A detection method integrating PCR amplification and capillary electrophoresis analysis

Abstract

The present invention relates to a clamp device and a detection method integrating PCR amplification and capillary electrophoresis analysis. The present invention adopts an integrated pipeline closed clamp, so the contamination source of high-concentration PCR products is effectively controlled, avoiding cross contamination between laboratory tests, and eliminating manual intervention between PCR amplification and capillary electrophoresis analysis, thereby improving test efficiency and having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical equipment, and in particular relates to a clamping device and a detection method integrating PCR amplification and capillary electrophoresis analysis. Background Art

[0002] Polymerase chain reaction (PCR) is an important method in nucleic acid research and is widely used in life science research, inspection and quarantine, justice, medical diagnosis and research and other related fields. The efficient integration of nucleic acid extraction, PCR amplification and capillary electrophoresis analysis is a new design concept that meets the requirements of the current medical field for nucleic acid detection devices with high analysis efficiency, high integration, high degree of automation and high accuracy.

[0003] Nucleic acid PCR amplification can be divided into two types: static fixed amplification PCR and continuous flow PCR (CFPCR). In the static fixed amplification PCR test platform, a metal block with a large heat capacity is used for thermal cycle temperature control. Its reaction speed is theoretically restricted by factors such as the heat capacity of the PCR reaction system, the heat capacity of the temperature control module material, the heater, and the heat capacity of the sensor. In the CFPCR test, the sample to be amplified flows continuously through three constant temperature zones with different temperatures. Since repeated heating or cooling is not required during the amplification process, its heating and cooling speed is generally not limited by the heat capacity of the equipment system, thereby simplifying the temperature control design, shortening the time required for amplification, and greatly improving the efficiency of PCR amplification.

[0004] Capillary electrophoresis is a type of chromatographic separation and analysis technology that uses capillaries as separation channels and high-voltage direct current electric fields as driving forces. The capillary electrophoresis system mainly includes capillary components, cathode cells, anode cells, and gel circuit systems. At present, capillary electrophoresis separation technology is often used for post-processing of samples after PCR amplification.

[0005] Patent CN106701566B discloses a mechanical device that integrates nucleic acid extraction, amplification, and detection. However, only nucleic acid extraction and PCR amplification can be completed in one device, and subsequent detection needs to be performed in another detection device. Therefore, manual intervention is required in the detection process after PCR amplification, and it does not truly integrate the extraction, amplification, and detection of molecular diagnosis. In addition, the problem of manual intervention makes the operation of the device prone to nucleic acid contamination, making it difficult to promote it on a large scale in clinical practice.

[0006] Patent CN208829682U discloses an integrated DNA analyzer. Although the DNA analyzer can realize the extraction, amplification and detection of molecular diagnosis in one, in the instrument, the high-concentration PCR product is exposed and placed inside the instrument, and the instrument only uses a cooling fan to remove pollutants. This method not only cannot avoid contamination by high-concentration PCR products, but is more likely to pollute the environment inside the entire device.

[0007] Patent US9889449B2 discloses an integrated system for multiplex amplification and detection of six or more dye-labeled fragments, in which PCR amplification and capillary electrophoresis analysis are integrated into a biochip to achieve the two-in-one amplification and detection through the biochip, but its high manufacturing cost and low test throughput restrict its clinical promotion.

[0008] The document "Study on Continuous Flow PCR Microfluidic Device" (Zhang Chunsun, Xing Da. "Acta Laser Biologica Sinica [J]. 2007, 16 (4): 501-508.) and patent CN102605065A both relate to continuous flow PCR microfluidic technology. The document "Study on Continuous Flow PCR Microfluidic Device" discloses a continuous flow PCR microfluidic device, but it does not realize the integration of PCR amplification and detection in one device. Patent CN102605065A discloses a continuous flow nested PCR microfluidic method, but the method includes two PCR amplifications, and manual intervention is required between the two amplifications, and automation cannot be achieved. Summary of the invention

[0009] The purpose of the present invention is to provide a card holder device and a detection method that integrates PCR amplification and capillary electrophoresis analysis. The present invention can realize the integration of PCR amplification and electrophoresis separation analysis in a card holder, and complete the detection of the target sample after amplification together with the supporting instrument. The purpose of the present invention is achieved by the following technical solutions:

[0010] The first aspect of the present invention relates to a clamping device integrating PCR amplification and capillary electrophoresis analysis, which includes a sample inlet 19 to be tested, a PCR amplification area 2, a liquid suction and transportation device 3, a liquid divider 5, a temperature control area 18 and a capillary electrophoresis system 21 connected in sequence, wherein the liquid divider 5 is also connected to an electrophoresis analysis reagent containing device 20, and the liquid suction and transportation device 3 is also connected to a water containing device 4 and a waste liquid bag 13 respectively.

[0011] In a preferred embodiment, a negative pressure degassing buffer device 1 is provided between the sample inlet 19 to be detected and the PCR amplification area 2 .

[0012] The function of the negative pressure degassing buffer device 1 is to precipitate the dissolved gas in the sample to be detected. This can avoid the situation where the liquid segments in the sample to be detected are segmented due to bubbles precipitated at high temperature during the denaturation process, which affects the amplification efficiency; on the other hand, it can enable the subsequent PCR amplification area 2 to accommodate more samples to be detected, greatly improving the amplification efficiency of the instrument; on the other hand, it can also improve the accuracy of controlling the movement and positioning of the fluid in the entire flow path.

[0013] In a preferred embodiment, the capillary electrophoresis system 21 includes a cathode pool 7, a capillary assembly, an anode pool 10 and a gel box 14 which are connected in sequence and fluidically connected; the cathode pool 7 is provided with two cathode electrodes 6, and the anode pool 10 is provided with an anode electrode 11; the capillary assembly includes a capillary 17, a capillary temperature control unit 16 arranged around the outer wall of the capillary 17 and a detection window 15 arranged on the outer wall of the capillary 17, and an optical detection assembly is provided at the detection window 15.

[0014] In a preferred embodiment, the cathode pool 7 is also connected to the temperature control zone 18 and the buffer storage device 9 respectively; the anode pool 10 is also connected to the overflow plastic bag 12.

[0015] In a preferred embodiment, the water in the water containing device 4 is double distilled water (ddH2O).

[0016] In a specific embodiment, the sample inlet 19 to be detected can be connected to a device such as a sample injector or a sample tray outside the clamping device that can accommodate the sample to be detected.

[0017] In a specific embodiment, the PCR amplification area 2 may use a static fixed amplification PCR device or a continuous flow PCR device, preferably a CFPCR device, and more preferably a pipeline CFPCR device.

