CN113684118A

CN113684118A - Integrated nucleic acid analysis chip

Info

Publication number: CN113684118A
Application number: CN202110797647.1A
Authority: CN
Inventors: 吴坚; 伍辉; 钱斯雯洁
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2021-11-23
Anticipated expiration: 2041-07-14
Also published as: CN113684118B

Abstract

The invention discloses an integrated nucleic acid analysis chip. The chip is provided with air holes, a reaction cavity, a waste liquid cavity and other cavities, a storage tube connecting interface, a slide rail interface, a liquid flowing pipeline, an exhaust pipeline and the like, and a plurality of pipeline ports, and the cavities are communicated with one pipeline port through respective pipelines; the storage tube is arranged on the chip through an interface to be used as a cavity; the slide rail and the chip are tightly fixed through a slide rail interface, and the conduction valve is positioned in the slide rail to slide and is used for selectively communicating the cavity and the piston pump; the piston pump is connected with the conduction valve in a sealing way through a guide pipe. The invention can complete the complete steps of nucleic acid extraction, amplification and detection in the chip, can effectively avoid possible pollution, and can better remove the reagent which has the inhibition effect on the subsequent amplification, thereby avoiding the influence on the amplification and improving the efficiency and the accuracy.

Description

Integrated nucleic acid analysis chip

Technical Field

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

Background

Nucleic acids are carriers of genetic information in organisms. By detecting nucleic acids, organisms can be accurately identified. At present, nucleic acid analysis and detection are widely applied to the fields of food safety detection, environmental monitoring, medical diagnosis and the like, and play an important role. Nucleic acid detection methods based on nucleic acid amplification technology are the most commonly used nucleic acid analysis detection methods, but the detection process often comprises complex nucleic acid extraction operation, repeated pipetting is needed, and cross contamination is easily caused in the process of transferring liquid. In order to simplify the nucleic acid extraction procedure, some researchers directly perform nucleic acid amplification on an untreated sample or perform nucleic acid amplification after treating the sample by a simple lysis method (heating, alkaline lysis). Although this can simplify the operation, many impurities still exist in the obtained nucleic acid sample, which may affect the efficiency and detection sensitivity of the subsequent nucleic acid amplification reaction. In addition, some reagents having inhibitory effect on subsequent amplification are used in the nucleic acid extraction process, and long operation time is also required for removing the reagents.

For the detection of amplification products, it is mainly relying on a precise fluorescence reading device to obtain a real-time fluorescence curve, and determining the result. Some end-point detection methods using turbidity of a reaction solution, color change of the reaction solution, and the like are convenient to operate, but have the problems of lack of specificity, insufficient distinction between positive and negative results, and the like. CRISPR (clustered regularly interspaced short palindromic repeats) is an immune system present in most bacteria and archaea. The system mainly comprises Cas protein and crRNA. Some Cas proteins (e.g., Cas12, Cas13, and Cas14) are also capable of cleaving non-target single-stranded DNA (i.e., a bypass cleavage effect) when cleaving a target nucleic acid sequence under the guidance of crRNA. Taking advantage of this property, some researchers have combined nucleic acid amplification reactions with CRISPR technology for achieving detection of target nucleic acids. However, because the two systems have the problem of temperature incompatibility, CRISPR reagents are often introduced after the nucleic acid amplification reaction is finished, and aerosol pollution is easily generated.

Currently, some integrated nucleic acid analysis and detection devices are established, such as the cobalt Liat of rochon, merriee, ipomatic system of saint xiang, and nucleic acid amplification detection analyzer of yousida, hang. However, these devices still have some defects such as large size, high price, and complicated design, which cannot fully satisfy the basic requirements. Therefore, it is important to develop an integrated nucleic acid analysis device which is low in cost, multifunctional, simple in operation, and easy in result judgment, and which enables the popularization of nucleic acid analysis and detection, particularly in resource-limited regions.

Disclosure of Invention

In order to solve the problems existing in the background art, the invention aims 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 detected nucleic acid and the nucleic acid amplification product from leaving the detection chip and entering the surrounding environment, thereby avoiding possible pollution. Meanwhile, the chip can remove some reagents which have inhibition effect on subsequent amplification and are used in the nucleic acid extraction process, so that the influence on amplification is avoided. The nucleic acid amplification process can adopt temperature-variable amplification or isothermal amplification. The detection of the amplification product can be performed by real-time fluorescence detection or end-point detection.

