CN113684118B - Integrated nucleic acid analysis chip - Google Patents

Abstract

The invention discloses an integrated nucleic acid analysis chip. The chip is provided with chambers such as an air vent, a reaction chamber, a waste liquid chamber and the like, a storage pipe connecting interface, a sliding rail interface, a liquid flow pipeline, an exhaust pipeline and the like, and a plurality of pipeline ports, wherein the chambers are communicated with one pipeline port through respective pipelines; the storage tube is arranged on the chip through an interface and used as a cavity; the slide rail is tightly fixed with the chip through a slide rail interface, and the conducting valve is positioned in the slide rail to slide and is used for selectively communicating the cavity with the piston pump; the piston pump is connected with the conduction valve in a sealing way through a conduit. The invention can complete the complete steps of nucleic acid extraction, amplification and detection in the chip, can effectively avoid pollution possibly generated, and can better remove the reagent with inhibiting effect on the subsequent amplification, thereby avoiding the influence on the amplification and improving the efficiency and the accuracy.

Description

An integrated nucleic acid analysis chip

Technical Field

The invention relates to a chip used for nucleic acid experiments in the field of nucleic acid analysis and detection, and in particular to an integrated nucleic acid analysis chip.

Background technique

Nucleic acid is the carrier of genetic information of organisms. By detecting nucleic acids, organisms can be accurately identified. At present, nucleic acid analysis and detection have been widely used in food safety testing, environmental monitoring, medical diagnosis and other fields, and play an important role. The nucleic acid detection method based on nucleic acid amplification technology is the most commonly used nucleic acid analysis and detection method, but its detection process often includes complex nucleic acid extraction operations, which require repeated pipetting and are prone to cross contamination during the liquid pipetting process. In order to simplify the nucleic acid extraction operation steps, some researchers directly perform nucleic acid amplification on untreated samples or use simple lysis methods (heating, alkaline solution lysis) to treat the samples before nucleic acid amplification. Although this can simplify the operation, there are still many impurities in the obtained nucleic acid samples, which will affect the subsequent nucleic acid amplification reaction efficiency and detection sensitivity. In addition, some reagents that have an inhibitory effect on subsequent amplification are used in the nucleic acid extraction process, and it also takes a long time to remove these reagents.

For the detection of amplification products, it is mainly dependent on precise fluorescence reading devices to obtain real-time fluorescence curves and judge the results. For some endpoint detection methods based on reaction solution turbidity, reaction solution color change, etc., although the operation is relatively convenient, there are problems such as lack of specificity or insufficient distinction between positive and negative results. CRISPR (clustered regularly spaced short palindromic repeats) is an immune system present in most bacteria and archaea. The system mainly includes Cas proteins and crRNA. Some Cas proteins (such as Cas12, Cas13 and Cas14) can also cut non-target single-stranded DNA (i.e., bypass cutting effect) when cutting target nucleic acid sequences under the guidance of crRNA. Using this property, some researchers combine nucleic acid amplification reactions with CRISPR technology to achieve detection of target nucleic acids. However, due to the temperature incompatibility problem between the two systems, it is often necessary to open the cover after the nucleic acid amplification reaction is completed and then introduce CRISPR reagents, which is prone to aerosol contamination.

At present, some integrated nucleic acid analysis and detection devices have been established, such as Roche's CobasLiat, BioMérieux's FilmArray, Shengxiang Bio's iPonatic system, and Hangzhou Yousida Bio's nucleic acid amplification detection analyzer. However, these devices still have some defects such as large size, high price, and complex design, and cannot fully meet the needs of the grassroots. Therefore, it is of great significance to develop a low-cost, multifunctional, simple to operate, and easy-to-judgment integrated nucleic acid analysis device, which can popularize nucleic acid analysis and detection, especially for areas with limited resources.

Summary of the invention

In order to solve the problems existing in the background technology, the purpose of the present invention is to provide an integrated nucleic acid analysis chip, which can complete the complete steps of nucleic acid extraction, amplification and detection in the chip; and can prevent the tested nucleic acid and nucleic acid amplification products from leaving the detection chip and entering the surrounding environment, thereby avoiding possible contamination. At the same time, the chip can remove some reagents used in the nucleic acid extraction process that have an inhibitory effect on subsequent amplification, thereby avoiding their influence on amplification. The nucleic acid amplification process can adopt variable temperature amplification or isothermal amplification. Amplification product detection can be carried out by real-time fluorescence detection or endpoint detection.

The present invention is achieved in that:

1. An integrated nucleic acid analysis chip:

The chip is provided with a pump and a slide rail structure, and the pump is controlled by the slide rail structure to connect different chambers, thereby realizing selective connection between the pump and different chambers on the chip.

The nucleic acid amplification process can adopt variable temperature amplification or isothermal amplification. The amplification product detection can be carried out by real-time fluorescence detection or endpoint detection.

The present invention can complete the complete steps of nucleic acid extraction, amplification and detection in a chip; and can prevent the detected nucleic acid and nucleic acid amplification products from leaving the detection chip and entering the surrounding environment, thereby avoiding possible contamination.

Multiple CRISPR detection chambers are set on the chip, each of which stores CRISPR detection reagents for different nucleic acid targets. After the nucleic acid amplification reaction is completed, the amplification products are pumped into each CRISPR detection chamber using a piston pump to achieve endpoint multiple detection. The present invention uses CRISPR technology to perform endpoint detection on nucleic acid amplification products.

The chip itself is made transparent at the chamber and is provided with an image sensor, which uses the image sensor to detect bubbles in the fluorescent signal in the chamber. When bubbles are found, the pump valve on the chip is further used to extract liquid from the chamber to eliminate the bubbles in the chamber, thereby eliminating the interference of bubbles and other factors on the fluorescent signal detection.

The chip is provided with a washing chamber, and the washing chamber contains chip washing reagents, which are used to wash the pipes in the chip and other chambers except the washing chamber, and the reagents used in the nucleic acid experiment process that have an impact on subsequent nucleic acid amplification are removed, thereby avoiding affecting nucleic acid amplification.

The chip is provided with an exhaust pipe with a diaphragm and an air hole connected to the exhaust pipe. Under the premise that there is no exchange of nucleic acid molecules between the chip and the outside world, clean gas from the outside world is sucked into the chip through the air hole through the exhaust pipe, thereby cleaning and blowing the pipes and chambers inside the chip.

The invention can accommodate and be applicable to all processes and reagents of nucleic acid extraction operation, and can be used for nucleic acid extraction of large-volume samples.