[0018] The function of the liquid divider 5 is to achieve proportional mixing of the electrophoresis analysis reagent and the PCR amplification solution.

[0019] The temperature control zone 18 is used to denature the double-stranded DNA into single-stranded DNA to facilitate subsequent capillary electrophoresis analysis.

[0020] In a specific embodiment, the capillary 17 can be a commercially available product, such as an elastic fused silica capillary produced by PolymicroTechnologies PT, which is generally drawn from an artificial fused silica semi-finished product. Similarly, natural hot quartz, borosilicate glass, a variety of plastics or molten silicon material synthetic substances can also be used to manufacture, which mainly depends on the actual application requirements. Further, the capillary 17 is preferably bent as a carrier for electrophoretic separation to form a smooth arc to achieve the best effect of electrophoretic separation. Of course, the capillary 17 can also be arranged in other ways, such as directly in a straight line. In addition, in a preferred embodiment, only one capillary 17 is arranged inside a capillary assembly, but according to actual needs, such as in order to improve the analysis throughput or analysis efficiency, multiple capillaries 17 can also be arranged in parallel in the form of a capillary bundle, such as 4, 8, 16, 24 or 32 capillaries 17 are arranged in the capillary assembly in the form of an array, and this is not specifically limited in the present invention.

[0021] In a preferred embodiment, the capillary temperature control unit 16 includes a heating element, a temperature sensor, a heat preservation element, etc., wherein the temperature sensor is used to provide real-time feedback of the temperature of the capillary 17 .

[0022] The detection window 15 and the optical detection component are used to implement detection and analysis of capillary electrophoresis.

[0023] The overflow plastic bag 12 functions to disperse the pressure in the anode cell 10 during the glue filling process.

[0024] The plastic box 14, the electrophoresis analysis reagent containing device 20 and the buffer containing device 9 are all connected to a power device that can transport the contents therein to the outside. The power device includes a device that can provide push-pull force or suction force by manpower, electricity, etc. For example, a piezoelectric pump 8 can be provided between the cathode pool 7 and the buffer containing device 9, between the plastic box 14 and the anode pool 10, and between the electrophoresis analysis reagent containing device 20 and the liquid divider 5, and the piezoelectric pump 8 provides power for the transportation of each liquid; or, the buffer containing device 9, the plastic box 14 and the electrophoresis analysis reagent containing device 20 are all connected to a plunger rod, and the plunger rod squeezes out each liquid after being squeezed by an external force; or a piezoelectric pump and a plunger rod can be used in combination in different devices.

[0025] In a preferred embodiment, the clamping device integrating PCR amplification and capillary electrophoresis analysis of the present invention comprises a sample inlet 19 to be detected, a PCR amplification area 2, a liquid suction and delivery device 3, a liquid divider 5, a temperature control area 18 and a capillary electrophoresis system 21 which are sequentially connected and fluidically connected, wherein

[0026] The liquid suction and delivery device 3 is also connected to the water containing device 4 and the waste liquid bag 13 respectively;

[0027] The liquid divider 5 is also connected to the electrophoresis analysis reagent containing device 20;

[0028] The capillary electrophoresis system 21 comprises a cathode pool 7, a capillary assembly, an anode pool 10 and a plastic box 14 which are sequentially connected and flow-connected; the capillary assembly comprises a capillary 17, a capillary temperature control unit 16 arranged around the outer wall of the capillary 17 and a detection window 15 arranged on the outer wall of the capillary 17, and an optical detection assembly connected thereto is arranged at the detection window 15; the cathode pool 7 is also connected to the temperature control area 18 and the buffer containing device 9 respectively, and two cathode electrodes 6 are arranged on the cathode pool 7; the anode pool 10 is also connected to the overflow plastic bag 12, and an anode electrode 11 is arranged on the anode pool 10;

[0029] The plastic box 14, the electrophoresis analysis reagent containing device 20 and the buffer containing device 9 are all connected with a power device that can transport the contents therein to the outside.

[0030] In a preferred embodiment, the liquid suction and delivery device 3 includes a pump valve integrated part 311 and a rotary dispensing valve 312;

[0031] The pump-valve integrated part 311 includes a rotary valve for controlling whether it is connected to the atmosphere and whether it is connected to the water containing device 4, and a plunger pump connected to the air path of the rotary valve;

[0032] The rotary dispensing valve 312 has four holes in four directions, the first hole is connected to the waste liquid bag 13, the second hole is connected to the liquid divider 5, the third hole is connected to the PCR amplification area 2, and the fourth hole is connected to the water containing device 4 through the pump-valve integrated part 311; the rotary dispensing valve 312 includes two flow channels connected to each other at the valve core, wherein the first flow channel is fixedly connected to the fourth hole, and the second flow channel is connected to one of the first hole, the second hole and the third hole through the rotation switching of the valve core.

[0033] In a specific embodiment, the rotation of the valve core of the rotary dispensing valve 312 is driven by its own power device (such as a stepper motor), thereby achieving communication between the second flow path channel and different holes.

[0034] In a preferred embodiment, the liquid suction and delivery device 3 includes a first column valve 321, a second column valve 322, a suction device 323, a first one-way valve 324 and a second one-way valve 325, wherein one side of the first column valve 321 can be switchably connected to the PCR amplification area 2 and the water containing device 4, and the other side is sequentially connected to the first one-way valve 324, the second one-way valve 325 and one side of the second column valve 322 through a flow path, the other side of the second column valve 322 can be switchably connected to the waste liquid bag 13 and the liquid divider 5, and a suction device 323 is connected between the first one-way valve 324 and the second one-way valve 325.

[0035] In a specific embodiment, the suction device 323 can be a small syringe. In the clamping device in this embodiment, since the liquid suction and delivery device 3 in the clamp is composed of passive devices (i.e., devices that do not require electricity), the manufacturing cost is lower, which further reduces the manufacturing cost of the clamping device of the present invention.

[0036] The second aspect of the present invention relates to a detection method integrating PCR amplification and capillary electrophoresis analysis, the method is performed using the clamping device as described in the first aspect of the present invention, and the method comprises the following steps:

[0037] 1) Filling the capillary electrophoresis system 21 with glue;

[0038] 2) Under the action of the liquid suction and delivery device 3, the sample to be tested enters the PCR amplification area 2 from the sample to be tested inlet 19 for PCR amplification;

[0039] 3) Under the action of the liquid divider 5, the PCR amplification solution and the electrophoresis analysis reagent are mixed in proportion and then enter the temperature control area 18 for heating and denaturation;

[0040] 4) After denaturation, the mixed solution enters the capillary electrophoresis system 21 for capillary electrophoresis analysis.