The invention is realized by the following steps:

an integrated nucleic acid analysis chip:

the chip is provided with a pump and a slide rail type structure, and the slide rail type structure is used for controlling the pump to be communicated with different cavities, so that the selective communication of the pump and the different cavities on the chip is realized.

The nucleic acid amplification process can adopt temperature-variable amplification or isothermal amplification. The detection of the amplification product can be performed by real-time fluorescence detection or end-point detection.

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

The chip is provided with a plurality of CRISPR detection cavities which respectively store CRISPR detection reagents aiming at different nucleic acid targets. After the nucleic acid amplification reaction is finished, the amplification products are respectively pumped into each CRISPR detection cavity by using a piston pump, so that the end point multiple detection is realized. The invention utilizes CRISPR technology to carry out end point detection on nucleic acid amplification products.

The chip is transparent at the cavity and is provided with an image sensor, and the image sensor is used for detecting bubbles of fluorescent signals in the cavity; when the existence of bubbles is found, the liquid in the chamber is further extracted by a pump valve on the chip to eliminate the bubbles in the chamber, so that the interference of the factors such as the bubbles on the detection of the fluorescence signal is eliminated.

The chip is provided with a washing chamber, a chip washing reagent is arranged in the washing chamber, a pipeline in the chip and other chambers except the washing chamber are washed, and the reagent which has an influence on subsequent nucleic acid amplification and is used in the nucleic acid experiment process is removed completely, so that the influence on the nucleic acid amplification is avoided.

The chip is equipped with the exhaust duct who takes the diaphragm and connects exhaust duct's bleeder vent, does not have the nucleic acid molecule exchange with the external world under the prerequisite, inhales external clean gas to the chip inside from the bleeder vent via exhaust duct from the external world, and then washes to the inside pipeline of chip and cavity and swing and blow.

The present invention can accommodate and be applied to all procedures and reagents for nucleic acid extraction operations, 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 air holes, a reaction cavity, a waste liquid cavity and other cavities, and is also provided with an interface, a slide rail interface, a liquid flow pipeline, an exhaust pipeline and the like which are connected with the storage pipes, 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 respective liquid flow pipeline, and the air holes are communicated with one respective pipeline port through the exhaust pipeline;

the reaction cavity and the waste liquid cavity on the chip are communicated with the air holes, and the air holes are internally provided with a diaphragm mechanism which is air permeable and can prevent nucleic acid molecules from passing through.

The storage tube is arranged on the chip through an interface to be used as a chamber and used for storing a detection sample or a reagent required in the reaction; and the bottom of the storage tube is provided with a through hole which is hermetically connected with an interface on the chip. Furthermore, the storage tube is provided with a tube cover, the tube cover is provided with a diaphragm which can be used for ventilating and preventing nucleic acid molecules from passing through, and the storage tube is connected with the tube cover in a sealing way.

The slide rail is tightly fixed with the chip through a slide rail interface and is used for moving the conduction valve, so that air holes, the reaction cavity, the waste liquid cavity and different storage tubes are selectively conducted with the interface on the conduction valve through a liquid flow pipeline/an exhaust pipeline of the chip;

the conduction valve is positioned in the slide rail and slides linearly in the slide rail, and a sealing gasket is arranged between the conduction valve and the chip to ensure the sealing property through sealing; furthermore, the sealing gasket is provided with circular through holes which are respectively communicated with the liquid flow pipeline openings on the chip.

And the piston pump is connected with the conduction valve in a sealing way through a guide pipe and is used for pumping or discharging liquid. Further, the piston is pushed and pulled in a reciprocating mode, and liquid can be mixed evenly.

Still include the seal membrane, reaction chamber, waste liquid chamber, liquid flow pipeline, exhaust duct, bleeder vent are all seted up through lining up at the chip top surface and are formed, the seal membrane be used for with the top surface opening of reaction chamber, waste liquid chamber, liquid flow pipeline, exhaust duct, bleeder vent on the chip is sealed. The sealing membrane may be a rigid solid or a flexible membrane.