The integrated nucleic acid analysis chip is more specifically:

The chip is provided with vent holes, reaction chambers, waste liquid chambers and other chambers, and is also provided with interfaces connected to the storage tubes, slide rail interfaces, liquid flow pipes, exhaust pipes and the like, and is provided with a plurality of pipe ports, the waste liquid chamber, the reaction chamber, and each storage tube are connected to a pipe port through a respective liquid flow pipe, and the vent holes are connected to a respective pipe port through an exhaust pipe;

The reaction chamber, waste liquid chamber and the like on the chip of the present invention are all connected to the air-permeable holes, and a diaphragm mechanism which can be both air-permeable and prevent nucleic acid molecules from passing through is arranged in the air-permeable holes.

The storage tube is installed on the chip through an interface as a chamber for storing test samples or reagents required for reactions; a through hole is provided at the bottom of the storage tube, which is sealed and connected to the interface on the chip. Furthermore, the storage tube is provided with a tube cover, which is provided with a diaphragm that is both breathable and can prevent nucleic acid molecules from passing through, and the storage tube is sealed and connected to the tube cover.

A slide rail, the slide rail and the chip are tightly fixed through a slide rail interface, and is used for the movement of the conduction valve, so as to realize the selective conduction between the vent hole, the reaction chamber, the waste liquid chamber, the different storage tubes through the liquid flow pipeline/exhaust pipeline of the chip and the interface on the conduction valve;

A conduction valve is located in the slide rail and slides linearly in the slide rail. A sealing gasket is provided between the conduction valve and the chip to ensure sealing performance through sealing. Furthermore, the sealing gasket is provided with circular through holes, which are respectively connected to the liquid flow pipeline openings on the chip.

A piston pump is sealed and connected to the conduction valve through a conduit, and is used to extract or discharge liquid. Furthermore, the liquid can be mixed by pushing and pulling the piston back and forth.

The chip further includes a sealing film, and the reaction chamber, waste liquid chamber, liquid flow pipe, exhaust pipe, and air vent are formed by opening through the top surface of the chip, and the sealing film is used to seal the top surface openings of the reaction chamber, waste liquid chamber, liquid flow pipe, exhaust pipe, and air vent on the chip. The sealing film can be a hard solid or a flexible film.

The conducting valve is provided with a movable handle and only one interface connected to the chip pipeline port. The conducting valve is moved by the movable handle on the conducting valve, and the only one interface on the conducting valve is aligned with a corresponding pipeline port connected to the chip, so that the only one interface on the conducting valve is always connected to the piston pump, thereby realizing selective connection between the air vent, the reaction chamber, the waste liquid chamber, the different storage tubes and the piston pump.

The chip is provided with a washing chamber, in which a chip washing reagent is contained. The washing chamber is connected to a pipe port through a respective liquid flow pipe, and then selectively connected to a conduction valve, so as to wash the pipes in the chip and other chambers except the washing chamber.

The air holes are connected to the external clean gas, and the external clean gas is sucked into the chip from the outside through the air holes through the exhaust pipe, thereby cleaning and blowing the pipes and chambers inside the chip.

The chip is made transparent in each chamber and is provided with an image sensor, which uses the image sensor to detect bubbles in the fluorescence signal in the chamber. When bubbles are found, the pump valve on the chip is further used to extract liquid from the chamber to eliminate the bubbles in the chamber, thereby eliminating the interference of bubbles and other factors on the fluorescence signal detection.

A buffer chamber is provided on the chip, and dilution storage is performed through the buffer chamber. Dilution storage through the buffer chamber avoids the extraction and transportation of trace liquid volume without reducing the detection sensitivity, thereby improving the reliability of detection.

The buffer chamber contains dilution reagents, which can dilute the amplified product to a certain extent before passing it into the storage tube containing the CRISPR detection reagent for detection. The dilution reagents stored in these buffer chambers can be the buffer required for CRISPR detection. Although this is a dilution process for the target nucleic acid, since the dilution is performed using the CRISPR detection buffer, the concentration of each active ingredient in the CRISPR system and the target nucleic acid to be detected can be ensured to remain unchanged by using concentrated reagents or freeze-dried reagents in the subsequent CRISPR detection chamber. In this way, while ensuring the detection sensitivity, the extraction and transportation of trace liquid volumes can be avoided.

When testing a sample, first, place the sample in the first storage tube of the chip for lysis. The storage tube contains lysis binding reagents and micro-nano magnetic balls, which are used to lyse the sample, release nucleic acid molecules, and bind nucleic acid molecules to the micro-nano magnetic balls. Move the conduction valve on the slide rail so that the first storage tube containing the sample is connected to the piston cylinder through the conduction valve, and the other end of the catheter is connected to the piston pump. Push and pull the piston back and forth to make the micro-nano magnetic balls and the sample fully contact and mix, so that as many nucleic acids as possible are adsorbed on the surface of the micro-nano magnetic balls. Furthermore, the lysis process can be heated and lysed according to sample requirements;

Next, use an external magnet or electromagnet to approach the wall of the first storage tube, adsorb the micro-nano magnetic balls in the first storage tube to the inner wall of the tube, and separate the micro-nano magnetic balls from the cleavage and binding reagent. Pull the piston outward to make the cleavage and binding reagent leave the storage tube and enter the piston cylinder. Move the conduction valve, and the waste liquid chamber is connected to the piston cylinder through the conduction valve. Push the piston inward to make the cleavage and binding reagent in the piston cylinder enter the waste liquid chamber;

Next, move the conduction valve, and the second storage tube is connected to the piston cylinder through the conduction valve. The second storage tube is pre-stored with a magnetic ball cleaning reagent, which is used to remove impurities such as proteins and polysaccharides that may be adsorbed on the surface of the magnetic balls. Pull the piston outward to allow a portion of the magnetic ball cleaning reagent to enter the piston cylinder. Move the conduction valve, and the first storage tube is connected to the piston cylinder through the conduction valve. Push the piston inward to allow the magnetic ball cleaning reagent in the piston cylinder to enter the first storage tube. Remove the external magnet or electromagnet, and push and pull the piston back and forth to allow the micro-nano magnetic balls to fully contact and mix with the magnetic ball cleaning reagent for a period of time, thereby achieving the first cleaning of the micro-nano magnetic balls;

Next, use an external magnet or electromagnet to adsorb the micro-nano magnetic balls in the first storage tube onto the inner wall of the tube, so that the micro-nano magnetic balls are separated from the magnetic ball cleaning reagent. Pull the piston outward to make the magnetic ball cleaning reagent leave the first storage tube and enter the piston cylinder. Move the conduction valve to connect the waste liquid chamber with the piston cylinder through the conduction valve. Push the piston inward to make the magnetic ball cleaning reagent enter the waste liquid chamber;

Next, the second storage tube is connected to the piston cylinder through the conduction valve. The piston is pulled outward to allow the remaining magnetic ball cleaning reagent in the second storage tube to enter the piston cylinder. Repeat the above steps to achieve the second cleaning of the micro-nano magnetic balls, and discharge the magnetic ball cleaning reagent into the waste liquid chamber. In practice, the specific number of cleaning times can be set as needed.

