A method integrating automatic cleaning, PCR amplification and detection

Abstract

The present invention relates to a flow path device and method integrating automatic cleaning, PCR amplification and detection. The flow path device and method of the present invention simplify the PCR detection process, realize automatic flow cleaning through multi-stage washing liquid between different test batches, thereby creating a PCR amplification assembly line operation, greatly improving the test throughput of the platform; and eliminating manual intervention in the cleaning, PCR amplification and detection processes, improving the test efficiency, and having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical devices, and in particular relates to a flow path device and method integrating automatic cleaning, PCR amplification and detection. 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 automatic cleaning, PCR amplification and detection 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 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, on the one hand, a plate reaction system is used, so a single batch of equipment can only complete a specific number of tests, and its test throughput is theoretically restricted by the basic configuration of the test platform; on the other hand, a metal block with a large heat capacity is used for thermal cycle temperature control, and 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 heat capacity of the heater and the heat capacity of the sensor. Therefore, it is necessary to optimize the heat capacity of the equipment system in order to shorten the reaction time and reduce energy consumption. In the CFPCR test, the DNA sample to be amplified and the reaction reagent flow continuously through three constant temperature zones with different temperatures, thereby achieving the purpose of thermal cycle amplification of DNA fragments. Since CFPCR does not require repeated heating or cooling, its heating and cooling speed is generally not limited by the heat capacity of the equipment system, thereby simplifying the temperature control design, greatly improving the accuracy of temperature control in the test platform, reducing the fluctuation of temperature control in the test platform, shortening the time required for amplification, and greatly improving the efficiency of PCR amplification.

[0004] At present, there are two main technologies for implementing CFPCR: chip-type and pipeline-type. Chip-type CFPCR uses micromachining technology to process "winding" or "spiral" microchannels on substrate materials. This method has a high degree of integration and occupies a small space, but it is difficult to make, the technology is complex, the production steps are many, and the cost is high. Pipeline-type CFPCR has a simple structure, low processing cost, and high test efficiency.

[0005] Patent CN108300656A discloses a method for thermal medium flow PCR amplification, in which a reaction container filled with a PCR reaction solution is inserted into or connected to a thermal medium container so that the reaction container can contact or be immersed in the thermal medium. A thermal medium driving device causes thermal media of different temperatures to flow through the thermal medium container, causing the temperature of the reaction container contacting the thermal medium to change. However, it does not achieve true flow, but only transforms the original static heating block into a dynamic heating block. Its flux per unit time still depends on the space of the detection device.

[0006] Patent CN103733074A discloses a system and method for a continuous flow PCR system, in which the liquid handling system of the PCR system is instructed to obtain a matrix of samples and reagents for a PCR experiment, and then complete PCR amplification at a fixed point, but it only achieves flow PCR amplification generated by droplets. In this system, multiple samples are analyzed through multiple microchannels, but only a single test is amplified in parallel to increase the throughput. In addition, multiple channels are detected at the same time, and there will be signal interference between channels during detection, which will eventually affect the detection sensitivity of the instrument. In addition, the system does not mention or realize the function of flow amplification processing that integrates automatic cleaning, PCR amplification and detection throughout the system.

[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.) discloses a continuous flow PCR microfluidic device, but the device does not integrate PCR amplification and detection in one device, and the device can only be used for a single test. The device does not mention or realize the function of automatic cleaning of the entire process, PCR amplification and detection in one flow amplification process.

[0009] In the devices and methods of the prior art, the test throughput of PCR amplification is still limited by the detection space of a single batch, and pipeline processing has not been truly realized, and there has been no innovative innovation in the test throughput. Summary of the invention

[0010] The purpose of the present invention is to provide a flow path device and method that integrates automatic cleaning, PCR amplification and detection, which can highly realize the assembly line operation of sample PCR amplification and greatly improve the test throughput per unit time. The purpose of the present invention is achieved through the following technical solutions:

[0011] The first aspect of the present invention relates to a flow path device integrating automatic cleaning, PCR amplification and detection, which includes a PCR sample storage tray 16, a first washing liquid bottle 15, a sample suction needle 17 located above them and movable freely, and a liquid suction and delivery device 14, wherein the liquid suction and delivery device 14 is provided with a plurality of holes, wherein the first hole is directly connected to the waste liquid bottle 11 to form a first flow path, the second hole is sequentially connected to the PCR amplification area 7, the detection system 8 and the waste liquid bottle 11 to form a detection flow path, and the third hole is connected to the sample suction needle 17.

[0012] In a preferred embodiment, the first washing liquid bottle 15 includes a first dilute washing liquid bottle 151 and a first concentrated washing liquid bottle 152 .

[0013] In a preferred embodiment, the PCR amplification area 7 and the detection system 8 are integrated into one device to form a PCR amplification detection assembly device 18. In this case, the device of the present invention is preferably used for real-time quantitative PCR testing, and the detection system 8 is used to complete the fluorescence signal detection after each cycle extension segment.

[0014] In a preferred embodiment, the PCR amplification zone 7 uses a static fixed amplification PCR device or a CFPCR device, preferably a CFPCR device, and more preferably a pipeline CFPCR device.

[0015] In a preferred embodiment, a negative pressure degassing buffer device 6 is provided upstream of the PCR amplification zone 7. The function of the negative pressure degassing buffer device 6 is to precipitate the gas dissolved in the mixed solution entering therein, so that on the one hand, it can avoid the situation that the solution segmentation affects the amplification efficiency due to the precipitation of bubbles in each liquid segment of the mixed solution during the denaturation process due to high temperature; on the other hand, it can make the subsequent PCR amplification zone 7 accommodate more samples to be amplified, 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.