The conduction valve is provided with a movable handle and only one interface communicated with a chip pipeline port, the conduction valve is moved by the movable handle on the conduction valve, only one interface on the conduction valve is aligned and connected with a corresponding pipeline port on the chip, only one interface on the conduction valve is communicated with the piston pump all the time, and therefore air holes, a reaction cavity, a waste liquid cavity and differences are achieved, and the storage pipe is selectively communicated with the piston pump.

The chip is provided with a washing chamber, a chip washing reagent is arranged in the washing chamber, the washing chamber is communicated with a pipeline port through a respective liquid flowing pipeline and then selectively communicated with a conducting valve, and the pipeline in the chip and other chambers except the washing chamber are washed.

The bleeder vent intercommunication external clean gas, from the external clean gas of external follow bleeder vent via exhaust duct inhale to the chip inside, and then wash and swing and blow the pipeline and the cavity of chip inside.

The chip is made transparent at each cavity and is provided with an image sensor, and the image sensor is used for detecting bubbles of fluorescent signals in the cavities; when the existence of bubbles is found, the liquid in the chamber is further extracted by a pump valve on the chip to eliminate the bubbles in the chamber, so that the interference of the factors such as the bubbles on the detection of the fluorescence signal is eliminated.

A buffer chamber is arranged on the chip, and diluted storage is carried out through the buffer chamber. Dilute nature storage is carried out through buffer chamber, under the prerequisite that does not reduce detectivity, avoids the extraction and the transportation of trace liquid volume to improve the reliability that detects.

The buffer chamber is internally provided with a diluting reagent, and the amplification product can be diluted to a certain degree and then is introduced into a storage tube which is provided with a CRISPR detection reagent for detection. The diluting reagents stored in these buffer chambers can be the buffer required for CRISPR detection. Although the method is a dilution process of the target nucleic acid, the concentration of each active ingredient in the CRISPR system and the concentration of the target nucleic acid to be detected are constant by using a concentrated reagent or a freeze-dried reagent in a subsequent CRISPR detection cavity because the CRISPR detection buffer is used for dilution. Therefore, the detection sensitivity is ensured, and the extraction and the transfer of the volume of the trace liquid can be avoided.

When a sample is detected, firstly, the sample is put into a first storage tube of a chip for cracking, and a cracking binding reagent and a micro-nano magnetic sphere are pre-stored in the storage tube and are used for cracking the sample, releasing nucleic acid molecules and binding the nucleic acid molecules with the micro-nano magnetic sphere. The conduction valve is moved on the slide rail, so that the first storage tube containing the sample is communicated with the piston cylinder through the conduction valve, the other end of the guide tube is communicated with the piston pump, and the piston is pushed and pulled in a reciprocating mode to enable the micro-nano magnetic ball and the sample to be in full contact and uniform mixing, so that as much nucleic acid as possible is adsorbed on the surface of the micro-nano magnetic ball. Further, heating cracking can be carried out according to the requirement of a sample in the cracking process;

and then, an external magnet or electromagnet is used for approaching the pipe wall of the first storage pipe, and the micro-nano magnetic ball in the first storage pipe is adsorbed on the inner wall of the pipe, so that the micro-nano magnetic ball is separated from the cracking binding reagent. The piston is pulled outward, causing the cleaved binding reagent to exit the storage tube and enter the piston barrel. The guide valve is moved, and the waste liquid cavity is communicated with the piston cylinder through the guide valve. Pushing the piston inwards to make the cracking combined reagent in the piston cylinder enter the waste liquid cavity;

then, the conduction valve is moved, and the second storage tube is communicated with the piston cylinder through the conduction valve. The second storage tube is pre-stored with magnetic ball cleaning reagent for removing protein, polysaccharide and other impurities possibly adsorbed on the surface of the magnetic ball. And the piston is pulled outwards, so that a part of the magnetic ball cleaning reagent enters the piston cylinder. The guide valve is moved, and the first storage tube is communicated with the piston cylinder through the guide valve. The piston is pushed inwards, so that the magnetic ball cleaning reagent in the piston cylinder enters the first storage tube. Moving away an external magnet or electromagnet, and pushing and pulling the piston in a reciprocating manner to fully contact and uniformly mix the micro-nano magnetic ball and the magnetic ball cleaning reagent for a period of time, so as to realize the first cleaning of the micro-nano magnetic ball;