Next, the third storage tube is pre-stored with a chip washing reagent, which is used to clean the magnetic ball washing reagent remaining in the liquid flow pipeline and the piston cylinder. Generally, the above-mentioned magnetic ball washing reagent contains organic reagents such as alcohol. These organic reagents will inhibit subsequent nucleic acid amplification. In the chip, these magnetic ball washing reagents may remain in the pipeline of the chip and enter the subsequent reagents, thereby inhibiting nucleic acid amplification. In the design of the chip of the present invention, a washing chamber, a chip washing reagent and a washing step are further innovatively set to clean the chip pipeline and the piston pump to eliminate the possible effects of the residual magnetic ball washing reagent. Specifically, the conduction valve is moved to connect the third storage tube to the piston cylinder through the conduction valve. The piston is pulled outward to allow a portion of the chip washing reagent to enter the piston cylinder, and the conduction valve is moved to connect the second storage tube to the piston cylinder through the conduction valve. The piston is pushed and pulled back and forth to allow the chip washing reagent to fully wash the piston pump. Finally, the piston is pulled outward to allow the washing reagent to leave the second storage tube and enter the piston cylinder. The conduction valve is moved to connect the waste liquid chamber to the piston cylinder through the conduction valve. The piston is pushed inwards to allow the chip washing reagent to enter the waste liquid chamber. Further, the washing process can be repeated for multiple times according to the actual washing situation.

Next, move the conduction valve to connect the third storage tube to the piston cylinder through the conduction valve. Pull the piston outward to allow a portion of the chip washing reagent to enter the piston cylinder, and move the conduction valve to connect the first storage tube to the piston cylinder through the conduction valve. Push and pull the piston back and forth to allow the chip washing reagent to fully wash the liquid flow channel connected to the first storage tube. Finally, pull the piston outward to allow the chip washing reagent to enter the piston cylinder. Move the conduction valve to connect the waste liquid chamber to the piston cylinder through the conduction valve. Push the piston inward to allow the chip washing reagent to enter the waste liquid chamber. Furthermore, during the washing process, care should be taken to avoid contact between the chip washing reagent and the micro-nano magnetic balls in the first storage tube. Furthermore, the washing process can be repeated and washed multiple times according to the actual washing situation.

After the pipes are washed, clean air can be sucked in from the outside through the diaphragm via the exhaust pipe using a piston pump to further clean and blow the pipes and micro-nano magnetic balls, thereby further eliminating any harmful residual substances that may exist on the surface of the pipes and magnetic balls.

Clean air is sucked from the outside through the diaphragm by a piston pump through the exhaust pipe, which can also be used for stirring and mixing the solution in the chamber.

Next, move the conduction valve to connect the fourth storage tube to the piston cylinder through the conduction valve. The fourth storage tube is pre-stored with an elution reagent for eluting the nucleic acid molecules adsorbed on the surface of the micro-nano magnetic ball. Pull the piston outward to allow the elution reagent to enter the piston cylinder. Move the conduction valve to connect the first storage tube to the piston cylinder through the conduction valve. Push and pull the piston back and forth to allow the micro-nano magnetic ball to fully contact and mix with the elution reagent for a period of time, so as to achieve the elution of the nucleic acid molecules adsorbed on the surface of the micro-nano magnetic ball and allow the nucleic acid molecules to enter the elution reagent.

Next, use an external magnet or electromagnet to adsorb the micro-nano magnetic balls in the first storage tube onto the inner wall of the first storage tube to separate the micro-nano magnetic balls from the elution reagent. Pull the piston outward to allow the elution reagent to leave the storage tube and enter the piston cylinder. Move the conduction valve to connect the reaction chamber to the piston cylinder through the conduction valve. Amplification reagents are pre-stored in the reaction chamber for nucleic acid amplification reactions. Push the piston inward to allow the elution reagent to enter the reaction chamber, and then perform the amplification reaction through an external temperature control device. Compared with the standard test kit nucleic acid extraction method, it avoids the need to transfer a trace amount of elution reagent for nucleic acid amplification, thereby improving the reliability of the test. Furthermore, one end of the reaction chamber is connected to an air hole that is breathable and prevents nucleic acid molecules from passing through.

It is also possible to set a buffer chamber on the chip as needed, in which a nucleic acid amplification buffer is stored, which is used to dilute the elution reagent containing the nucleic acid and then pump it into the reaction chamber for amplification. For example, the original reaction system is 50 microliters, of which 5 microliters are the nucleic acid eluate containing the test sample. In order to avoid errors and fluctuations that may be caused by extracting a tiny volume on the chip, 50 microliters of the nucleic acid eluate can be extracted and pumped into a buffer chamber containing 450 microliters of amplification buffer. After mixing evenly, 50 microliters of the mixed solution can be extracted and pumped into the reaction chamber. In this way, while ensuring the detection sensitivity, the extraction and transportation of trace liquid volumes can be avoided, reducing possible detection errors;

Next, after the nucleic acid amplification is completed, the piston is pulled outward to allow the amplified product to leave the reaction chamber and enter the piston cylinder. The conduction valve is moved to connect the fifth storage tube to the piston cylinder through the conduction valve. The fifth storage tube is pre-stored with CRISPR detection reagents for endpoint detection of the amplified product. The piston is pushed inward to allow the amplified product to enter the storage tube for reaction. Furthermore, multiple storage tubes can be set on the chip to store CRISPR detection reagents for different nucleic acid targets to achieve multiple nucleic acid detection.

Furthermore, the CRISPR detection reagent contains Cas protein, buffer, guide RNA, single-stranded nucleic acid fluorescent probe, RNase inhibitor, sterile water, etc. When the target nucleic acid is present, it will perform base complementary pairing with the guide RNA, thereby activating the activity of the Cas protein, cutting the single-stranded nucleic acid fluorescent probe, and generating a fluorescent signal. Furthermore, the CRISPR detection reagent can be solidified (such as freeze-dried) for easy storage.