[0016] In a preferred embodiment, the fourth hole of the liquid suction and delivery device 14 is connected to the second washing liquid bottle, which is sequentially connected to the peristaltic pump 10, the cleaning swab 12 arranged around the outer wall of the sample suction needle 17, the diaphragm pump 13 and the waste liquid bottle 11; the optional fifth hole is connected to the isolation liquid bottle 1. Preferably, the second washing liquid bottle includes a second dilute washing liquid bottle 2 and / or a second concentrated washing liquid bottle 3. The diaphragm pump 13 is used to provide a continuous negative pressure for the flow path so that the washing liquid can flow along the flow path.

[0017] In a preferred embodiment, the liquid suction and delivery device 14 includes a pump-valve integrated part 4 and a rotary dispensing valve 5, wherein the rotary dispensing valve 5 is provided with a plurality of holes, the number of holes being ≥4, wherein the first hole sequentially connects the pump-valve integrated part 4 and the waste liquid bottle 11 to form a first flow path, the second hole sequentially connects the PCR amplification area 7, the detection system 8 and the waste liquid bottle 11 to form a detection flow path, the third hole connects the sample suction needle 17, the fourth hole connects the second washing liquid bottle, and the optional fifth hole connects the isolation liquid bottle 1. The rotary dispensing valve 5 includes two flow channels connected at the valve core, wherein the first flow channel is fixedly connected to the first hole, and the second flow channel is connected to one of the other holes through the rotation switching of the valve core.

[0018] In a specific embodiment, the rotation of the valve core of the rotary dispensing valve 5 is driven by its own power device (such as a stepping motor), thereby achieving communication between the second flow path channel and different holes.

[0019] In a preferred embodiment, the flow path device integrating automatic cleaning, PCR amplification and detection of the present invention comprises a PCR sample storage tray 16, a first dilute washing solution bottle 151 and a first concentrated washing solution bottle 152 placed adjacent to the PCR sample storage tray 16, a sample suction needle 17 located above them and freely movable, a cleaning swab 12 arranged around the outer wall of the sample suction needle 17, and a six-way rotary dispensing valve 5, wherein

[0020] The six-way rotary dispensing valve 5 has six holes in six directions, the first hole sequentially connects the pump-valve integrated part 4 and the waste liquid bottle 11, the second hole sequentially connects the PCR amplification area 7, the detection system 8 and the waste liquid bottle 11, the third hole connects the sample suction needle 17, the fourth hole connects the second concentrated washing liquid bottle 3, the fifth hole connects the second diluted washing liquid bottle 2, and the sixth hole connects the isolation liquid bottle 1; the six-way rotary dispensing valve 5 includes two flow channels connected at the valve core, wherein the first flow channel is fixedly connected to the first hole, and the second flow channel is connected to one of the second hole to the sixth hole through the rotation switching of the valve core;

[0021] The second dilute washing liquid bottle 2 and the second concentrated washing liquid bottle 3 are connected to the first hole and the second hole of the three-way valve 9 through pipelines respectively, and the third hole of the three-way valve 9 is connected to the peristaltic pump 10, the cleaning swab 12, the diaphragm pump 13 and the waste liquid bottle 11 in sequence through pipelines.

[0022] In a preferred embodiment, the detection system 8 includes a detection window 81 arranged at the pipeline between the PCR amplification area 7 and the waste liquid bottle 11, an excitation light unit 82 located on one side of the detection window 81 and a signal receiving unit 83 located on the other side of the detection window 81.

[0023] The second aspect of the present invention relates to a method integrating automatic cleaning, PCR amplification and detection, which is performed using a flow path device in which the PCR amplification zone 7 and the detection system 8 are sequentially connected as described in the first aspect of the present invention, and comprises the following steps:

[0024] 1) Insert the sample suction needle 17 into the first washing liquid bottle 15, and under the action of the liquid suction and delivery device 14, make the first washing liquid flow along the first flow path and be discharged into the waste liquid bottle 11;

[0025] 2) inserting the sample aspirating needle 17 into the PCR sample storage tray 16, and under the action of the liquid suction and delivery device 14, aspirating the PCR sample and quantitatively injecting it into the PCR amplification area 7 for PCR amplification;

[0026] 3) optionally repeating steps 1 and 2 to achieve multiple consecutive injections of PCR samples;

[0027] 4) Under the action of the liquid suction and delivery device 14, the amplified mixed liquid is passed through the detection system 8 for detection, and the detected mixture is discharged into the waste liquid bottle 11. In a preferred embodiment, the method includes the following steps:

[0028] 1) Insert the sample suction needle 17 into the first washing liquid bottle 15, and under the action of the liquid suction and delivery device 14, make the first washing liquid flow along the first flow path and be discharged into the waste liquid bottle 11 to clean the inner wall of the sample suction needle 17; under the action of the peristaltic pump 10 and the diaphragm pump 13, make the second washing liquid flow along the entire flow path and clean the outer wall of the sample suction needle 17 through the up and down movement of the washing swab 12 and be discharged into the waste liquid bottle 11;

[0029] 2) Under the action of the liquid suction and delivery device 14, the second washing liquid, the isolation liquid, the PCR sample, the isolation liquid and the second washing liquid are successively sucked into it to obtain a layered mixed liquid, and then the mixed liquid is quantitatively injected into the PCR amplification area 7 for PCR amplification;

[0030] 3) Optionally, repeating steps 1 and 2 to achieve multiple consecutive injections of PCR samples;

[0031] 4) Under the action of the liquid suction and delivery device 14, the amplified mixed liquid is passed through the detection system 8 for detection, and the detected mixture is discharged into the waste liquid bottle 11;

[0032] 5) Optionally, under the action of the liquid suction and delivery device 14 , the second washing liquid flows through the first flow path and / or the detection flow path and is discharged into the waste liquid bottle 11 .