and then, adsorbing the micro-nano magnetic balls in the first storage tube on the inner wall of the tube by using an external magnet or electromagnet to separate the micro-nano magnetic balls from the magnetic ball cleaning reagent. The piston is pulled outwards, so that the magnetic ball cleaning reagent leaves the first storage tube and enters the piston cylinder. And moving the conduction valve to enable the waste liquid cavity to be communicated with the piston cylinder through the conduction valve. Pushing the piston inwards to make the magnetic ball cleaning reagent enter the waste liquid cavity;

then, the second storage tube communicates with the piston cylinder through a conduction valve. And the piston is pulled outwards, so that the residual magnetic ball cleaning reagent in the second storage pipe enters the piston cylinder. And repeating the steps to realize secondary cleaning of the micro-nano magnetic balls, and discharging the magnetic ball cleaning reagent into a waste liquid cavity. In practice, the specific number of times of cleaning can be set as required.

And then, a chip washing reagent is pre-stored in the third storage tube and is used for washing the liquid flow pipeline and the residual magnetic ball cleaning reagent in the piston cylinder. In general, the magnetic ball cleaning reagent contains an organic reagent such as alcohol. These organic reagents may inhibit subsequent nucleic acid amplification. In the chip, these magnetic bead washing reagents may remain in the channel of the chip and enter subsequent reagents, thereby causing an influence such as inhibition of nucleic acid amplification. In the design of the chip, a washing cavity, a chip washing reagent and a washing step are further innovatively arranged, and a chip pipeline and a piston pump are cleaned so as to eliminate the possible influence caused by the residual magnetic ball washing reagent. Specifically, the conduction valve is moved to communicate the third storage tube with the piston cylinder via the conduction valve. And pulling the piston outwards to make a part of the chip washing reagent enter the piston cylinder, and moving the conduction valve to make the second storage tube communicated with the piston cylinder through the conduction valve. And the piston is pushed and pulled in a reciprocating manner, so that the chip washing reagent can fully wash the piston pump. Finally, the piston is pulled outwards, so that the washing reagent leaves the second storage tube and enters the piston cylinder. And moving the conduction valve to enable the waste liquid cavity to be communicated with the piston cylinder through the conduction valve. The piston is pushed inwards to make the chip washing reagent enter the waste liquid cavity. Further, the washing process can be repeated for multiple times according to the actual washing condition.

Then, the conduction valve is moved to communicate the third storage tube with the piston cylinder through the conduction valve. And pulling the piston outwards to make a part of the chip washing reagent enter the piston cylinder, and moving the conduction valve to make the first storage tube communicated with the piston cylinder through the conduction valve. And pushing and pulling the piston back and forth to ensure that the chip washing reagent fully washes the liquid flow channel connected with the first storage tube. And finally, pulling the piston outwards to enable the chip washing reagent to enter the piston cylinder. And moving the conduction valve to enable the waste liquid cavity to be communicated with the piston cylinder through the conduction valve. The piston is pushed inwards to make the chip washing reagent enter the waste liquid cavity. Further, during the washing process, care should be taken to avoid the chip washing reagent from contacting the micro-nano magnetic ball in the first storage tube. Further, the washing process can be repeated for multiple times according to the actual washing condition.

After the pipeline and the like are washed, clean air can be sucked from the outside through the diaphragm by utilizing the piston pump through the exhaust pipeline, and the pipeline and the micro-nano magnetic ball are further washed, swung and blown, so that harmful residual substances possibly existing on the surfaces of the pipeline and the magnetic ball are further eliminated.

Clean air is sucked from the outside through the diaphragm by the piston pump through the exhaust pipeline, and the operation of stirring, uniformly mixing and the like of the solution in the cavity can also be performed.