Furthermore, when performing optical detection, there may be factors such as bubbles and particles in the reaction liquid that affect the detection. Bubbles may be generated by the detection reaction, or they may be generated by various factors such as the dissolution of the freeze-dried reagent and the action of pumps and valves during liquid transportation. If the common single-beam real-time fluorescence signal reading is performed, if the light beam happens to fall on the bubble, the bubble will affect the reading of the fluorescence signal, thereby affecting the judgment of the experimental results. Therefore, the present invention introduces an image sensor to perform multi-point detection of the fluorescence signal in the detection cavity during the detection process. By realizing the spatial distribution detection of the fluorescence signal of the reaction liquid, the fluorescence signal at the bubble position is avoided, thereby avoiding the interference of possible bubbles on the fluorescence signal detection;

The specific detection algorithm is as follows:

1. Use the image sensor to read the fluorescence signal of the area in the detection chamber that is illuminated by the incident light and the excitation light, and record and save the fluorescence signal of each pixel in the image sensor respectively;

2. According to the fluorescence signal of each pixel, the average signal intensity of the pixel is calculated;

3. Set certain discrimination criteria, such as taking the average signal intensity plus or minus 10% as the normal signal selection range; for pixels whose fluorescence value deviation is greater than this range, the fluorescence signal can be considered to be caused by bubbles, particles, etc., and is judged as an abnormal point; in practice, the selection of the specific deviation range of the signal of the normal pixel point can be determined according to the chip and detection cavity design, etc.;

4. After eliminating the signals of abnormal points, recalculate the average fluorescence intensity of normal signal points as the detection signal;

5. Furthermore, if foreign matter such as bubbles is detected in step 3, the pump valve of the chip can be further used to extract liquid from the detection chamber to eliminate the bubbles in the chamber, thereby eliminating the interference of factors such as bubbles on the fluorescence signal detection.

Furthermore, the position where the magnet or electromagnet adsorbs the micro-nano magnetic ball is close to the through hole at the bottom of the storage tube;

Furthermore, during the movement of the conduction valve in the slide rail, when the interface on the conduction valve that is in communication with the conduit is not in communication with the pipeline opening at the bottom of the conduction valve on the chip, the pipeline opening at the bottom of the conduction valve is in a sealed state; when the interface on the conduction valve that is in communication with the conduit is in communication with a pipeline opening at the bottom of the conduction valve on the chip, the other pipeline openings at the bottom of the conduction valve are also in a sealed state;

Furthermore, the control of the magnet or electromagnet, the movement of the conduction valve, and the push and pull of the piston pump can be controlled by an external mechanical device;

Furthermore, the selected chip washing reagent will not inhibit the subsequent amplification reaction;

Furthermore, the chip is provided with an exhaust pipe connected to an air hole that is both air-permeable and prevents nucleic acid molecules from passing through. It can be used to form a clean air column with the piston pump, and then push the piston inward to ensure that some liquid reagents remaining in the liquid flow pipe on the chip are completely and cleanly discharged. It can also be used to promote the volatilization of organic reagents remaining on the surface of the micro-nano magnetic ball or in the chip;

Furthermore, the micro-nano magnetic spheres can be replaced by other materials that have the ability to capture nucleic acids, such as silica spheres, filter paper, silica gel films, etc.

The present invention cleverly designs and arranges a slide rail structure, cooperates with the piston movement, controls each reaction process and treatment in sequence, can conveniently implement experimental operation, avoids contamination, can better and thoroughly remove reagents that have an inhibitory effect on subsequent amplification, and improves reaction efficiency and reaction accuracy.

The present invention provides a washing chamber in combination with a slide rail, so that the complete washing of the chip can be well achieved instead of just cleaning the magnetic ball.

The present invention can also realize the effect and function of air intake cleaning by arranging the slide rail in combination with the air vent and the exhaust pipe.

The nucleic acid analysis chip of the present invention is characterized in that it can complete the complete steps of nucleic acid extraction, amplification and detection; and it can prevent the tested nucleic acid and nucleic acid amplification products from leaving the detection chip and entering the surrounding environment, thereby avoiding possible contamination. At the same time, the chip can remove some reagents used in the nucleic acid extraction process that have an inhibitory effect on subsequent amplification, thereby avoiding their influence on amplification. In some commercial devices, micro-nano magnetic balls will enter the nucleic acid amplification reaction solution for reaction, which will affect the efficiency of the amplification reaction and put forward higher requirements for the micro-nano magnetic ball process. The nucleic acid analysis chip can avoid the introduction of nano-magnetic balls into the nucleic acid reaction reagent to affect the reaction. In addition, the chip is flexibly configured, and the nucleic acid amplification process can adopt variable temperature amplification or isothermal amplification. Amplification product detection can be performed by real-time fluorescence detection or endpoint detection. Combined with CRISPR technology for analysis of amplification products, the specificity and sensitivity of detection are improved, and multiple detection is realized, the requirements for supporting instruments are simplified, and the results are easy to read.

The chip of the present invention can be used not only for nucleic acid analysis, but also for other molecular biology detection experiments or immune detection experiments with similar operation requirements.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: A schematic diagram of the structure of a nucleic acid analysis chip;

Figure 2: A schematic diagram of the structure of a nucleic acid analysis chip;

Figure 3: A top view of a nucleic acid analysis chip;

Figure 4: Top view of the chip;

Figure 5: Schematic diagram of the conduction valve structure.

Description of the drawings:

100-chip, 101-liquid flow pipeline, 102-waste liquid chamber, 103-air vent, 104-reaction chamber, 105-storage tube interface, 106-slide rail interface, 107-pipeline port, 108-exhaust pipeline,

200-slide rail, 201-slide rail fixing rod,

300-piston pump, 301-piston cylinder, 302-piston rod, 303-piston pump interface,

400-catheter,

500-storage tube, 501-storage tube cover, 502-storage tube body, 503-storage tube through hole,

600-sealing film,

700-sealing gasket, 701-round through hole,

800-conduction valve, 801-moving handle, 802-conduction valve interface, 803-handle fixing rod.

Detailed ways

The present invention will be further explained below in conjunction with specific embodiments and drawings. The following embodiments are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Embodiments of the present invention are as follows:

Example 1

Specifically, as shown in FIG. 1 to FIG. 5 , the nucleic acid analysis chip includes: a chip 100 , a slide rail 200 , a piston pump 300 , a catheter 400 , a storage tube 500 , a sealing film 600 , a sealing gasket 700 , and a conduction valve 800 .

Each storage tube 500 is used to store related reagents; a piston pump 300 is used to transfer and stir the reagents in the storage tube 500; and a slide rail 200 is used to move the conduction valve 800.