[0041] In a specific embodiment, the denatured mixed solution first enters the cathode pool 7 in the capillary electrophoresis system 21 and completes the timed injection of the mixed solution through the feedback of the two cathode electrodes 6; then, under the joint action of the anode electrode 11 and the cathode electrode 6 in the capillary electrophoresis system 21, the mixed solution enters the capillary 17 for separation. The timed injection is achieved in the following manner: the capillary electrophoresis system 21 controls the time for applying the high voltage electric field to the anode and the cathode to achieve the timed injection of the sample. Due to the charged polarity of the sample, the sample moves in a directional manner under the action of the electric field, thereby achieving the timed injection.

[0042] In a preferred embodiment, step 1) includes the following steps: injecting the gel from the gel box 14 into the anode pool 10 under power drive, and then completing the gel filling process of the capillary 17 by quickly squeezing the gel in the anode pool 10, wherein the gel overflowing from the anode pool 10 enters the overflow gel bag 12, and the gel overflowing from the capillary 17 enters the waste liquid bag 13, and then under power drive, the buffer in the buffer containing device 9 flushes the gel overflowing into the cathode pool 7 into the waste liquid bag 13 and ensures that the cathode pool 7 is filled with buffer through feedback from the two cathode electrodes 6.

[0043] In a preferred embodiment, after step 4), the following steps are also included:

[0044] 5) Under the action of the liquid suction and delivery device 3, the water in the water containing device 4 pushes the uninjected denatured mixed liquid into the waste liquid bag 13;

[0045] 6) The cathode cell 7 is filled with the buffer solution in the buffer solution containing device 9 under power driving;

[0046] 7) Under the action of the liquid suction and conveying device 3, the water in the water containing device 4 cleans the liquid suction and conveying device 3 and the pipeline, and pushes the cleaned waste liquid into the waste liquid bag 13 to prepare for the next detection.

[0047] Optionally, between steps 1) and 2), a step a) is further included in which the front section of the PCR amplified solution is pushed into the waste liquid bag 13 under the action of the liquid suction and delivery device 3; and / or

[0048] Optionally, between steps 6) and 7), the method further includes the step b) of pushing the remaining post-PCR amplification solution into the waste liquid bag 13 under the action of the liquid suction and delivery device 3 .

[0049] In a preferred embodiment, the method is performed using the clamping device as described in the first aspect of the present invention, and the method comprises the following steps:

[0050] 1) Glue filling: The gel is injected into the anode pool 10 from the gel box 14 under power drive, and then the gel filling process of the capillary 17 is completed by quickly squeezing the gel in the anode pool 10, wherein the gel overflowing from the anode pool 10 enters the overflow gel bag 12, and the gel overflowing from the capillary 17 enters the waste liquid bag 13, and then the buffer in the buffer storage device 9 is driven by power to flush the gel overflowing into the cathode pool 7 into the waste liquid bag 13 and ensure that the cathode pool 7 is filled with buffer through the feedback of the two cathode electrodes 6. According to the actual situation, the glue filling process can be completed once or repeatedly completed. Preferably, the glue filling process is repeated multiple times. In the case of multiple glue filling, the cathode pool 7 can be rinsed with buffer after each glue filling, or the cathode pool 7 can be rinsed with buffer only after the last or last two glue fillings; in addition, glue filling and flushing can be performed successively or simultaneously. Preferably, the piezoelectric pump 8 provides power to pump out the buffer in the buffer storage device 9. Wherein, the buffer can be a solution containing an acid-base stabilizing system during electrophoresis, and common nucleic acid electrophoresis buffers include TAE, TBE, TPE, Tris-glycine and MOPS, etc., preferably TBE, more preferably 0.5XTBE buffer, which contains 0.045mol / L Tris-boric acid and 0.001mol / LEDTA (pH 8.0). Wherein, the gel used in the capillary electrophoresis analysis can be a gel commonly used in the art for analyzing products after PCR amplification, such as polyacrylamide gel, currently commonly used POP-7 and POP-4, preferably POP-7, which contains the following substances: 7% non-crossed linear polyacrylamide molecules, 8M urea (for keeping the DNA molecules in the capillary in a continuously denatured state), 5% 2-pyrrolidone, 10mM N-Tris-(hydroxymethyl)methyl-3-acidic sulfate-aminopropanol (TAPS), and the pH is adjusted to 8.0 with NaOH.

[0051] 2) Under the action of the liquid suction and delivery device 3, the sample to be detected enters the PCR amplification area 2 from the sample to be detected inlet 19 for PCR amplification.

[0052] 3) Optionally, under the action of the liquid suction and delivery device 3, the front section of the solution after PCR amplification is pushed into the waste liquid bag 13.

[0053] 4) Under the action of the liquid divider 5, the PCR amplified solution and the electrophoresis analysis reagent are mixed in proportion and enter the temperature control area 18 for heating denaturation, and then the denatured mixed solution enters the cathode pool 7 and the timed injection of the mixed solution is completed through the feedback of the two cathode electrodes 6. In a preferred embodiment, the sample to be detected is first degassed by negative pressure through the negative pressure degassing buffer device 1, and then enters the PCR amplification area 2 for PCR amplification. The sample to be detected includes a PCR amplification sample (which includes nucleic acids, enzymes and primers, etc.), and may also include a cleaning liquid cathode electrode and / or an isolation liquid; in the case of including a cleaning liquid and / or an isolation liquid, the PCR amplification sample and the cleaning liquid and / or the isolation liquid sequentially enter the PCR amplification area 2. The electrophoresis analysis reagent includes a reference reagent and highly deionized formamide (HIDI), and the reference reagent is a solution containing standard molecular weight DNA. In a specific embodiment, the volume of the PCR amplified sample in the sample to be tested each time is 1 μL-60 μL, preferably 2 μL-40 μL, more preferably 5 μL-30 μL, more preferably 10 μL-25 μL. In a specific embodiment, the volume of the PCR amplified solution entering the liquid divider 5 is 0.5 μL-5 μL, more preferably 0.8 μL-2 μL, more preferably 1 μL-1.5 μL.

[0054] 5) Under the joint action of the anode electrode 11 and the cathode electrode 6 in the capillary electrophoresis system 21, the mixed solution enters the capillary electrophoresis system 21 for separation. During the separation process, the capillary temperature control unit 16 provides a reliable and stable temperature environment for the capillary 17, and the entire separation process is detected at the detection window 15 by the laser induced fluorescence system in the optical detection component.