Then, the conduction valve is moved to communicate the fourth storage tube with the piston cylinder through the conduction valve. And an elution reagent is prestored in the fourth storage tube and is used for eluting the nucleic acid molecules adsorbed on the surfaces of the micro-nano magnetic spheres. The piston is pulled outwards, so that the elution reagent enters the piston cylinder. The guide valve is moved to communicate the first storage tube with the piston cylinder through the guide valve. And pushing and pulling the piston back and forth to make the micro-nano magnetic ball and the elution reagent fully contact and uniformly mix for a period of time, so that the elution of the nucleic acid molecules adsorbed on the surface of the micro-nano magnetic ball is realized, and the nucleic acid molecules enter the elution reagent.

And then, adsorbing the micro-nano magnetic balls in the first storage tube on the inner wall of the first storage tube by using an external magnet or electromagnet to separate the micro-nano magnetic balls from the elution reagent. The plunger is pulled outward, causing the elution reagent to exit the storage tube and enter the plunger barrel. And moving the conduction valve to enable the reaction cavity to be communicated with the piston cylinder through the conduction valve. The reaction chamber is pre-stored with amplification reagents for performing a nucleic acid amplification reaction. The piston is pushed inwards to make the elution reagent enter the reaction chamber, and then the amplification reaction is carried out through an external temperature control device. Compared with a standard kit nucleic acid extraction method, the method avoids transferring trace elution reagents for nucleic acid amplification, and improves the reliability of detection. Further, one end of the reaction chamber is connected with a vent hole which is permeable to air and can prevent nucleic acid molecules from passing through.

Or a buffer chamber can be arranged on the chip according to the requirement, nucleic acid amplification buffer solution is stored in the buffer chamber, and the elution reagent containing nucleic acid is diluted to a certain extent and then pumped into the reaction cavity for amplification. The reaction system is 50 microliter as designed, and the nucleic acid eluent containing the sample to be detected is 5 microliter. In order to avoid the error and fluctuation caused by the tiny volume of the chip, 50 microliters of nucleic acid eluent can be pumped into the buffer chamber containing 450 microliters of amplification buffer solution, and after the nucleic acid eluent is uniformly mixed, 50 microliters of mixed solution is pumped into the reaction chamber. Thus, the detection sensitivity is ensured, the extraction and the transfer of the volume of the trace liquid can be avoided, and the possible detection error is reduced;

then, after the nucleic acid amplification is finished, the piston is pulled outwards, so that the amplified product leaves the reaction chamber and enters the piston cylinder. The conduction valve is moved to communicate the fifth storage tube with the piston cylinder through the conduction valve. And a CRISPR detection reagent is prestored in the fifth storage tube and is used for carrying out end point detection on the amplification product. The piston is pushed inwards, so that the amplification product enters the storage tube for reaction. Furthermore, a plurality of storage tubes can be arranged on the chip and used for storing CRISPR detection reagents aiming at different nucleic acid targets, so that multiple nucleic acid detection is realized.

Further, the CRISPR detection reagent comprises a Cas protein, a buffer, a guide RNA, a single-stranded nucleic acid fluorescent probe, an rnase inhibitor, sterile water, and the like. When the target nucleic acid exists, the target nucleic acid and the guide RNA carry out base complementary pairing, so that the activity of the Cas protein is activated, the single-stranded nucleic acid fluorescent probe is cut, and a fluorescent signal is generated. Further, the CRISPR detection reagent can be cured (e.g., freeze-dried) for storage.

Further, in the optical detection, detection may be affected by the presence of air bubbles, particles, and the like in the reaction solution. The air bubbles may be generated by the detection reaction, or may be generated by various factors such as dissolution of the lyophilized reagent and pump and valve action during liquid transport. If the common single-beam real-time fluorescent signal reading is carried out, if the light beam just falls on the bubble, the bubble can influence the reading of the fluorescent signal, thereby influencing the judgment of the experimental result. Therefore, the invention introduces an image sensor to carry out multi-point detection on the fluorescence signal in the detection cavity in the detection process. By realizing the detection of the spatial distribution of the fluorescence signals of the reaction liquid, the fluorescence signals at the bubble positions are prevented from being collected, so that the interference of the possibly existing bubbles on the detection of the fluorescence signals is avoided;

the specific detection algorithm is as follows:

1. reading a fluorescent signal of an area irradiated by incident light excitation light in the detection chamber by using an image sensor, and respectively recording and storing the fluorescent signal of each pixel point in the image sensor;