The chip 100 is provided with a waste liquid chamber 102 and a reaction chamber 104, which are used to pre-store waste liquid and reaction reagents respectively. The waste liquid chamber 102 is set as a larger capacity chamber, which is only for discharge but not discharge. The chip 100 is provided with a vent hole 103. The storage tube 500 is fixed to the chip 100 using a storage tube interface 105. The top of the storage tube 500 is provided with a tube cover 501 to ensure the sealing of the storage tube, and the bottom is provided with a through hole 503 to connect with the chip 100 through the storage tube interface 105. The storage tube cover 501 is provided with a diaphragm that is both breathable and can prevent nucleic acid molecules from passing through.

The slide rail 200 is tightly connected to the slide rail interface 106 on the chip 100 through the slide rail fixing rod 201, so that the slide rail 200 is fixed on the chip 100. The conduction valve 800 is fixedly installed in the slide rail 200 and can slide linearly in the slide rail.

A plurality of pipe openings 107 are provided on the chip 100 in the slide rail 200. The waste liquid chamber 102, the reaction chamber 104 and each storage tube 500 are connected to each other through a respective liquid flow pipe 101 and a respective pipe opening 107. The air vents 103 are connected to each other through an exhaust pipe 108 and a respective pipe opening 107. The exhaust pipe 108 may be provided with a diaphragm.

The conduction valve 800 includes a movable handle 801 and a conduction valve interface 802. The lower end of the conduction valve interface 802 can be connected to a storage tube interface 105 through a pipeline port 107 inside the chip 100 and a liquid flow pipeline 101, and then connected to the storage tube 500 corresponding to the storage tube interface 105. The upper end of the conduction valve interface 802 is tightly connected to one end of the catheter 400, and the other end of the catheter 400 is tightly connected to the piston pump 300 through the piston pump interface 303.

A sealing gasket 700 is provided on the chip 7 at the bottom of the conduction valve 800. The sealing gasket 700 is kept fixed to ensure the sealing between the conduction valve 800 and the chip 100 in the slide rail 200. There are circular through holes 701 on the sealing gasket 700. The number of the circular through holes 701 is the same as the number of the pipeline openings 107 and corresponds one to one. Each circular through hole 701 corresponds to each pipeline opening 107 provided on the chip 100 in the slide rail 200.

At the same time, the reaction chamber 104 and the waste liquid chamber 102 on the chip 100 are connected to the air holes 103 which are both air permeable and can prevent nucleic acid molecules from passing through.

Furthermore, in actual application, the shape and size of each storage tube 500 can be adjusted according to respective needs, and do not need to be the same size and shape. The material of each storage tube 500 can be glass, plastic, ceramic or metal, etc. according to respective needs. Furthermore, the number of storage tubes 500 can also be adjusted according to actual needs. Furthermore, the shape and size of the storage tube cover 501 can be adjusted according to respective needs, but it is necessary to ensure that the storage tube cover 501 can be tightly connected to the storage tube 500 to ensure sealing. The storage tube cover 501 is provided with a diaphragm that is both breathable and prevents nucleic acid molecules from passing through. An elastic tube cover can also be used. On the one hand, it can prevent the macromolecules (such as nucleic acids) in the device from diffusing to the outside and causing contamination; on the other hand, it can also maintain air pressure balance.

Furthermore, a membrane that is both breathable and can prevent nucleic acid molecules from passing through is provided in the air hole 103, thereby preventing large molecules (such as nucleic acids) in the device from diffusing to the outside and causing pollution; at the same time, it can also maintain air pressure balance or inhale clean gas that does not contain nucleic acid molecules from the outside.

Further, the size of the slide rail 200 can be adjusted as needed, but it is necessary to ensure that the pipe openings 107 opened on the chip 100 are all inside the slide rail 200. The material of the slide rail 200 can be metal, plastic, etc. according to the needs, and the slide rail 200 needs to have a certain rigidity. The design of the slide rail 200 must ensure that the conduction valve 800 can be tightly fitted with it, and the conduction valve 800 can slide linearly along the slide rail 200 in the slide rail 200. Further, a groove can be set in the slide rail 200 so that the conduction valve 800 is embedded in the groove to achieve a tight fit and enable the conduction valve 800 to slide linearly. Steel balls and steel ball rolling tracks can also be used to achieve a tight fit between the slide rail 200 and the conduction valve 800 and enable the conduction valve 800 to slide linearly.

Furthermore, the size of the conduction valve 800 can be adjusted as needed, but the pipe opening 107 must always be sealed by the conduction valve 800 during the movement of the conduction valve 800 in the slide rail 200. This can prevent the reagent in the storage tube 500 from being sucked back into the conduction valve 800 through the liquid flow pipe 101 during the movement of the conduction valve 800, causing contamination. The material of the conduction valve 800 can be metal, glass, ceramic, plastic, etc. as needed.

Furthermore, the material of the sealing gasket 700 is an elastic polymer material, such as rubber, silicone, etc. Furthermore, the material of the sealing gasket 700 should be able to ensure both elasticity and friction resistance.

Furthermore, the sealing film 600 can be strongly adhered to the chip 100 to ensure sealing. Preferably, the surface of the sealing film 600 is hydrophobic and can be made of a transparent material or an opaque material. Preferably, the sealing film can still maintain close adhesion to the chip 100 in a high temperature environment (about 98°C).

Furthermore, the material of the chip 100 may be metal, plastic, glass, etc. Preferably, the chip 100 needs to have a certain rigidity. The shape and size of the liquid flow conduit 101 on the chip 100 can be designed according to actual requirements while ensuring that the storage tube 500 can be connected to the conduction valve 800.

The nucleic acid analysis chip can ensure the sealing of the entire detection process, and different pre-stored reagents will not mix and cause contamination, which is conducive to transportation and storage. The transfer and stirring of different reagents can be easily achieved by combining the conduction valve with the piston pump. In addition, the movement of the conduction valve and the reciprocating push of the piston pump can be automatically controlled by simple mechanical devices without the need for a complex operating system. Therefore, the nucleic acid analysis chip of the present invention has great potential for promotion and use at the grassroots level.

Example 2

The following describes the specific operation process of using the nucleic acid analysis chip to achieve integrated nucleic acid analysis:

As shown in FIG3 , the nucleic acid analysis chip comprises a total of seven storage tubes 500, which are numbered 500a to 500g from left to right. As shown in FIG4 , the liquid flow pipe 101 connected to the storage tube interface 105, the reaction chamber 104, the waste liquid chamber 102, and the air vent 103 are numbered 101a to 101i from left to right.

