which is used to accurately detect the target pathogens.

3. The pathogen detection kit based on microfluidics and CRISPR / Cas system according to claim 1, characterized in that: The number of nucleic acid release chambers (4) in the nucleic acid release agent layer (1) is multiple, so as to meet the demand of processing multiple samples simultaneously. The nucleic acid release agent layer (1), the RPA reaction chamber layer (2) and the CRISPR detection chamber layer (3) are all made of transparent materials, so as to facilitate observation of the reaction process.

4. The pathogen detection kit based on microfluidics and CRISPR / Cas system according to claim 1, characterized in that: The nucleic acid release chamber (4) is used to place the sample and add a nucleic acid release agent therein to achieve rapid release of the nucleic acid in the sample. The RPA amplification chamber (7) is used to perform a recombinase polymerase amplification reaction, receive the nucleic acid transferred from the nucleic acid release chamber, and amplify the nucleic acid under suitable conditions to provide a sufficient amount of nucleic acid for subsequent detection.

5. The pathogen detection kit based on microfluidics and CRISPR / Cas system according to claim 1, characterized in that: The CRISPR detection chamber (10) is used to receive the amplification product from the RPA amplification chamber and perform a CRISPR shearing reaction therein.

6. A detection method, characterized in that: The detection is performed using the pathogen detection kit based on microfluidics and CRISPR / Cas system according to any one of claims 1 to 5, comprising the following steps: S1: Preparation before testing, check all components of the test kit, including the nucleic acid release agent layer, the RPA reaction chamber layer and the CRISPR detection chamber layer, and check that the first slider and the second slider between the layers are connected normally with the first and second slide rails, and the three-electrode electrochemical detection contacts function normally. Check whether the nucleic acid release agent, RPA reagent and CRISPR reagent in the kit are sufficient and not expired, and that each chamber is well sealed and ready for use; S2: Sample collection and processing: collect samples, place the samples in a nucleic acid release chamber, add an appropriate amount of nucleic acid release agent, immerse the samples in the nucleic acid release agent, seal the nucleic acid release chamber containing the samples and the nucleic acid release agent, and place it in an instrument for nucleic acid release; S3: Nucleic acid amplification. After the nucleic acid is released, the nucleic acid release agent layer is rotated slowly and steadily, so that the first slider slides uniformly from one end to the other end along the first slide rail, so that the nucleic acid release agent layer is accurately connected with the RPA reaction chamber layer, and the RPA amplification chamber is connected with the nucleic acid release chamber. The entire detection kit is instantaneously centrifuged using a centrifuge, and the centrifugation parameters are adjusted so that the nucleic acid is transferred from the nucleic acid release chamber into the RPA amplification chamber under the action of centrifugal force. The device is placed in a constant temperature device, and the RPA reagent in the RPA amplification chamber performs an amplification reaction on the transferred nucleic acid; S4: CRISPR shearing reaction. After the amplification reaction is completed, the nucleic acid release agent layer and the RPA reaction chamber layer are rotated. At this time, the nucleic acid release agent layer and the RPA reaction chamber layer are regarded as a whole, and the second slider is smoothly slid from one end to the other end along the second slide rail, so that the RPA amplification chamber is accurately connected with the CRISPR detection chamber. In the CRISPR detection chamber, the CRISPR reagent performs a CRISPR shearing reaction on the amplification product entering; S5: Detection and result analysis. After the CRISPR reaction is completed, use an electrochemical detection device to connect the three-electrode electrochemical detection contacts on the top surface of the CRISPR detection chamber layer for electrochemical signal detection. Before detection, ensure that the detection equipment has been calibrated and that the connection between the electrodes and the detection contacts is firm and in good contact. Record the changes in the electrochemical signal, including the change curves of the current and potential parameters. Based on the electrochemical signal data obtained from the test and combined with the standard curve or reference value provided by the kit, analyze and determine whether the target pathogen exists in the sample and the content information of the pathogen. Record the test results to complete the test.

7. The detection method according to claim 6, characterized in that: The sample collected in S2 is a swab or saliva. For respiratory pathogen detection, a throat swab or nasal swab collection method can be used. For saliva samples, the subject should be required to rinse the mouth thoroughly before collection, and then naturally spit out an appropriate amount of saliva into a special collection container.

8. The detection method according to claim 6, characterized in that: The first sliding block slides along the first sliding rail from one end to the other end at a constant speed to ensure that the connection between the layers remains stable during the rotation process without dislocation or separation.

9. The detection method according to claim 6, characterized in that: The centrifugation parameters in S3 and S4 are that the centrifugation speed is set at 2500-3500 rpm and the centrifugation time is 1-2 minutes. The amplification reaction time in S3 is adjusted according to the requirements of the nucleic acid amount and amplification efficiency, specifically 15-30 minutes.

10. The detection method according to claim 6, characterized in that: The shear reaction temperature in S4 is between 30-37°C and the time is 10-20 minutes. During the nucleic acid release process in S2, the temperature is incubated at a constant temperature of 37°C for 15-30 minutes to promote the full release of the nucleic acid of the pathogen in the sample into the solution.

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