2. according to the fluorescence signals of all the pixel points, the average signal intensity of the pixels is obtained;

3. setting a certain judgment standard, such as taking the plus or minus 10% of the average signal intensity as a normal signal selection range; for the pixel points with the fluorescence value deviation larger than the range, the fluorescence signals can be considered to be caused by bubbles, particles and the like and are judged as abnormal points; in practice, the selection of the specific signal deviation range of the normal pixel point can be determined according to the design of a chip, a detection cavity and the like;

4. after removing the signals of the abnormal points, recalculating the average fluorescence intensity of the normal signal points as detection signals;

5. furthermore, after the foreign matter such as bubbles is detected in step 3, the pump valve of the chip may be further used to pump liquid into the detection chamber to eliminate the bubbles in the chamber, thereby eliminating the interference of the factors such as bubbles on the detection of the fluorescent signal.

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

furthermore, in the process that the conduction valve moves in the slide rail, when an interface communicated with the guide pipe on the conduction valve is not communicated with a pipeline port at the bottom of the conduction valve on the chip, the pipeline port at the bottom of the conduction valve is in a sealed state; when the interface communicated with the guide pipe on the conduction valve is communicated with one pipeline port at the bottom of the conduction valve on the chip, other pipeline ports at the bottom of the conduction valve are also in a sealed state;

further, the control of the magnet or electromagnet, the movement of the conduction valve and the pushing and pulling of the piston pump can be controlled by external mechanical devices;

furthermore, the selected chip washing reagent does not inhibit the subsequent amplification reaction;

furthermore, the chip is provided with an exhaust pipeline which is connected with an air vent which is air permeable and can prevent nucleic acid molecules from passing through. The device can be used for enabling the piston pump to form a clean air column, then pushing the piston inwards, ensuring that some liquid reagents remained in a liquid flow pipeline on the chip are thoroughly and cleanly discharged, and simultaneously can also be used for promoting the volatilization of organic reagents remained 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 with the capability of capturing nucleic acid, such as silicon dioxide spheres, filter paper, silica gel films and the like.

The invention skillfully designs and arranges the slide rail structure, and controls each reaction process and treatment in turn by matching with the motion of the piston, thereby being capable of well and conveniently realizing experimental operation, avoiding pollution, being capable of better and thoroughly removing the reagent which has the inhibiting effect on the subsequent amplification, and improving the reaction efficiency and the reaction accuracy.

According to the invention, the slide rail is arranged to be matched with the washing chamber, so that the chip can be well washed completely, rather than only the magnetic ball is cleaned.

The air inlet cleaning device is provided with the air holes and the exhaust pipeline in a matched manner by arranging the slide rails, and the function of air inlet cleaning can be realized.

The nucleic acid analysis chip of the invention is characterized in that: can complete the complete steps of nucleic acid extraction, amplification and detection; and can prevent the detected nucleic acid and the nucleic acid amplification product from leaving the detection chip and entering the surrounding environment, thereby avoiding possible pollution. Meanwhile, the chip can remove some reagents which have inhibition effect on subsequent amplification and are used in the nucleic acid extraction process, so that the influence on amplification is avoided. In some commercial devices, the micro-nano magnetic spheres enter the nucleic acid amplification reaction solution for reaction, which affects the amplification reaction efficiency and puts higher requirements on the micro-nano magnetic sphere process. The nucleic acid analysis chip can avoid the influence on the reaction caused by introducing the nano magnetic ball into the nucleic acid reaction reagent. The chip is flexible in configuration, and the nucleic acid amplification process can adopt temperature-variable amplification or isothermal amplification. The detection of the amplification product can be performed by real-time fluorescence detection or end-point detection. The CRISPR technology is combined for analyzing the amplification product, so that the specificity and the sensitivity of detection are improved, multiple detection is realized, the requirement on a matched instrument is simplified, and the result is easy to read.

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

Drawings

FIG. 1: a schematic structure diagram of a nucleic acid analysis chip;

FIG. 2: a schematic structure diagram of a nucleic acid analysis chip;

FIG. 3: a top view of a nucleic acid analysis chip;

FIG. 4: a top view of the chip;

FIG. 5: the structure of the conduction valve is schematically shown.