[0033] In a preferred embodiment, the method comprises the following steps:

[0034] 1) Initial washing of the sample suction needle 17: connect the second flow path of the six-way rotary dispensing valve 5 with the third hole, insert the sample suction needle 17 into the first concentrated washing liquid bottle 152, and under the action of the pump-valve integrated part 4, make the first concentrated washing liquid flow along the entire flow path and be discharged into the waste liquid bottle 11, and initially wash the inner wall of the sample suction needle 17; close the first hole of the three-way valve 9, open the second hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, make the second concentrated washing liquid flow along the entire flow path and initially wash the outer wall of the sample suction needle 17 through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0035] 2) Re-washing the sample suction needle 17: connecting the second flow path of the six-way rotary dispensing valve 5 with the third hole, inserting the sample suction needle 17 into the first dilute washing liquid bottle 151, and under the action of the pump-valve integrated part 4, making the first dilute washing liquid flow along the entire flow path and discharged into the waste liquid bottle 11, and washing the inner wall of the sample suction needle 17 again; closing the second hole of the three-way valve 9, opening the first hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, making the second dilute washing liquid flow along the entire flow path and washing the outer wall of the sample suction needle 17 again through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0036] 3) Liquid aspiration: The second flow channel of the six-way rotary dispensing valve 5 is connected to the fifth hole, the sixth hole, the fourth hole, and the sixth hole in sequence, and under the action of the pump-valve integrated part 4, the second dilute washing liquid, the isolation liquid, the second concentrated washing liquid, and the isolation liquid are aspirated into the second flow channel in sequence; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the third hole, the sample aspiration needle 17 is inserted into the PCR sample storage tray 16, and under the action of the pump-valve integrated part 4, the PCR sample is aspirated into the second flow channel; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the sixth hole and the second hole in sequence, and under the action of the pump-valve integrated part 4, the isolation liquid and the second dilute washing liquid are aspirated into the second flow channel in sequence;

[0037] 4) Injection and PCR amplification: The second flow channel of the six-way rotary dispensing valve 5 is connected to the second hole, and under the action of the pump valve integrated part 4, the mixed solution in the second flow channel is quantitatively injected into the PCR amplification area 7 for PCR amplification. The PCR sample is a mixed solution to be amplified, which includes a template, an enzyme, a primer, a buffer, etc. Preferably, the mixed solution passes through a negative pressure degassing buffer device 6 before being injected into the PCR amplification area 7.

[0038] 5) optionally repeating steps 1) to 4) to achieve multiple consecutive injections of PCR samples;

[0039] 6) Detection: Under the continuous action of the pump-valve integrated part 4, the amplified mixed solution is passed through the detection system 8 for detection, and the detected mixture is discharged into the waste liquid bottle 11;

[0040] 7) Preliminary cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fourth hole, and the second concentrated washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second concentrated washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0041] 8) Re-cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fifth hole, and the second dilute washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0042] 9) The second flow channel of the six-way rotary dispensing valve 5 is connected to the fourth hole, and under the action of the pump-valve integrated part 4, the second concentrated washing liquid is sucked into the second flow channel, so that the second concentrated washing liquid flows along the entire flow channel and is discharged into the waste liquid bottle 11;

[0043] 10) The second flow path of the six-way rotary dispensing valve 5 is connected to the fifth hole. Under the action of the pump-valve integrated component 4, the second dilute washing liquid is absorbed into the second flow path, so that the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11, preparing for the next detection.

[0044] Preferably, steps 7) to 10) are performed only before and after testing a batch of samples to clean and maintain the entire flow path device and improve test accuracy.

[0045] Preferably, the mixed liquid in the second flow channel that has not been injected in step 4) is discharged into the waste liquid bottle 11 via the first flow channel, and then steps 9) and / or 10) are performed to complete the cleaning of the first flow channel to avoid cross contamination between PCR samples.

[0046] In a preferred embodiment, the method is performed using a flow path device as described in the first aspect of the present invention, in which the PCR amplification zone 7 and the detection system 8 are integrated into one device to form a PCR amplification detection assembly device 18, and comprises the following steps:

[0047] 1) Insert the sample suction needle 17 into the first washing liquid bottle 15, and under the action of the liquid suction and delivery device 14, make the first washing liquid flow along the first flow path and be discharged into the waste liquid bottle 11;

[0048] 2) Insert the sample suction needle 17 into the PCR sample storage tray 16, and under the action of the liquid suction and delivery device 14, suck the PCR sample and quantitatively inject it into the PCR amplification and detection collection device 18 composed of the PCR amplification area 7 and the detection system 8 for PCR amplification and detection, and discharge the detected mixture into the waste liquid bottle 11.

[0049] In a preferred embodiment, the method comprises the following steps:

[0050] 1) Insert the sample suction needle 17 into the first washing liquid bottle 15, and under the action of the liquid suction and delivery device 14, make the first washing liquid flow along the first flow path and be discharged into the waste liquid bottle 11 to clean the inner wall of the sample suction needle 17; under the action of the peristaltic pump 10 and the diaphragm pump 13, make the second washing liquid flow along the entire flow path and clean the outer wall of the sample suction needle 17 through the up and down movement of the washing swab 12 and be discharged into the waste liquid bottle 11;

[0051] 2) Under the action of the liquid suction and delivery device 14, the second washing liquid, the isolation liquid, the PCR sample, the isolation liquid and the second washing liquid are sucked into it in turn to obtain a layered mixed liquid, and then the mixed liquid is quantitatively sampled into the PCR amplification detection integrated device 18 composed of the PCR amplification area 7 and the detection system 8 for PCR amplification and detection, and the detected mixture is discharged into the waste liquid bottle 11

[0052] 3) Optionally, under the action of the liquid suction and delivery device 14 , the second washing liquid flows through the first flow path and / or the detection flow path and is discharged into the waste liquid bottle 11 .