As shown in Figures 1 to 5, before use, samples, lysis and binding reagents and micro-nano magnetic balls are placed in the storage tube 500a, and a magnet can be placed next to the storage tube 500a to adsorb the micro-nano magnetic balls; magnetic ball cleaning reagents are placed in the storage tube 500b, chip washing reagents are placed in the storage tube 500c, elution reagents are placed in the storage tube 500d, dilution reagents are placed in the storage tube 500e, solidified CRISPR detection reagents for the first target are placed in the storage tube 500f, solidified CRISPR detection reagents for the second target are placed in the storage tube 500g, and solidified nucleic acid amplification reagents are pre-placed in the reaction chamber 104.

Furthermore, the chip 100, the slide rail 200, the storage tube 500, the sealing film 600, and the conduction valve 800 used in the embodiment of the present invention are all made of transparent polymer materials, such as polymethyl methacrylate or polycarbonate.

Furthermore, the CRISPR detection reagent used in the embodiment of the present invention is a CRISPR/Cas12a detection reagent, which includes Cas12a protein, guide RNA, buffer, single-stranded DNA fluorescent probe, RNase inhibitor, sterile water, etc. When the target DNA is present, the guide RNA will accurately pair with the target DNA, thereby activating the cutting activity of the Cas12a protein, cutting a large number of single-stranded DNA fluorescent probes, and generating fluorescent signals.

The nucleic acid analysis test process specifically implemented by the present invention is as follows, but not limited thereto:

1) Unscrew the cap of the storage tube 500a, add the sample to be tested into the storage tube 500a and cover the cap to ensure sealing. At this time, the sample to be tested is cleaved by the cleavage binding reagent to release nucleic acid molecules, thereby allowing the nucleic acid molecules to bind to the micro-nano magnetic balls. Move the conduction valve 800 on the slide rail 200 to drive the conduction valve interface 802 on the conduction valve 800 to align with the liquid flow pipeline 101a of the storage tube 500a, so that the storage tube 500a is connected to the piston cylinder 302 in the piston pump 300 through the liquid flow pipeline 101a and the conduction valve interface 802, and reciprocate to push the piston rod 302, so that the micro-nano magnetic balls can be fully mixed with the nucleic acid molecules released by the sample, ensuring that the nucleic acid molecules can be captured by the micro-nano magnetic balls;

2) Use an external magnet to approach the storage tube 500a, so that the micro-nano magnetic ball is adsorbed on the tube wall and separated from the cleavage binding reagent. Pull out the piston rod 302 from the piston cylinder 302 to allow the cleavage binding reagent to enter the piston cylinder 302 of the piston pump 300, move the conduction valve 800, drive the conduction valve interface 802 to align with the liquid flow pipeline 101g of the waste liquid chamber 102, so that the waste liquid chamber 102 is connected to the piston cylinder 302 in the piston pump 300 through the liquid flow pipeline 101g and the conduction valve interface 802, and push the piston rod 302 to discharge the cleavage binding reagent into the waste liquid chamber 102;

3) Move the conduction valve 800 to align the conduction valve interface 802 with the liquid flow pipe 101b of the storage tube 500b, so that the storage tube 500b is connected with the piston cylinder 302 in the piston pump 300 through the liquid flow pipe 101b and the conduction valve interface 802, and pull out the piston rod 302 to allow a portion of the magnetic ball cleaning reagent in the storage tube 500b to enter the piston cylinder 302 of the piston pump 300;

4) Move the conduction valve 800, drive the conduction valve interface 802 to align with the liquid flow pipe 101a of the storage tube 500a, so that the storage tube 500a is connected to the piston cylinder 302 in the piston pump 300 again after passing through the liquid flow pipe 101b and the conduction valve interface 802, remove the external magnet of the storage tube 500a, push the piston rod 302 back and forth, so that the magnetic ball cleaning reagent enters the storage tube 500a and is fully mixed with the micro-nano magnetic balls, and then use the external magnet to approach the storage tube 500a, so that the micro-nano magnetic balls are adsorbed on the tube wall of the storage tube 500a and separated from the magnetic ball cleaning reagent. Then pull out the piston rod 302 to allow the magnetic ball cleaning reagent to enter the piston cylinder 302 of the piston pump 300;

5) Move the conduction valve 800 to align the conduction valve interface 802 with the liquid flow pipe 101g of the waste liquid chamber 102, so that the waste liquid chamber 102 is connected with the piston cylinder 302 in the piston pump 300 again through the liquid flow pipe 101g and the conduction valve interface 802, and pull out the piston rod 302 to discharge the magnetic ball cleaning reagent into the waste liquid chamber 102;

6) Repeat the above steps 3), 4) and 5), and use the remaining magnetic ball cleaning reagent in the storage tube 500b to clean the micro-nano magnetic balls again; make the magnetic ball cleaning reagent repeatedly enter and flow out of the storage tube 500a, and then use up the magnetic ball cleaning reagent and fully mix with the micro-nano magnetic balls before cleaning and discharging;

7) Move the conduction valve 800 to align the conduction valve interface 802 with the liquid flow pipe 101c of the storage tube 500c, so that the storage tube 500c is connected with the piston cylinder 302 in the piston pump 300 through the liquid flow pipe 101c and the conduction valve interface 802, and pull out the piston rod 302 to allow a portion of the chip washing reagent in the storage tube 500c to enter the piston cylinder 302 of the piston pump 300;

8) Move the conducting valve 800 to align the conducting valve interface 802 with the liquid flow pipe 101b of the storage tube 500b, so that the storage tube 500b is connected again with the piston cylinder 302 in the piston pump 300 through the liquid flow pipe 101b and the conducting valve interface 802, and the piston rod 302 is pushed in and pulled out to discharge the chip washing reagent into the storage tube 500b; then pull out the piston rod 302 to allow the chip washing reagent to enter the piston cylinder 302 of the piston pump 300.

By pushing the piston rod 302 back and forth so quickly, the chip washing reagent cleans the piston pump 300, the catheter 400, the storage tube 500b and the liquid flow pipe 101b, so as to eliminate the influence of the components (such as alcohol) that may be contained in the previous residual magnetic ball washing reagent on the subsequent nucleic acid amplification.

9) After the piston pump 300, the catheter 400, the storage tube 500b and the liquid flow pipe 101b are cleaned, the conducting valve 800 is moved to align the conducting valve interface 802 with the liquid flow pipe 101a of the storage tube 500a, so that the storage tube 500a is connected with the piston cylinder 302 in the piston pump 300 again through the liquid flow pipe 101a and the conducting valve interface 802, and the piston rod 302 is pushed in to discharge the chip washing reagent into the bottom of the storage tube 500a (the liquid cannot enter the tube here, as it will come into contact with the magnetic beads); then the piston rod 302 is pulled out to allow the chip washing reagent to enter the piston cylinder 302 of the piston pump 300.