Description of the drawings:

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

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

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

400-a guide pipe, wherein the guide pipe is arranged on the guide pipe,

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

600-a sealing film, wherein the sealing film,

700-a sealing gasket, 701-a circular through hole,

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

Detailed Description

The invention is further explained below with reference to specific embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

The examples of the invention are as follows:

example 1

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

Wherein the respective storage tubes 500 are used to store the relevant reagents; the piston pump 300 is used for transferring and stirring the reagent in the storage tube 500; the movement of the conduction valve 800 is accomplished using the sliding rail 200.

The chip 100 is provided with a waste liquid chamber 102 and a reaction chamber 104 for storing waste liquid and reaction reagent in advance, respectively, and the waste liquid chamber 102 is configured as a larger-capacity chamber for only discharging, not discharging. The chip 100 is provided with a vent 103. The storage tube 500 is secured to the chip 100 using the storage tube interface 105. The storage tube 500 is provided with a tube cover 501 at the top to ensure the storage tube is sealed, and a through hole 503 at the bottom to enable connection with the chip 100 through the storage tube interface 105. The lid 501 is provided with a membrane that is both gas permeable and prevents passage of nucleic acid molecules.

The slide rail 200 is tightly connected to the slide rail interface 106 of 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.

The chip 100 in the slide rail 200 is provided with a plurality of pipe ports 107, the waste liquid chamber 102, the reaction chamber 104, and each storage pipe 500 are communicated with a respective one of the pipe ports 107 through a respective one of the liquid flow pipes 101, the air vents 103 are communicated with a respective one of the pipe ports 107 through the exhaust pipe 108, and the exhaust pipe 108 may be provided with a diaphragm.

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

The chip 7 at the bottom of the conduction valve 800 is provided with a sealing gasket 700, and the sealing gasket 700 is kept fixed, so that the sealing performance between the conduction valve 800 and the chip 100 in the slide rail 200 can be ensured. The sealing gasket 700 is provided with circular through holes 701, the number of the circular through holes 701 is the same as that of the pipeline ports 107, and the circular through holes 701 correspond to the pipeline ports 107 arranged on the chip 100 in the slide rail 200.

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

Further, in practical application, the shape and size of each storage tube 500 can be adjusted according to their own needs, and do not need to be the same. The material of each storage tube 500 may be glass, plastic, ceramic, metal, or the like, as required. Further, the number of the storage tubes 500 can be adjusted according to actual requirements. Further, the shape and size of the storage tube cover 501 may be adjusted according to the needs, but it is necessary to ensure that the storage tube cover 501 can be tightly connected to the storage tube 500, thereby ensuring the sealing performance. A diaphragm which is air permeable and can prevent nucleic acid molecules from passing through is arranged in the storage tube cover 501, and an elastic tube cover can also be adopted, so that on one hand, macromolecules (such as nucleic acid) in the device can be prevented from diffusing to the outside to cause pollution; on the other hand, the air pressure can be kept balanced.

Furthermore, the air holes 103 are provided with air-permeable membranes which can prevent nucleic acid molecules from passing through, so that macromolecules (such as nucleic acid) in the device can be prevented from diffusing to the outside to cause pollution; at the same time, the pressure balance can be maintained, or the clean gas without nucleic acid molecules can be sucked from the outside.

Further, the size of the slide rail 200 can be adjusted as required, but it is necessary to ensure that the pipe openings 107 formed in the chip 100 are all inside the slide rail 200. The material of the slide rail 200 may be metal, plastic, etc. according to the requirement, and the slide rail 200 needs to have a certain rigidity. The design of the sliding rail 200 is to ensure that the conduction valve 800 can be tightly fitted with the sliding rail 200, and the conduction valve 800 can slide linearly along the sliding rail 200 in the sliding rail 200. Further, a groove may be formed in the slide rail 200 to allow the conduction valve 800 to be inserted into the groove to achieve tight fitting and allow the conduction valve 800 to slide linearly. The steel ball and the steel ball rolling rail can be used to realize the tight embedding of the slide rail 200 and the conduction valve 800 and the linear sliding of the conduction valve 800.