[0053] In a preferred embodiment, the method comprises the following steps:

[0054] 1) Initial washing of the sample suction needle 17: connect the second flow path of the six-way rotary dispensing valve 5 with the third hole, insert the sample suction needle 17 into the first concentrated washing liquid bottle 152, and under the action of the pump-valve integrated part 4, make the first concentrated washing liquid flow along the entire flow path and be discharged into the waste liquid bottle 11, and initially wash the inner wall of the sample suction needle 17; close the first hole of the three-way valve 9, open the second hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, make the second concentrated washing liquid flow along the entire flow path and initially wash the outer wall of the sample suction needle 17 through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0055] 2) Re-washing the sample suction needle 17: connecting the second flow path of the six-way rotary dispensing valve 5 with the third hole, inserting the sample suction needle 17 into the first dilute washing liquid bottle 151, and under the action of the pump-valve integrated part 4, making the first dilute washing liquid flow along the entire flow path and discharged into the waste liquid bottle 11, and washing the inner wall of the sample suction needle 17 again; closing the second hole of the three-way valve 9, opening the first hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, making the second dilute washing liquid flow along the entire flow path and washing the outer wall of the sample suction needle 17 again through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0056] 3) Liquid aspiration: The second flow channel of the six-way rotary dispensing valve 5 is connected to the fifth hole, the sixth hole, the fourth hole, and the sixth hole in sequence, and under the action of the pump-valve integrated part 4, the second dilute washing liquid, the isolation liquid, the second concentrated washing liquid, and the isolation liquid are aspirated into the second flow channel in sequence; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the third hole, the sample aspiration needle 17 is inserted into the PCR sample storage tray 16, and under the action of the pump-valve integrated part 4, the PCR sample is aspirated into the second flow channel; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the sixth hole and the second hole in sequence, and under the action of the pump-valve integrated part 4, the isolation liquid and the second dilute washing liquid are aspirated into the second flow channel in sequence;

[0057] 4) Injection and PCR amplification: The second flow channel of the six-way rotary dispensing valve 5 is connected to the second hole, and under the action of the pump-valve integrated part 4, the mixed liquid in the second flow channel is quantitatively injected into the PCR amplification detection assembly device 18 composed of the PCR amplification area 7 and the detection system 8 for PCR amplification and detection, and the detected mixture is discharged into the waste liquid bottle 11. The PCR sample is a mixed liquid to be amplified, which includes a template, an enzyme, a primer, a buffer, etc. Preferably, the mixed liquid passes through the negative pressure degassing buffer device 6 before being injected into the PCR amplification area 7.

[0058] 5) Preliminary cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fourth hole, and the second concentrated washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second concentrated washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0059] 6) Re-cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fifth hole, and the second dilute washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0060] 7) The second flow channel of the six-way rotary dispensing valve 5 is connected to the fourth hole, and under the action of the pump-valve integrated part 4, the second concentrated washing liquid is sucked into the second flow channel, so that the second concentrated washing liquid flows along the entire flow channel and is discharged into the waste liquid bottle 11;

[0061] 8) Make the second flow channel of the six-way rotary dispensing valve 5 connected to the fifth hole. Under the action of the pump-valve integrated part 4, the second dilute washing liquid is absorbed into the second flow channel, so that the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11, preparing for the next detection.

[0062] Preferably, steps 5) to 8) are performed only before and after testing a batch of samples to clean and maintain the entire flow path device and improve test accuracy.

[0063] Preferably, the mixed liquid in the second flow channel that has not been injected in step 4) is discharged into the waste liquid bottle 11 via the first flow channel, and then steps 7) and / or 8) are performed to complete the cleaning of the first flow channel to avoid cross contamination between PCR samples.

[0064] In a preferred embodiment, wherein

[0065] The isolation liquid is mineral oil, 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.

[0066] The first washing liquid and the second washing liquid are independently ethanol solution, deionized water or DNA strong oxidizing liquid. The ethanol solution is preferably 95% ethanol, preferably AR grade ethanol; the deionized water is preferably double distilled water (ddH2O); the DNA strong oxidizing liquid is preferably sodium hypochlorite solution, preferably sodium hypochlorite solution with a mass fraction of 5%-10%.

[0067] The PCR sample contains template, enzyme, primer, dNTP (deoxyribonucleoside triphosphate) and buffer. The template, enzyme, primer and buffer used include those commonly used in current PCR amplification and detection. When the probe method is used for fluorescent quantitative PCR, the PCR sample contains template, enzyme (such as Taq enzyme), probe (such as taqMan probe), primer, dNTP and buffer (such as containing Mg 2+ , K + When the dye method is used for fluorescent quantitative PCR, the PCR sample contains a template, an enzyme (such as Taq enzyme), a fluorescent dye, a primer, dNTP and a buffer (such as Mg 2+ , K +, such as 100 mM KCl solution). The fluorescent dye can be selected according to the actual application requirements, such as propidium iodide (PI), DAPI, Hoechst 33342, thiazole orange or acridine orange.