The piston rod 302 is pushed back and forth quickly to allow the chip washing reagent to clean the liquid flow channel 101a. The number of washing times can be determined according to actual conditions during the entire washing process, while preventing the chip washing reagent from contacting the micro-nano magnetic ball.

In the embodiment of the present invention, the washing is repeated three times.

10) After completing the washing process, move the conduction valve 800 to connect the conduction valve interface 802 and the exhaust pipe 108, so that the external clean gas is connected with the piston cylinder 302 in the piston pump 300 through the air vent 103 and the conduction valve interface 802, and pull out the piston rod 302 to allow the external clean gas to be sucked into the piston cylinder 302 of the piston pump 300; move the conduction valve 800 to align the conduction valve interface 802 with the liquid flow pipe 101a of the storage tube 500a, and then push in the piston rod 302 to allow the external clean gas to be discharged from the piston cylinder 302 of the piston pump 300.

The whole process is to inhale clean air from the air vent, then pump it into the storage tube 500a, and then inhale clean air from the air vent again, and then pump it into the storage tube 500a, and repeat this process to achieve the purpose of inhaling clean gas from the outside to cleanse and blow the chip pipeline and storage tube for further cleaning.

11) After the washing process is completed, the conduction valve 800 is moved to align the conduction valve interface 802 with the liquid flow pipe 101d of the storage tube 500d, so that the storage tube 500d is connected with the piston cylinder 302 in the piston pump 300 through the liquid flow pipe 101d and the conduction valve interface 802, and the piston rod 302 is pulled out to allow the elution reagent in the storage tube 500d to enter the piston cylinder 302 of the piston pump 300;

12) Move the conduction valve 800, drive the conduction valve interface 802 to align with the liquid flow pipeline 101a of the storage tube 500a, so that the storage tube 500a is connected to the piston cylinder 302 in the piston pump 300 again after passing through the liquid flow pipeline 101a and the conduction valve interface 802, remove the external magnet near the storage tube 500a, and quickly push and pull the piston rod 302 back and forth, so that the elution reagent in the piston cylinder 302 repeatedly enters and flows out of the storage tube 500a and is fully mixed with the micro-nano magnetic balls. Afterwards, use an external magnet to approach the storage tube 500a, so that the micro-nano magnetic balls are adsorbed on the tube wall of the storage tube 500a and separated from the elution reagent. At this time, the nucleic acid molecules are eluted by the elution reagent and have entered the elution reagent; pull out the piston rod 302, so that the elution reagent containing the nucleic acid molecules enters the piston cylinder 302 of the piston pump 300;

13) Move the conduction valve 800 to align the conduction valve interface 802 with the liquid flow pipe 101f of the reaction chamber 104, so that the reaction chamber 104 is connected to the piston cylinder 302 in the piston pump 300 through the liquid flow pipe 101f and the conduction valve interface 802, and the piston rod 302 is pushed in, so that the elution reagent containing nucleic acid molecules in the piston cylinder 302 enters the dissolved and solidified nucleic acid amplification reagent in the reaction chamber 104. The reaction chamber 104 performs nucleic acid amplification with the help of an external temperature control device. After the amplification reaction is completed, the piston rod 302 is pulled out to allow the amplification product in the reaction chamber 104 to enter the piston cylinder 302 of the piston pump 300.

14) Move the conducting valve 800 to align the conducting valve interface 802 with the liquid flow pipe 101e of the storage tube 500e, so that the storage tube 500e is connected with the piston cylinder 302 in the piston pump 300 through the liquid flow pipe 101e and the conducting valve interface 802, and the piston rod 302 is pushed in and pulled out quickly and reciprocally, so that the amplification product in the piston cylinder 302 repeatedly enters and flows out of the storage tube 500e and is evenly mixed with the dilution reagent in the storage tube 500e.

15) Move the conduction valve 800, drive the conduction valve interface 802 to align with the liquid flow pipes 101h and 101i of the storage tubes 500f and 500g in turn, and divide the diluted amplified products into the storage tubes 500f and 500g respectively, so that the solidified CRISPR reagent is dissolved for product detection. Although the amplified products are diluted to a certain extent by the dilution reagent, the amount of amplified products after nucleic acid amplification is in the millions, and CRISPR detection is very sensitive, so it will not affect the overall detection sensitivity. At the same time, in the case of high requirements for detection sensitivity, the buffer required for CRISPR detection can be stored in the buffer chamber as a dilution reagent. Although this is a dilution process for the target nucleic acid, since it is diluted using the CRISPR detection buffer, the concentration of each active component in the CRISPR system and the detection target nucleic acid can be ensured to remain unchanged by using a concentrated reagent or a freeze-dried reagent in the subsequent CRISPR detection chamber. In this way, while ensuring the detection sensitivity, the extraction and transportation of trace liquid volumes can be avoided. There have been many reports on the specific components of the buffer for CRISPR detection systems (e.g., Science 360 (2018) 436-439), which can also be further optimized according to the reagent conditions.

During operation, if there is liquid remaining in the pipeline or the volatilization of organic reagents needs to be promoted, the conduction valve 800 can be moved to align the interface 802 with the exhaust pipeline 108, and the piston rod 302 can be pulled outward to form a clean air column by the piston pump 300. Then, the piston rod 302 is quickly pushed inward to drain the residual liquid in the channel or volatilize the organic reagents.

When detecting the amplification product, endpoint detection can be performed to observe whether there is a fluorescent signal to judge the result; real-time fluorescent signal detection can also be performed, but if there are bubbles in the reaction solution, it will interfere with the reading of the fluorescent signal. The image sensor is used to identify the bubbles in the reaction solution to avoid collecting the fluorescent signal at the bubble position, and to achieve spatial distribution detection of the fluorescent signal of the reaction solution, thereby avoiding the interference of possible bubbles on the fluorescent signal detection. If bubbles are detected, the chip's pump valve can be further used to extract liquid from the detection chamber to eliminate the bubbles in the chamber, thereby eliminating the interference of bubbles and other factors on the fluorescent signal detection.

The image sensor here can be a CMOS or CCD type product commonly seen on the market, and the pixel specifications selected can be determined according to the requirements for the detection accuracy of bubbles and impurities, etc. In general, common megapixel-level image sensors can meet the detection requirements.

Example 3

Take the dual detection of CaMV35S sequence and Lectin sequence in transgenic soybean leaves as an example.

As shown in FIG. 1 to FIG. 5 , the material used for most of the chip is polymethyl methacrylate, the piston pump is a 1 ml disposable medical syringe, and the material used for the storage tube is polypropylene.