[0055] 6) Under the action of the liquid suction and delivery device 3, the water in the water containing device 4 pushes the uninjected denatured mixed liquid into the waste liquid bag 13.

[0056] 7) The buffer solution in the buffer solution holding device 9 is driven by power to fill the cathode tank 7.

[0057] 8) Optionally, under the action of the liquid suction and delivery device 3, the remaining PCR amplification solution is pushed into the waste liquid bag 13.

[0058] 9) Under the action of the liquid suction and conveying device 3, the water in the water containing device 4 cleans the liquid suction and conveying device 3 and the pipeline, and pushes the cleaned waste liquid into the waste liquid bag 13 to prepare for the next detection.

[0059] In a preferred embodiment, the method is performed using a clamping device as described in the first aspect of the present invention, wherein the liquid suction and delivery device 3 includes a pump-valve integrated part 311 and a rotary dispensing valve 312, and the method comprises the following steps:

[0060] 1) Glue filling: The gel is injected from the gel box 14 into the anode cell 10 under power drive, and then the gel filling process of the capillary 17 is completed by quickly squeezing the gel in the anode cell 10, wherein the gel overflowing from the anode cell 10 enters the overflow gel bag 12, and the gel overflowing from the capillary 17 enters the waste liquid bag 13, and then under power drive, the buffer in the buffer storage device 9 flushes the gel overflowing from the cathode cell 7 into the waste liquid bag 13 and ensures that the cathode cell 7 is filled with buffer through feedback from the two cathode electrodes 6.

[0061] 2) Switch the second flow path channel in the rotary dispensing valve 312 to connect with the third hole. Under the suction action of the pump-valve integrated part 311, the sample to be tested enters the PCR amplification area 2 from the sample inlet 19 for PCR amplification, and then the solution after PCR amplification enters the second flow path channel.

[0062] 3) Optionally, the second flow path is switched to be connected to the first hole, and the front section of the post-PCR amplification solution is pushed into the waste liquid bag 13 under the thrust of the pump-valve integrated part 311 .

[0063] 4) Switch the second flow path channel to connect with the second hole. Under the thrust of the pump-valve integrated part 311 and through the action of the liquid divider 5, the PCR amplification solution and the electrophoresis analysis reagent are mixed in proportion and enter the temperature control area 18 for heating denaturation. Then, the denatured mixed solution enters the cathode pool 7 and the timed injection of the mixed solution is completed through the feedback of the two cathode electrodes 6.

[0064] 5) Under the joint action of the anode electrode 11 and the cathode electrode 6 in the capillary electrophoresis system 21 , the mixed solution enters the capillary electrophoresis system 21 for separation.

[0065] 6) Under the action of the pump-valve integrated part 311 , the water in the water containing device 4 pushes the uninjected mixed liquid into the waste liquid bag 13 .

[0066] 7) The buffer solution in the buffer solution holding device 9 is driven by power to fill the cathode tank 7.

[0067] 8) Optionally, the second flow path channel is switched to be connected to the third hole and the first hole in sequence, and under the action of the pump-valve integrated part 311, the remaining PCR amplification solution is pushed into the waste liquid bag 13.

[0068] 9) Switch the second flow path in the rotary dispensing valve 312 to connect with the first hole. Under the action of the pump-valve integrated part 311, the water in the water containing device 4 cleans the liquid suction and conveying device 3 and the pipeline, and pushes the cleaned waste liquid into the waste liquid bag 13 to prepare for the next detection.

[0069] In a preferred embodiment, the method is performed using a clamping device as described in the first aspect of the present invention, wherein the liquid suction delivery device 3 includes a first column valve 321, a second column valve 322, a suction device 323, a first check valve 324 and a second check valve 325, and the method comprises the following steps:

[0070] 1) Glue filling: The gel is injected from the gel box 14 into the anode cell 10 under power drive, and then the gel filling process of the capillary 17 is completed by quickly squeezing the gel in the anode cell 10, wherein the gel overflowing from the anode cell 10 enters the overflow gel bag 12, and the gel overflowing from the capillary 17 enters the waste liquid bag 13, and then under power drive, the buffer in the buffer storage device 9 flushes the gel overflowing from the cathode cell 7 into the waste liquid bag 13 and ensures that the cathode cell 7 is filled with buffer through feedback from the two cathode electrodes 6.

[0071] 2) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the PCR amplification area 2. Under the suction action of the suction device 323, the sample to be detected enters the PCR amplification area 2 from the sample inlet 19 to be detected for PCR amplification, and then the solution after PCR amplification enters the suction device 323.

[0072] 3) Optionally, close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the waste liquid bag 13, and under the thrust of the suction device 323, push the front part of the solution after PCR amplification into the waste liquid bag 13.

[0073] 4) Close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side of it is connected to the liquid divider 5. Under the thrust of the suction device 323 and through the action of the liquid divider 5, the PCR amplification solution and the electrophoresis analysis reagent are mixed in proportion and enter the temperature control area 18 for heating denaturation. Then, the denatured mixed solution enters the cathode pool 7 and the timed injection of the mixed solution is completed through the feedback of the two cathode electrodes 6.

[0074] 5) Under the joint action of the anode electrode 11 and the cathode electrode 6 in the capillary electrophoresis system 21 , the mixed solution enters the capillary electrophoresis system 21 for separation.

[0075] 6) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the water containing device 4, and under the suction action of the suction device 323, the water in the water containing device 4 enters the suction device 323; close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the liquid divider 5, and under the thrust action of the suction device 323, the unsampled mixed liquid is pushed into the waste liquid bag 13.

[0076] 7) The buffer solution in the buffer solution holding device 9 is driven by power to fill the cathode tank 7.

[0077] 8) Optionally, open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the PCR amplification area 2, and under the suction action of the suction device 323, the post-PCR amplification solution enters the suction device 323; close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the waste liquid bag 13, and under the thrust action of the suction device 323, the remaining post-PCR amplification solution is pushed into the waste liquid bag 13.

[0078] 9) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the water containing device 4, and under the suction action of the suction device 323, the water in the water containing device 4 enters the suction device 323; close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the waste liquid bag 13, and under the thrust action of the suction device 323, the water cleans the components and pipes flowing through, and pushes the cleaned waste liquid into the waste liquid bag 13, in preparation for the next detection.