[0068] In a preferred embodiment, the volume of the PCR sample in the mixed solution injected each time is 1 μL-60 μL, preferably 2 μL-40 μL, more preferably 5 μL-30 μL, more preferably 10 μL-25 μL.

[0069] A third aspect of the present invention relates to a mixed solution with a self-cleaning function for a PCR amplification flow path, comprising a PCR sample layer, an isolation liquid layer disposed on both sides of the PCR sample layer, and a washing liquid layer disposed outside the isolation liquid layer.

[0070] In a preferred embodiment, the mixed solution is a layered liquid mixture comprising the following solutions in sequence: a layer of dilute wash solution, a layer of spacer solution, a layer of concentrated wash solution, a second layer of spacer solution, a PCR sample layer, a third layer of spacer solution, and a second layer of dilute wash solution;

[0071] In a preferred embodiment, wherein

[0072] Isolation liquid 1, isolation liquid 2 and isolation liquid 3 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.

[0073] The dilute washing solution 1 and the dilute washing solution 2 are independently selected from an ethanol solution or deionized water. The ethanol solution is preferably 95% ethanol, preferably AR grade ethanol; and the deionized water is preferably double distilled water.

[0074] The concentrated washing solution is a strong DNA oxidizing solution, which is preferably a sodium hypochlorite solution, preferably a sodium hypochlorite solution with a mass fraction of 5%-10%.

[0075] The PCR sample contains template, enzyme, primer, dNTP and buffer. When the probe method is used for fluorescence quantitative PCR, the PCR sample contains template, enzyme (such as Taq enzyme), probe (such as TaqMan probe), primer, dNTP and buffer (such as Mg 2 + , K + When the dye method is used for fluorescent quantitative PCR, the PCR sample contains a template, an enzyme (such as Taq enzyme), a fluorescent dye, a primer, dNTP and a buffer (such as Mg 2+ , K +The fluorescent dye can be selected according to the actual application requirements, such as propidium iodide (PI), DAPI, Hoechst33342, thiazole orange or acridine orange.

[0076] The fourth aspect of the present invention relates to the use of the mixed solution described in the third aspect of the present invention for self-cleaning of PCR amplification flow path in continuous flow PCR amplification.

[0077] The present invention has the following advantages:

[0078] 1) The flow path device and method of the present invention, which integrates automatic cleaning, PCR amplification and detection, simplifies the PCR detection process, and proposes to achieve automatic flow cleaning through multiple washes between different test batches, thereby creating a PCR amplification assembly line operation, greatly improving the test throughput of the platform;

[0079] 2) The flow path device and method of the present invention eliminates manual intervention in the cleaning, PCR amplification and detection processes, thereby improving the test efficiency;

[0080] 3) 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 also allows the subsequent PCR amplification area 7 to accommodate more samples to be tested, greatly improving the amplification efficiency of the instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a schematic diagram of the flow path device of the present invention that integrates automatic cleaning, PCR amplification and detection.

[0082] Figure 2 This is a schematic diagram of a flow path device integrating automatic cleaning, PCR amplification and detection in Example 1 of the present invention.

[0083] Figure 3 This is a schematic diagram of a flow path device integrating automatic cleaning, PCR amplification and detection in Example 2 of the present invention.

[0084] The reference numerals have the following meanings:

[0085] 1-isolation liquid bottle, 2-second dilute washing liquid bottle, 3-second concentrated washing liquid bottle, 4-pump-valve integrated part, 5-rotary dispensing valve, 6-negative pressure degassing buffer device, 7-PCR amplification area, 8-detection system, 81-detection window, 82-excitation light unit, 83-signal receiving unit, 9-three-way valve, 10-peristaltic pump, 11-waste liquid bottle, 12-cleaning swab, 13-diaphragm pump, 14-liquid suction and conveying device, 15-first washing liquid bottle, 151-first dilute washing liquid bottle, 152-first concentrated washing liquid bottle, 16-PCR sample storage tray, 17-sample suction needle, 18-PCR amplification detection collection device. DETAILED DESCRIPTION

[0086] 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.

[0087] Example 1

[0088] like Figure 2 The flow path device shown in the figure integrates automatic cleaning, PCR amplification and detection, which includes a PCR sample storage tray 16, a first dilute washing solution bottle 151 and a first concentrated washing solution bottle 152 placed adjacent to the PCR sample storage tray 16, a sample suction needle 17 located above them and freely movable, a cleaning swab 12 arranged around the outer wall of the sample suction needle 17, and a six-way rotary dispensing valve 5, wherein

[0089] The six-way rotary dispensing valve 5 has six holes in six directions, the first hole sequentially connects the pump-valve integrated part 4 and the waste liquid bottle 11, the second hole sequentially connects the negative pressure degassing buffer device 6, the PCR amplification area 7, the detection system 8 and the waste liquid bottle 11, the third hole connects the sample suction needle 17, the fourth hole connects the second concentrated washing liquid bottle 3, the fifth hole connects the second diluted washing liquid bottle 2, and the sixth hole connects the isolation liquid bottle 1; the six-way rotary dispensing valve 5 includes two flow channels connected at the valve core, wherein the first flow channel is fixedly connected to the first hole, and the second flow channel is connected to one of the second hole to the sixth hole through the rotation switching of the valve core;

[0090] The second dilute washing liquid bottle 2 and the second concentrated washing liquid bottle 3 are connected to the first hole and the second hole of the three-way valve 9 through pipelines respectively, and the third hole of the three-way valve 9 is connected to the peristaltic pump 10, the cleaning swab 12, the diaphragm pump 13 and the waste liquid bottle 11 in sequence through pipelines.