Nucleic acid amplification adopts isothermal amplification method, and nucleic acid amplification reagents are: Bst DNA polymerase 16 units, 10xThermopol reaction buffer 5 microliters, betaine 0.8 mol/L, dNTP 0.35 millimoles per liter, magnesium sulfate 2 millimoles per liter, primer mixture (including 4 inner primers, 2 inner primers of 1.6 micromol per liter each, 2 inner primers of 0.8 micromol per liter each; 4 outer primers, 2 outer primers of 0.2 micromol per liter each, 2 outer primers of 0.1 micromol per liter each; 4 loop primers, 2 loop primers of 0.4 micromol per liter each, 2 loop primers of 0.2 micromol per liter each). These reagents are freeze-dried to form solids and placed in the reaction chamber 104 in advance.

The CRISPR detection reagent used is the CRISPR/Cas12a detection reagent, whose main components are: 200 nanomoles per liter of Cas12a protein, 10 microliters of 10x NEBuffer 2.1 buffer, 2.5 micromoles per liter of single-stranded DNA probe, 600 nanomoles per liter of guide RNA, and 20 units of RNase inhibitor. These reagents are freeze-dried to form solids and placed in storage tubes 500f and 500g in advance. Storage tube 500f detects the CaMV35S sequence, and storage tube 500g detects the Lectin sequence.

The nano magnetic spheres are SeraSil-MagTM SpeedBeads carboxyl magnetic spheres from GE Company, and other commercial magnetic spheres or self-made ones can also be used.

Before use, place 200 μL of lysis reagent (4 mol/L guanidine thiocyanate, 50 mmol/L tris(hydroxymethyl)aminomethane hydrochloride, 20 mmol/L ethylenediaminetetraacetic acid, pH 7.6-8.0), 200 μL of isopropanol and 10 μL of nanomagnetic balls in storage tube 500a, place 800 μL of magnetic ball cleaning reagent (80% ethanol) in storage tube 500b, place 1.2 ml of chip washing reagent (sterile water) in storage tube 500c, place 50 μL of elution reagent (sterile water) in storage tube 500d, and place 50 μL of dilution reagent (sterile water or CRISPR detection buffer, such as NEBuffer 2.1 buffer) in storage tube 500e.

The soybean leaves were crushed, and 100 μl of the supernatant was added to the storage tube 500a. The tube was capped, and then the piston pump was reciprocated to achieve full mixing of the sample to be tested and the nanomagnetic spheres, and incubated for 10 minutes.

The operation process is referred to Example 2, wherein, in order to ensure that the liquid is evenly mixed, the number of times the syringe is pushed back and forth is at least ten times. When the micro-nano magnetic ball is cleaned with the magnetic ball cleaning reagent, 400 microliters are drawn each time for cleaning, and a total of two cleanings are performed. When the piston pump 300 and the catheter 400 are washed with the chip washing reagent, 300 microliters are drawn each time for washing, and a total of three cleanings are performed. When washing the liquid flow conduit 101a, 100 microliters are drawn each time for washing, and a total of three cleanings are performed.

When performing CRISPR detection, a portable fluorescence observation device can be used to observe the storage tubes 500f and 500g. Positive amplification will produce a fluorescent signal, while negative amplification will not produce a fluorescent signal.

For experimental information such as primer sequence design and guide RNA sequence design of this detection, please refer to reference (Biosensors and Bioelectronics 157 (2020) 112153).

The specific implementation scheme of the nucleic acid analysis chip of the present invention for nucleic acid analysis is described in detail above, but the above embodiments are exemplary, and the chip with integrated nucleic acid analysis described in the present invention is not limited to the specific details in the above embodiments, and cannot be understood as a limitation of the present invention. In the design of the present invention, similar modifications, replacements, variations, etc. can be made to the technical solution of the present invention, which all belong to the protection scope of the present invention.

Claims (3)

1. An integrated nucleic acid analysis chip, comprising:

the chip is provided with an air vent, a reaction cavity, a waste liquid cavity, an interface connected with the storage pipe, a sliding rail interface, a liquid flow pipeline and an exhaust pipeline, and a plurality of pipeline ports, wherein the waste liquid cavity, the reaction cavity and each storage pipe are respectively communicated with one pipeline port after passing through one liquid flow pipeline respectively, and the air vent is communicated with one pipeline port through the exhaust pipeline;

the storage tube is arranged on the chip through an interface and used as a cavity and used for storing a detection sample or a reagent required in a reaction;

The slide rail is tightly fixed with the chip through a slide rail interface and is used for moving the conduction valve, so that selective conduction between the vent holes, the reaction cavity, the waste liquid cavity and different storage pipes and the conduction valve through a liquid flow pipeline/an exhaust pipeline of the chip is realized;

the guide valve is positioned in the sliding rail and slides in the sliding rail;

The piston pump is in sealing connection with the conducting valve through a conduit and is used for pumping or discharging liquid;

The on-valve is provided with a movable handle and only one interface communicated with the pipeline opening of the chip, the on-valve is moved by the movable handle, the only one interface on the on-valve is aligned to be connected with the corresponding pipeline opening on the chip, and the only one interface on the on-valve is always communicated with the piston pump, so that selective communication among the air holes, the reaction cavity, the waste liquid cavity, different storage pipes and the piston pump is realized;

The chip is provided with a washing cavity, a chip washing reagent is arranged in the washing cavity, the washing cavity is communicated with a pipeline port after passing through a liquid flowing pipeline, and then is selectively communicated with a conducting valve to wash the pipeline in the chip and other cavities;

arranging a plurality of CRISPR detection cavities on a chip, and after the nucleic acid amplification reaction is finished, pumping amplification products into each CRISPR detection cavity respectively to realize the multiple detection of the end point;

The chip is provided with an exhaust pipeline with a diaphragm and an air vent connected with the exhaust pipeline, and on the premise that the chip and the outside are not subjected to nucleic acid molecule exchange, the air vent is used for sucking the outside cleaning gas into the chip through the exhaust pipeline, so that the pipeline and the cavity in the chip are scrubbed and blown.

2. The integrated nucleic acid analysis chip of claim 1, wherein:

The chip is made transparent at each cavity and is provided with an image sensor, and the image sensor is used for detecting bubbles by using fluorescent signals in the cavities; in case that the existence of bubbles is found, the pump valve on the chip is further utilized to pump the liquid from the chamber so as to eliminate the bubbles in the chamber.

3. The integrated nucleic acid analysis chip of claim 1, wherein:

And a buffer chamber is arranged on the chip, and dilution storage is carried out through the buffer chamber.