[0079] In the specific operation process, step 5) and step 6) in all the above methods must be performed in sequence, and step b) (if this step is required) and step 7) must be performed in sequence. In a specific implementation scheme, step 4), step 5), step 6), step b) and step 7) can be performed successively, or step 5), step 6), step b) and step 7) can be performed in sequence while step 4), or step b), step 7), step 6) and step 6) can be performed in sequence, or step 5) and step 6) can be performed first, and / or step b) and step 7) before step 4). Preferably, step 5), step 6), step b) and step 7) are performed in sequence while step 4). Preferably, in the case of continuous detection, step 1) of the next detection can also be performed simultaneously with one or more of steps 5), step 6), step b) and step 7) of the previous detection to improve the detection efficiency of the equipment.

[0080] In a preferred embodiment, the sample to be detected is a layered liquid mixture comprising the following solutions in sequence: a first dilute washing solution, a first isolation solution, a PCR amplification sample, a second isolation solution, a concentrated washing solution, a third isolation solution, and a second dilute washing solution, wherein

[0081] The first spacer liquid, the second spacer liquid and the third spacer liquid are independently selected from one or more mineral oils, preferably mineral oil with a kinematic viscosity of 10-14 centistokes, which is a colorless translucent liquid, insoluble in water and ethanol, and miscible with ether, petroleum ether and oil-soluble dyes.

[0082] The first dilute washing liquid and the second dilute washing liquid are independently selected from ethanol solution or deionized water. The ethanol solution is preferably 95% ethanol, preferably AR grade ethanol; the deionized water is preferably double distilled water.

[0083] The concentrated washing solution is a strong DNA oxidizing solution, preferably a sodium hypochlorite solution, preferably a sodium hypochlorite solution with a mass fraction of 5%-10%.

[0084] The PCR amplification sample contains template, enzyme (such as Taq enzyme), primer, dNTP (deoxyribonucleoside triphosphate) and buffer (such as Mg-containing 2+ , K + The template, enzyme, primer and buffer used therein include those commonly used in current PCR amplification and detection, and the primers therein are labeled with fluorescent groups.

[0085] In this embodiment, steps a) and b) in all the above detection method implementation schemes are performed, that is, during the detection process, only the PCR amplification sample liquid segment in the layered liquid mixture is subjected to subsequent capillary electrophoresis analysis, and the liquid segments before and after the PCR amplification sample liquid segment are both entered into the waste liquid bag 13 by the action of the liquid suction and delivery device 3, and the PCR amplification area 2 and the flow-through elements and pipelines are cleaned at the same time.

[0086] In a preferred embodiment, the detection method is performed 200-500 times in a continuous manner in the same clamping device. Preferably, the detection method is performed 200-400 times in a continuous manner in the same clamping device, more preferably 200-300 times, and further preferably 200 times.

[0087] The present invention has the following advantages:

[0088] 1) The clamp device integrating PCR amplification and capillary electrophoresis analysis of the present invention is an integrated pipeline closed clamp, so the contamination source of high-concentration PCR products is effectively controlled, avoiding cross contamination between laboratory tests;

[0089] 2) The clamp device of the present invention also has the reagents for capillary electrophoresis analysis built in, so it can support multiple repeated tests, fully ensuring the number of continuous tests on the clamp, reducing the cost of a single test, and increasing the possibility of clinical promotion;

[0090] 3) The clamping device of the present invention can support flexible insertion of multiple clamps on one instrument at the same time, thereby improving the test throughput of the entire instrument;

[0091] 4) The clamping device and method integrating PCR amplification and capillary electrophoresis analysis of the present invention eliminates the manual intervention between PCR amplification and capillary electrophoresis analysis, thereby improving the test efficiency;

[0092] 5) The clamping device of the present invention adopts single-channel capillary electrophoresis analysis, which effectively avoids signal interference between test batches during the current multi-channel capillary electrophoresis analysis and detection, and greatly improves the sensitivity and resolution of the instrument;

[0093] 6) The present invention proposes for the first time to perform negative pressure degassing of the sample to be tested before PCR amplification, which effectively avoids the situation in which the liquid segments in the sample to be tested are segmented due to bubbles precipitated at high temperature during the denaturation process, affecting the amplification efficiency, and can also allow the subsequent PCR amplification area 2 to accommodate more samples to be tested, greatly improving the amplification efficiency of the instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 It is a schematic diagram of the clamping device integrating PCR amplification and capillary electrophoresis analysis of the present invention.

[0095] Figure 2 This is a schematic diagram of the clamping device integrating PCR amplification and capillary electrophoresis analysis described in Example 1 of the present invention.

[0096] Figure 3 Schematic diagram of the clamping device integrating PCR amplification and capillary electrophoresis analysis described in Example 3 of the present invention.

[0097] The reference numerals have the following meanings:

[0098] 1-negative pressure degassing buffer device, 2-PCR amplification area, 3-liquid suction and delivery device, 311-pump valve integrated part, 312-rotary dispensing valve, 321-first column valve, 322-second column valve, 323-suction device, 324-first one-way valve, 325-second one-way valve, 4-water containing device, 5-liquid divider, 6-cathode electrode, 7-cathode pool, 8-piezoelectric pump, 9-buffer containing device, 10-anode pool, 11-anode electrode, 12-overflow plastic bag, 13-waste liquid bag, 14-plastic box, 15-detection window, 16-capillary temperature control unit, 17-capillary, 18-temperature control zone, 19-sample inlet to be detected, 20-electrophoresis analysis reagent containing device, 21-capillary electrophoresis system. DETAILED DESCRIPTION

[0099] The present invention is further described in detail below in conjunction with examples and drawings, but it should be understood that the examples are merely illustrative rather than restrictive.

[0100] Example 1

[0101] like Figure 2 The clamping device shown integrates PCR amplification and capillary electrophoresis analysis, which includes a sample inlet 19 to be tested, a negative pressure degassing buffer device 1, a PCR amplification area 2, a liquid suction and delivery device 3, a liquid divider 5, a temperature control area 18 and a capillary electrophoresis system 21, which are sequentially connected and flow-connected.