[0091] The detection system 8 includes a detection window 81 arranged at the pipeline between the PCR amplification area 7 and the waste liquid bottle 11, an excitation light unit 82 located on one side of the detection window 81 and a signal receiving unit 83 located on the other side of the detection window 81.

[0092] The PCR amplification area 7 uses a pipeline CFPCR device.

[0093] Example 2

[0094] like Figure 3 The flow path device shown integrates automatic cleaning, PCR amplification and detection. It is different from the flow path device in Example 1 in that, in this embodiment, the PCR amplification area 7 and the detection system 8 are integrated into one device to form a PCR amplification detection collection device 18.

[0095] Example 3

[0096] A method integrating automatic cleaning, PCR amplification and detection, the method is performed using the flow path device as described in Example 1, and comprises the following steps:

[0097] 1) Initial washing of the sample suction needle 17: connect the second flow path of the six-way rotary dispensing valve 5 with the third hole, insert the sample suction needle 17 into the first concentrated washing liquid bottle 152, and under the action of the pump-valve integrated part 4, make the first concentrated washing liquid flow along the entire flow path and be discharged into the waste liquid bottle 11, and initially wash the inner wall of the sample suction needle 17; close the first hole of the three-way valve 9, open the second hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, make the second concentrated washing liquid flow along the entire flow path and initially wash the outer wall of the sample suction needle 17 through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0098] 2) Re-washing the sample suction needle 17: connecting the second flow path of the six-way rotary dispensing valve 5 with the third hole, inserting the sample suction needle 17 into the first dilute washing liquid bottle 151, and under the action of the pump-valve integrated part 4, making the first dilute washing liquid flow along the entire flow path and discharged into the waste liquid bottle 11, and washing the inner wall of the sample suction needle 17 again; closing the second hole of the three-way valve 9, opening the first hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, making the second dilute washing liquid flow along the entire flow path and washing the outer wall of the sample suction needle 17 again through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0099] 3) Liquid aspiration: The second flow channel of the six-way rotary dispensing valve 5 is connected to the fifth hole, the sixth hole, the fourth hole, and the sixth hole in sequence, and under the action of the pump-valve integrated part 4, the second dilute washing liquid, the isolation liquid, the second concentrated washing liquid, and the isolation liquid are aspirated into the second flow channel in sequence; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the third hole, the sample aspiration needle 17 is inserted into the PCR sample storage tray 16, and under the action of the pump-valve integrated part 4, the PCR sample is aspirated into the second flow channel; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the sixth hole and the second hole in sequence, and under the action of the pump-valve integrated part 4, the isolation liquid and the second dilute washing liquid are aspirated into the second flow channel in sequence;

[0100] 4) Injection and PCR amplification: The second flow channel of the six-way rotary dispensing valve 5 is connected to the second hole, and under the action of the pump valve integrated part 4, the mixed solution in the second flow channel passes through the negative pressure degassing buffer device 6, and then quantitatively injected into the PCR amplification area 7 for PCR amplification. The PCR sample is a mixed solution to be amplified, which includes a template, an enzyme, a primer, and a buffer, etc. The volume of the PCR sample in the mixed solution injected each time is 15 μL.

[0101] 5) Repeating steps 1) to 4) to achieve multiple continuous injections of PCR samples;

[0102] 6) Detection: Under the continuous action of the pump-valve integrated part 4, the amplified mixed solution is passed through the detection system 8 for detection, and the detected mixture is discharged into the waste liquid bottle 11;

[0103] 7) Preliminary cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fourth hole, and the second concentrated washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second concentrated washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0104] 8) Re-cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fifth hole, and the second dilute washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0105] 9) The second flow channel of the six-way rotary dispensing valve 5 is connected to the fourth hole, and under the action of the pump-valve integrated part 4, the second concentrated washing liquid is sucked into the second flow channel, so that the second concentrated washing liquid flows along the entire flow channel and is discharged into the waste liquid bottle 11;

[0106] 10) The second flow path of the six-way rotary dispensing valve 5 is connected to the fifth hole. Under the action of the pump-valve integrated component 4, the second dilute washing liquid is absorbed into the second flow path, so that the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11, preparing for the next detection.

[0107] The isolation liquid is mineral oil; the first dilute washing liquid is 95% ethanol solution; the first concentrated washing liquid is 6% sodium hypochlorite solution; the second dilute washing liquid is double distilled water; the second concentrated washing liquid is 8% sodium hypochlorite solution; the PCR sample contains template, Taq enzyme, taqMan probe, primer, dNTP and 100mM KCl solution as buffer.

[0108] Example 4

[0109] A method integrating automatic cleaning, PCR amplification and detection, the method is performed using the flow path device as described in Example 2, and comprises the following steps:

[0110] 1) Initial washing of the sample suction needle 17: connect the second flow path of the six-way rotary dispensing valve 5 with the third hole, insert the sample suction needle 17 into the first concentrated washing liquid bottle 152, and under the action of the pump-valve integrated part 4, make the first concentrated washing liquid flow along the entire flow path and be discharged into the waste liquid bottle 11, and initially wash the inner wall of the sample suction needle 17; close the first hole of the three-way valve 9, open the second hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, make the second concentrated washing liquid flow along the entire flow path and initially wash the outer wall of the sample suction needle 17 through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0111] 2) Re-washing the sample suction needle 17: connecting the second flow path of the six-way rotary dispensing valve 5 with the third hole, inserting the sample suction needle 17 into the first dilute washing liquid bottle 151, and under the action of the pump-valve integrated part 4, making the first dilute washing liquid flow along the entire flow path and discharged into the waste liquid bottle 11, and washing the inner wall of the sample suction needle 17 again; closing the second hole of the three-way valve 9, opening the first hole and the third hole, and under the action of the peristaltic pump 10 and the diaphragm pump 13, making the second dilute washing liquid flow along the entire flow path and washing the outer wall of the sample suction needle 17 again through the up and down movement of the washing swab 12, and the waste liquid is discharged into the waste liquid bottle 11;

[0112] 3) Liquid aspiration: The second flow channel of the six-way rotary dispensing valve 5 is connected to the fifth hole, the sixth hole, the fourth hole, and the sixth hole in sequence, and under the action of the pump-valve integrated part 4, the second dilute washing liquid, the isolation liquid, the second concentrated washing liquid, and the isolation liquid are aspirated into the second flow channel in sequence; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the third hole, the sample aspiration needle 17 is inserted into the PCR sample storage tray 16, and under the action of the pump-valve integrated part 4, the PCR sample is aspirated into the second flow channel; the second flow channel of the six-way rotary dispensing valve 5 is then connected to the sixth hole and the second hole in sequence, and under the action of the pump-valve integrated part 4, the isolation liquid and the second dilute washing liquid are aspirated into the second flow channel in sequence;

[0113] 4) Injection and PCR amplification: The second flow channel of the six-way rotary dispensing valve 5 is connected to the second hole. Under the action of the pump-valve integrated part 4, the mixed liquid in the second flow channel passes through the negative pressure degassing buffer device 6, and then quantitatively injected into the PCR amplification detection assembly device 18 composed of the PCR amplification area 7 and the detection system 8 for PCR amplification and detection, and the detected mixture is discharged into the waste liquid bottle 11. The PCR sample is a mixed liquid to be amplified, which includes a template, an enzyme, a primer, a buffer, etc. The volume of the PCR sample in the mixed liquid injected each time is 15μL.

[0114] 5) Preliminary cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fourth hole, and the second concentrated washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second concentrated washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0115] 6) Re-cleaning of the detection flow path: the second flow path of the six-way rotary dispensing valve 5 is connected to the fifth hole, and the second dilute washing liquid is sucked into the second flow path under the action of the pump-valve integrated part 4; then the second flow path of the six-way rotary dispensing valve 5 is connected to the second hole, and the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11 under the action of the pump-valve integrated part 4;

[0116] 7) The second flow channel of the six-way rotary dispensing valve 5 is connected to the fourth hole, and under the action of the pump-valve integrated part 4, the second concentrated washing liquid is sucked into the second flow channel, so that the second concentrated washing liquid flows along the entire flow channel and is discharged into the waste liquid bottle 11;

[0117] 8) Make the second flow channel of the six-way rotary dispensing valve 5 connected to the fifth hole. Under the action of the pump-valve integrated part 4, the second dilute washing liquid is absorbed into the second flow channel, so that the second dilute washing liquid flows along the entire flow path and is discharged into the waste liquid bottle 11, preparing for the next detection.

[0118] The isolation liquid is mineral oil; the first dilute washing liquid is 95% ethanol solution; the first concentrated washing liquid is 6% sodium hypochlorite solution; the second dilute washing liquid is double distilled water; the second concentrated washing liquid is 8% sodium hypochlorite solution; the PCR sample contains template, Taq enzyme, taqMan probe, primer, dNTP and 100mM KCl solution as buffer.

[0119] Example 5

[0120] A mixed solution with a self-cleaning function for a PCR amplification flow path, which is a layered liquid mixture comprising the following solutions in sequence: a layer of a dilute washing solution, a layer of an isolation solution, a layer of a concentrated washing solution, a second layer of an isolation solution, a PCR sample layer, a third layer of an isolation solution, and a second layer of a dilute washing solution;

[0121] The dilute washing solution 1 is a 95% ethanol solution, the dilute washing solution 2 is deionized water; the isolation solution 1, the isolation solution 2 and the isolation solution 3 are independently selected from one or more mineral oils; the concentrated washing solution is an 8% sodium hypochlorite solution; the PCR sample contains a template, Taq enzyme, fluorescent dye propidium iodide, primers, dNTPs and a 100mM KCl solution as a buffer.

[0122] 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 method integrating automatic cleaning, PCR amplification and detection, characterized in that: The method was performed using a flow path device as described below: A flow path device, comprising a PCR sample storage tray (16), a first washing liquid bottle (15), a sample suction needle (17) located above the tray and freely movable, and a liquid suction and delivery device (14), wherein the liquid suction and delivery device (14) is provided with a plurality of holes, wherein the first hole is directly connected to a waste liquid bottle (11) to form a first flow path, the second hole is sequentially connected to a PCR amplification area (7), a detection system (8) and the waste liquid bottle (11) to form a detection flow path, and the third hole is connected to the sample suction needle (17); A negative pressure degassing buffer device (6) is provided upstream of the PCR amplification zone (7); The fourth hole of the liquid suction and delivery device (14) is connected to the second washing liquid bottle, which is sequentially connected to the peristaltic pump (10), the washing swab (12) arranged around the outer wall of the sample suction needle (17), the diaphragm pump (13) and the waste liquid bottle (11); the fifth hole is connected to the isolation liquid bottle (1); The method comprises the following steps: 1) inserting a sample suction needle (17) into a first washing liquid bottle (15), and under the action of a liquid suction and delivery device (14), causing a first washing liquid to flow along a first flow path and be discharged into a waste liquid bottle (11), thereby cleaning the inner wall of the sample suction needle (17); under the action of a peristaltic pump (10) and a diaphragm pump (13), causing a second washing liquid to flow along the entire flow path and, through the up and down movement of a washing swab (12), cleaning the outer wall of the sample suction needle (17) and being discharged into the waste liquid bottle (11); 2) Under the action of the liquid suction and delivery device (14), the second washing liquid, the isolation liquid, the PCR sample, the isolation liquid and the second washing liquid are successively sucked into it to obtain a layered mixed liquid, and then the mixed liquid is quantitatively injected into the PCR amplification area (7) through the negative pressure degassing buffer device (6) to perform PCR amplification; 3) Repeating steps 1) and 2) to achieve multiple continuous injections of PCR samples; 4) Under the action of the liquid suction and delivery device (14), the amplified mixed liquid is passed through the detection system (8) for detection, and the detected mixture is discharged into the waste liquid bottle (11); 5) Under the action of the liquid suction and delivery device (14), the second washing liquid flows through the first flow path and / or the detection flow path and is discharged into the waste liquid bottle (11).