[0102] The liquid suction and delivery device 3 includes a pump-valve integrated part 311 and a rotary dispensing valve 312; wherein the pump-valve integrated part 311 includes a rotary valve for controlling whether it is connected to the atmosphere and whether it is connected to the water containing device 4, and a plunger pump connected to the air path of the rotary valve; the rotary dispensing valve 312 has four holes in four directions, the first hole is connected to the waste liquid bag 13, the second hole is connected to the liquid divider 5, the third hole is connected to the PCR amplification area 2, and the fourth hole is connected to the water containing device 4 through the pump-valve integrated part 311; the rotary dispensing valve 312 includes two flow channels connected at the valve core, wherein the first flow channel is fixedly connected to the fourth hole, and the second flow channel is connected to one of the first hole, the second hole and the third hole through the rotation switching of the valve core; the liquid suction and delivery device 3 is also connected to the water containing device 4 and the waste liquid bag 13 respectively;

[0103] The capillary electrophoresis system 21 comprises a cathode pool 7, a capillary assembly, an anode pool 10 and a plastic box 14 which are sequentially connected and flow-connected; the capillary assembly comprises a capillary 17, a capillary temperature control unit 16 arranged around the outer wall of the capillary 17 and a detection window 15 arranged on the outer wall of the capillary 17, and an optical detection component is arranged at the detection window 15; the cathode pool 7 is also connected to the temperature control area 18 and the buffer storage device 9 respectively, and two cathode electrodes 6 are arranged on the cathode pool 7; the anode pool 10 is also connected to the overflow plastic bag 12, and an anode electrode 11 is arranged on the anode pool 10;

[0104] The plastic box 14, the electrophoresis analysis reagent containing device 20 and the buffer containing device 9 are all connected with a power device that can transport the contents therein to the outside.

[0105] The sample inlet 19 to be tested is connected to a sample injector outside the clamping device.

[0106] The PCR amplification area 2 uses a continuous flow PCR device.

[0107] The capillary 17 can be an elastic fused silica capillary produced by Polymicro Technologies PT. The capillary 17 is directly arranged in a straight line and only one capillary 17 is arranged inside a capillary assembly. The capillary temperature control unit 16 includes a heating element, a temperature sensor, a heat preservation element, etc., wherein the temperature sensor is used to feedback the temperature of the capillary 17 in real time.

[0108] The power device is a piezoelectric pump 8 disposed between the cathode pool 7 and the buffer solution containing device 9, and the plastic box 14 and the electrophoresis analysis reagent containing device 20 are both connected with a plunger rod, and the plunger rod squeezes out each liquid after being squeezed by an external force.

[0109] Example 2

[0110] A method for detecting using the clamp device integrating PCR amplification and capillary electrophoresis analysis as described in Example 1, the method comprising the following steps:

[0111] 1) Glue filling: The gel is injected into the anode pool 10 from the gel box 14 under power drive, and then the gel filling process of the capillary 17 is completed by quickly squeezing the gel in the anode pool 10, wherein the gel overflowing from the anode pool 10 enters the overflow gel bag 12, and the gel overflowing from the capillary 17 enters the waste liquid bag 13, and then the buffer in the buffer storage device 9 is driven by power to flush the gel overflowing into the cathode pool 7 into the waste liquid bag 13 and ensure that the cathode pool 7 is filled with buffer through feedback from the two cathode electrodes 6. The glue filling process is repeated for multiple times and the cathode pool 7 is rinsed with buffer after each glue filling. In addition, the glue filling and rinsing are performed successively. The buffer used is 0.5XTBE buffer, which contains 0.045mol / L Tris-boric acid and 0.001mol / L EDTA (pH 8.0), and the gel used is POP-7.

[0112] 2) Switch the second flow path channel in the rotary dispensing valve 312 to connect with the third hole. Under the suction action of the pump-valve integrated part 311, the sample to be tested enters the negative pressure degassing buffer device 1 from the sample inlet 19 for negative pressure degassing, and then enters the PCR amplification area 2 for PCR amplification. After that, the solution after PCR amplification enters the second flow path channel.

[0113] 3) The second flow path is switched to be connected to the first hole, and the front part of the solution after PCR amplification is pushed into the waste liquid bag 13 under the thrust of the pump-valve integrated part 311 .

[0114] 4) Switch the second flow path channel to connect with the second hole. Under the thrust of the pump-valve integrated part 311 and through the action of the liquid divider 5, the PCR amplification solution and the electrophoresis analysis reagent are mixed in proportion and enter the temperature control area 18 for heating denaturation. Then, the denatured mixed solution enters the cathode pool 7 and the timed injection of the mixed solution is completed through the feedback of the two cathode electrodes 6.

[0115] The sample to be detected is a layered liquid mixture including the following solutions in sequence: a first dilute washing solution, an isolation solution, a PCR amplification sample, an isolation solution, a concentrated washing solution, an isolation solution, and a second dilute washing solution, wherein the isolation solution is a mineral oil with a kinematic viscosity of 10-14 centistokes, the first dilute washing solution is double distilled water, the second dilute washing solution is 95% ethanol, and the concentrated washing solution is a 5% sodium hypochlorite solution. The PCR amplification sample contains a template, Taq enzyme, primers, dNTPs, and a 100mM MgCl2 solution. The volume of the PCR amplification sample in the sample to be detected each time is 20μL, and the volume of the PCR amplification solution entering the liquid divider 5 is 1μL.

[0116] 5) Under the joint action of the anode electrode 11 and the cathode electrode 6 in the capillary electrophoresis system 21, the mixed solution enters the capillary electrophoresis system 21 for separation. During the separation process, the capillary temperature control unit 16 provides a reliable and stable temperature environment for the capillary 17, and the entire separation process is detected at the detection window 15 by the laser induced fluorescence system in the optical detection component.

[0117] 6) Under the action of the pump-valve integrated part 311 , the water in the water containing device 4 pushes the uninjected mixed liquid into the waste liquid bag 13 .

[0118] 7) The buffer solution in the buffer solution holding device 9 is driven by power to fill the cathode tank 7.

[0119] 8) The second flow path is switched to be connected to the third hole and the first hole in sequence, and under the action of the pump-valve integrated part 311 , the remaining PCR amplification solution is pushed into the waste liquid bag 13 .

[0120] 9) Switch the second flow path in the rotary dispensing valve 312 to connect with the first hole. Under the action of the pump-valve integrated part 311, the water in the water containing device 4 cleans the liquid suction and conveying device 3 and the pipeline, and pushes the cleaned waste liquid into the waste liquid bag 13 to prepare for the next detection.

[0121] The above detection method performs step 6), step 7), step 8) and step 9) in sequence while performing step 5), and the above detection method is performed continuously 200 times in the same clamping device before replacing the consumable clamping device in the equipment.

[0122] Example 3

[0123] like Figure 3 The clamping device shown integrates PCR amplification and capillary electrophoresis analysis, which includes a sample inlet 19 to be tested, a negative pressure degassing buffer device 1, a PCR amplification area 2, a liquid suction and delivery device 3, a liquid divider 5, a temperature control area 18 and a capillary electrophoresis system 21, which are sequentially connected and flow-connected.

[0124] The liquid suction and delivery device 3 includes a first column valve 321, a second column valve 322, a suction device 323, a first check valve 324 and a second check valve 325, wherein one side of the first column valve 321 can be switchably connected to the PCR amplification area 2 and the water containing device 4, and the other side is sequentially connected to the first check valve 324, the second check valve 325 and one side of the second column valve 322 through a flow path, and the other side of the second column valve 322 can be switchably connected to the waste liquid bag 13 and the liquid divider 5, and the suction device 323 is connected between the first check valve 324 and the second check valve 325. The suction device 323 is a small syringe.

[0125] The liquid divider 5 is also connected to the electrophoresis analysis reagent containing device 20;

[0126] The capillary electrophoresis system 21 comprises a cathode pool 7, a capillary assembly, an anode pool 10 and a plastic box 14 which are sequentially connected and flow-connected; the capillary assembly comprises a capillary 17, a capillary temperature control unit 16 arranged around the outer wall of the capillary 17 and a detection window 15 arranged on the outer wall of the capillary 17, and an optical detection component is arranged at the detection window 15; the cathode pool 7 is also connected to the temperature control area 18 and the buffer storage device 9 respectively, and two cathode electrodes 6 are arranged on the cathode pool 7; the anode pool 10 is also connected to the overflow plastic bag 12, and an anode electrode 11 is arranged on the anode pool 10;

[0127] The plastic box 14, the electrophoresis analysis reagent containing device 20 and the buffer containing device 9 are all connected with a power device that can transport the contents therein to the outside.

[0128] The sample inlet 19 to be detected is connected to a sample tray outside the clamping device.

[0129] The PCR amplification area 2 uses a continuous flow PCR device.

[0130] The capillary 17 can be an elastic fused silica capillary produced by Polymicro Technologies PT. The capillary 17 is directly arranged in a straight line and only one capillary 17 is arranged inside a capillary assembly. The capillary temperature control unit 16 includes a heating element, a temperature sensor, a heat preservation element, etc., wherein the temperature sensor is used to feedback the temperature of the capillary 17 in real time.

[0131] The power device is a piezoelectric pump 8 disposed between the cathode pool 7 and the buffer solution containing device 9, and the plastic box 14 and the electrophoresis analysis reagent containing device 20 are both connected with a plunger rod, and the plunger rod squeezes out each liquid after being squeezed by an external force.

[0132] Example 4

[0133] A method for detecting using the clamp device integrating PCR amplification and capillary electrophoresis analysis as described in Example 3, the method comprising the following steps:

[0134] 1) Glue filling: The gel is injected into the anode pool 10 from the gel box 14 under power drive, and then the gel filling process of the capillary 17 is completed by quickly squeezing the gel in the anode pool 10, wherein the gel overflowing from the anode pool 10 enters the overflow gel bag 12, and the gel overflowing from the capillary 17 enters the waste liquid bag 13, and then the buffer in the buffer storage device 9 is driven by power to flush the gel overflowing into the cathode pool 7 into the waste liquid bag 13 and ensure that the cathode pool 7 is filled with buffer through feedback from the two cathode electrodes 6. The glue filling process is repeated for multiple times and the cathode pool 7 is rinsed with buffer after each glue filling. In addition, the glue filling and rinsing are performed successively. The buffer used is 0.5XTBE buffer, which contains 0.045mol / L Tris-boric acid and 0.001mol / L EDTA (pH 8.0), and the gel used is POP-7.

[0135] 2) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the PCR amplification area 2. Under the suction action of the suction device 323, the sample to be tested enters the negative pressure degassing buffer device 1 from the sample inlet 19 to be tested for negative pressure degassing, and then enters the PCR amplification area 2 for PCR amplification. After that, the solution after PCR amplification enters the suction device 323.

[0136] 3) Close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the waste liquid bag 13, and under the thrust of the suction device 323, push the front part of the solution after PCR amplification into the waste liquid bag 13.

[0137] 4) Close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side of it is connected to the liquid divider 5. Under the thrust of the suction device 323 and through the action of the liquid divider 5, the PCR amplification solution and the electrophoresis analysis reagent are mixed in proportion and enter the temperature control area 18 for heating denaturation. Then, the denatured mixed solution enters the cathode pool 7 and the timed injection of the mixed solution is completed through the feedback of the two cathode electrodes 6.

[0138] The sample to be detected is a layered liquid mixture including the following solutions in sequence: a first dilute washing solution, an isolation solution, a PCR amplification sample, an isolation solution, a concentrated washing solution, an isolation solution, and a second dilute washing solution, wherein the isolation solution is a mineral oil with a kinematic viscosity of 10-14 centistokes, the first dilute washing solution is 95% ethanol and the second dilute washing solution is double distilled water, the concentrated washing solution is a 10% sodium hypochlorite solution, and the PCR amplification sample contains a template, Taq enzyme, primers, dNTPs and a 100mM KCl solution. The volume of the PCR amplification sample in the sample to be detected each time is 15μL, and the volume of the PCR amplification solution entering the liquid divider 5 is 1.2μL.

[0139] 5) Under the joint action of the anode electrode 11 and the cathode electrode 6 in the capillary electrophoresis system 21, the mixed solution enters the capillary electrophoresis system 21 for separation. During the separation process, the capillary temperature control unit 16 provides a reliable and stable temperature environment for the capillary 17, and the entire separation process is detected at the detection window 15 by the laser induced fluorescence system in the optical detection component.

[0140] 6) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the water containing device 4, and under the suction action of the suction device 323, the water in the water containing device 4 enters the suction device 323; close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the liquid divider 5, and under the thrust action of the suction device 323, the unsampled mixed liquid is pushed into the waste liquid bag 13.

[0141] 7) The buffer solution in the buffer solution holding device 9 is driven by power to fill the cathode tank 7.

[0142] 8) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the PCR amplification area 2, and under the suction action of the suction device 323, the post-PCR amplification solution enters the suction device 323; close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the waste liquid bag 13, and under the thrust action of the suction device 323, the remaining post-PCR amplification solution is pushed into the waste liquid bag 13.

[0143] 9) Open the first one-way valve 324, close the second one-way valve 325 and switch the first column valve 321 so that one side thereof is connected to the water containing device 4, and under the suction action of the suction device 323, the water in the water containing device 4 enters the suction device 323; close the first one-way valve 324, open the second one-way valve 325 and switch the second column valve 322 so that one side thereof is connected to the waste liquid bag 13, and under the thrust action of the suction device 323, the water cleans the components and pipes flowing through, and pushes the cleaned waste liquid into the waste liquid bag 13, in preparation for the next detection.