2. A method integrating automatic cleaning, PCR amplification and detection, characterized in that: The method was performed using a flow path device as described below: A flow path device, comprising a PCR sample storage tray (16), a first washing liquid bottle (15), a sample suction needle (17) located above the tray and freely movable, and a liquid suction and delivery device (14), wherein the liquid suction and delivery device (14) is provided with a plurality of holes, wherein the first hole is directly connected to a waste liquid bottle (11) to form a first flow path, the second hole is sequentially connected to a PCR amplification area (7), a detection system (8) and the waste liquid bottle (11) to form a detection flow path, and the third hole is connected to the sample suction needle (17); The PCR amplification area (7) and the detection system (8) are integrated together to form a PCR amplification detection assembly device (18); A negative pressure degassing buffer device (6) is provided upstream of the PCR amplification zone (7); The fourth hole of the liquid suction and delivery device (14) is connected to the second washing liquid bottle, which is sequentially connected to the peristaltic pump (10), the washing swab (12) arranged around the outer wall of the sample suction needle (17), the diaphragm pump (13) and the waste liquid bottle (11); the fifth hole is connected to the isolation liquid bottle (1); The method comprises the following steps: 1) inserting a sample suction needle (17) into a first washing liquid bottle (15), and under the action of a liquid suction and delivery device (14), causing a first washing liquid to flow along a first flow path and be discharged into a waste liquid bottle (11), thereby cleaning the inner wall of the sample suction needle (17); under the action of a peristaltic pump (10) and a diaphragm pump (13), causing a second washing liquid to flow along the entire flow path and, through the up and down movement of a washing swab (12), cleaning the outer wall of the sample suction needle (17) and being discharged into the waste liquid bottle (11); 2) Under the action of the liquid suction and delivery device (14), the second washing liquid, the isolation liquid, the PCR sample, the isolation liquid and the second washing liquid are sucked into it in sequence to obtain a layered mixed liquid, and then the mixed liquid is quantitatively injected into the PCR amplification detection assembly device (18) composed of the PCR amplification area (7) and the detection system (8) through the negative pressure degassing buffer device (6) to perform PCR amplification and detection of the amplified mixed liquid, and the detected mixture is discharged into the waste liquid bottle (11); 3) Under the action of the liquid suction and delivery device (14), the second washing liquid flows through the first flow path and / or the detection flow path and is discharged into the waste liquid bottle (11).

3. The method according to claim 1 or 2, characterized in that: in The isolation fluid is mineral oil; The first washing solution and the second washing solution are independently ethanol solution, deionized water or DNA strong oxidizing solution; PCR samples contain template, enzyme, primers, dNTPs and buffer.

4. The method according to claim 3, characterized in that in The mineral oil is a mineral oil with a kinematic viscosity of 10-14 centistokes; The ethanol solution is 95% ethanol; The deionized water is double distilled water; and / or The DNA strong oxidizing liquid is a sodium hypochlorite solution.

5. The method according to claim 4, characterized in that in The sodium hypochlorite solution is a sodium hypochlorite solution with a mass fraction of 5%-10%.

6. A mixed solution with a self-cleaning function for a PCR amplification flow path, characterized in that: It comprises a PCR sample layer, an isolation liquid layer arranged on both sides of the PCR sample layer, and a washing liquid layer arranged outside the isolation liquid layer.

7. The mixed solution according to claim 6, characterized in that The mixed solution is a layered liquid mixture comprising the following solutions in sequence: a layer of dilute washing solution, a layer of spacer solution, a layer of concentrated washing solution, a second layer of spacer solution, a PCR sample layer, a third layer of spacer solution, and a second layer of dilute washing solution; in The spacer liquid 1, the spacer liquid 2 and the spacer liquid 3 are independently selected from one or more mineral oils; The dilute washing solution 1 and the dilute washing solution 2 are independently selected from an ethanol solution or deionized water; The concentrated wash solution is a strong DNA oxidizing solution; PCR samples contain template, enzyme, primers, dNTPs and buffer.

8. The mixed solution according to claim 7, characterized in that: in The mineral oil is a mineral oil with a kinematic viscosity of 10-14 centistokes; The ethanol solution is 95% ethanol; The deionized water is double distilled water; and / or The DNA strong oxidizing liquid is a sodium hypochlorite solution.

9. The mixed solution according to claim 8, characterized in that: in The sodium hypochlorite solution is a sodium hypochlorite solution with a mass fraction of 5%-10%.

10. Use of the mixed solution according to any one of claims 6 to 9 for self-cleaning of PCR amplification flow path in continuous flow PCR amplification.

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