[0144] In the above detection method, steps 5), 6), 7), 8) and 9) are performed sequentially and the above detection method is performed continuously 300 times in the same clamping device before replacing the consumable clamping device in the equipment.

[0145] While the present invention has been described with reference to specific embodiments, those skilled in the art will recognize that changes or modifications may be made to the embodiments without departing from the spirit and scope of the present invention, which is defined by the appended claims.

Claims

1. A detection method integrating PCR amplification and capillary electrophoresis analysis, characterized in that: The method is carried out using a clamping device and comprises the following steps: 1) Filling the capillary electrophoresis system (21) with gel; 2) Under the action of the liquid suction and delivery device (3), the sample to be tested enters the PCR amplification area (2) from the sample to be tested inlet (19) to perform PCR amplification; 3) Under the action of the liquid divider (5), the PCR amplification solution and the electrophoresis analysis reagent are mixed in proportion and then enter the temperature control area (18) for heating and denaturation; 4) the denatured mixed solution enters the capillary electrophoresis system (21) for capillary electrophoresis analysis; The sample to be detected is a layered liquid mixture including the following solutions in sequence: a first dilute washing solution, a first isolation solution, a PCR amplification sample, a second isolation solution, a concentrated washing solution, a third isolation solution, and a second dilute washing solution. The first spacer liquid, the second spacer liquid and the third spacer liquid are independently selected from one or more of mineral oils; The first dilute washing liquid and the second dilute washing liquid are independently selected from an ethanol solution or deionized water; The concentrated wash solution is a strong DNA oxidizing solution; The PCR amplification sample contains template, enzyme, primer, dNTP and buffer; The clamping device comprises a sample inlet (19) to be tested, a PCR amplification area (2), a liquid suction and delivery device (3), a liquid divider (5), a temperature control area (18) and a capillary electrophoresis system (21) which are sequentially connected, wherein the liquid divider (5) is also connected to an electrophoresis analysis reagent containing device (20), and the liquid suction and delivery device (3) is also respectively connected to a water containing device (4) and a waste liquid bag (13); A negative pressure degassing buffer device (1) is provided between the sample inlet (19) to be detected and the PCR amplification area (2); The capillary electrophoresis system (21) comprises a cathode pool (7), a capillary assembly, an anode pool (10) and a plastic box (14) which are sequentially connected and flow-connected; the cathode pool (7) is provided with two cathode electrodes (6), and the anode pool (10) is provided with an anode electrode (11); the capillary assembly comprises a capillary (17), a capillary temperature control unit (16) arranged around the outer wall of the capillary (17), and a detection window (15) arranged on the outer wall of the capillary (17); the detection window (15) is provided with an optical detection assembly; The cathode pool (7) is also connected to the temperature control zone (18) and the buffer storage device (9) respectively; the anode pool (10) is also connected to the overflow plastic bag (12).

2. The detection method according to claim 1, characterized in that: The liquid suction and delivery device (3) comprises a pump-valve integrated part (311) and a rotary dispensing valve (312); wherein The pump-valve integrated part (311) comprises a rotary valve for controlling whether it is connected to the atmosphere and whether it is connected to the water containing device (4), and a plunger pump connected to the air path of the rotary valve; The rotary dispensing valve (312) has four holes in four directions, the first hole is connected to the waste liquid bag (13), the second hole is connected to the liquid divider (5), the third hole is connected to the PCR amplification area (2), and the fourth hole is connected to the water containing device (4) through the pump valve integrated part (311); the rotary dispensing valve (312) includes two flow channels connected to each other at the valve core, wherein the first flow channel is fixedly connected to the fourth hole, and the second flow channel is connected to one of the first hole, the second hole and the third hole through the rotation switching of the valve core.

3. The detection method according to claim 1, characterized in that: The liquid suction and delivery device (3) comprises a first column valve (321), a second column valve (322), a suction device (323), a first one-way valve (324) and a second one-way valve (325), wherein one side of the first column valve (321) can be switchably connected to the PCR amplification area (2) and the water containing device (4), and the other side is sequentially connected to the first one-way valve (324), the second one-way valve (325) and one side of the second column valve (322) through a flow path, and the other side of the second column valve (322) can be switchably connected to a waste liquid bag (13) and a liquid divider (5), and a suction device (323) is connected between the first one-way valve (324) and the second one-way valve (325).

4. The detection method according to any one of claims 1 to 3, wherein step 1) comprises the following steps: Under power drive, the gel is injected from the gel box (14) into the anode cell (10), and then the gel in the anode cell (10) is quickly squeezed to complete the gel filling process of the capillary (17), wherein the gel overflowing from the anode cell (10) enters the overflow gel bag (12), and the gel overflowing from the capillary (17) enters the waste liquid bag (13), and then under power drive, the buffer in the buffer storage device (9) flushes the gel overflowing into the cathode cell (7) into the waste liquid bag (13) and ensures that the cathode cell (7) is filled with the buffer through feedback from the two cathode electrodes (6).

5. The detection method according to any one of claims 1 to 3, further comprising the following steps after step 4): 5) Under the action of the liquid suction and delivery device (3), the water in the water containing device (4) pushes the uninjected denatured mixed liquid into the waste liquid bag (13); 6) driving the cathode tank (7) with the buffer solution in the buffer solution containing device (9) under power; 7) Under the action of the liquid suction and delivery device (3), the water in the water containing device (4) cleans the liquid suction and delivery device (3) and the pipeline, and pushes the cleaned waste liquid into the waste liquid bag (13) to prepare for the next test; Between steps 1) and 2), the method further includes the step of pushing the front portion of the solution after PCR amplification into the waste liquid bag (13) under the action of the liquid suction and delivery device (3); and / or Between steps 6) and 7), there is also included a step of pushing the remaining post-PCR amplification solution into a waste liquid bag (13) under the action of the liquid suction and delivery device (3).

6. The detection method according to any one of claims 1 to 3, wherein the first spacer liquid, the second spacer liquid and the third spacer liquid are mineral oils with a kinematic viscosity of 10-14 centistokes.

7. The detection method according to any one of claims 1 to 3, wherein the ethanol solution is 95% ethanol.

8. The detection method according to any one of claims 1 to 3, wherein the deionized water is double distilled water.

9. The detection method according to any one of claims 1 to 3, wherein the concentrated washing solution is a sodium hypochlorite solution.

10. The detection method according to claim 9, wherein the concentrated washing solution is a sodium hypochlorite solution with a mass fraction of 5%-10%.

Images