Further, the size of the conduction valve 800 can be adjusted as required, but during the movement of the conduction valve 800 in the slide rail 200, it is necessary to ensure that the pipe opening 107 can be sealed by the conduction valve 800 at all times. This prevents the reagent in the storage tube 500 from being sucked back into the conduction valve 800 through the liquid flow pipe 101 and contaminating the conduction valve 800 during the movement of the conduction valve 800. The conduction valve 800 may be made of metal, glass, ceramic, plastic, etc. as required.

Further, the sealing gasket 700 is made of elastic polymer material, such as rubber, silica gel, etc. Further, the material of the sealing gasket 700 should ensure both elasticity and friction resistance.

Further, the sealing film 600 can be strongly adhered to the chip 100, and the sealing property can be ensured. Preferably, the surface of the sealing film 600 is hydrophobic, and may be a transparent material or an opaque material. Preferably, the sealing film can remain tightly adhered to the chip 100 even in a high temperature environment (about 98 ℃).

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

The nucleic acid analysis chip can ensure the sealing performance of the whole detection process, and meanwhile, different pre-stored reagents cannot be mixed to cause pollution, so that the nucleic acid analysis chip is favorable for transportation and storage. Transfer and stirring of different reagents can be easily realized by combining the conduction valve with the piston pump. And the movement of the conduction valve and the reciprocating pushing of the piston pump can be automatically controlled by a simple mechanical device without a complex operating system, so that the nucleic acid analysis chip has potential to be popularized and used in a base layer.

Example 2

The specific operation process for realizing integrated nucleic acid analysis by using the nucleic acid analysis chip is described as follows:

as shown in FIG. 3, the nucleic acid analysis chip comprises seven storage tubes 500 in total, which are numbered from left to right as 500a to 500g in this order. As shown in fig. 4, the liquid flow tube 101, which is connected to the storage tube interface 105, the reaction chamber 104, the waste liquid chamber 102, and the air vent 103, is numbered from left to right as 101a to 101 i.

As shown in fig. 1 to 5, before use, a sample, a lysis binding reagent and a micro-nano magnetic sphere are placed in a storage tube 500a, and a magnet can be placed beside the storage tube 500a for adsorbing the micro-nano magnetic sphere; a magnetic bead washing reagent is placed in the storage tube 500b, a chip washing reagent is placed in the storage tube 500c, an elution reagent is placed in the storage tube 500d, a diluting reagent is placed in the storage tube 500e, a solidified CRISPR detection reagent for a first target is placed in the storage tube 500f, a solidified CRISPR detection reagent for a second target is placed in the storage tube 500g, and a solidified nucleic acid amplification reagent is placed in the reaction chamber 104 in advance.

Further, 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 made of a transparent polymer material, such as polymethyl methacrylate or polycarbonate.

Further, the CRISPR detection reagent employed in the embodiments of the present invention is a CRISPR/Cas12a detection reagent comprising Cas12a protein, guide RNA, buffer, single-stranded DNA fluorescent probe, rnase inhibitor, sterile water, and the like. When the target DNA exists, the guide RNA can be accurately paired with the target DNA, so that the cleavage activity of the Cas12a protein is activated, a large amount of single-stranded DNA fluorescent probes are cleaved, and a fluorescent signal is generated.

The nucleic acid analysis test procedure embodied in the present invention is as follows, but is not limited thereto:

1) unscrewing a tube cover of the storage tube 500a, adding a sample to be detected into the storage tube 500a, covering the tube cover to ensure sealing, and cracking the sample to be detected by a cracking binding reagent to release nucleic acid molecules so as to combine the nucleic acid molecules with the micro-nano magnetic spheres. Moving the conduction valve 800 on the slide rail 200, driving a conduction valve interface 802 on the conduction valve 800 to align with a liquid flow pipeline 101a of the storage tube 500a, enabling the storage tube 500a to be communicated with a piston cylinder 302 in the piston pump 300 after passing through the liquid flow pipeline 101a and the conduction valve interface 802, and pushing the piston rod 302 in a reciprocating manner, so that the micro-nano magnetic ball can be fully mixed with the nucleic acid molecules released by the sample, and the nucleic acid molecules can be captured by the micro-nano magnetic ball;

2) an external magnet is close to the storage tube 500a, so that the micro-nano magnetic spheres are adsorbed on the tube wall and separated from the cracking binding reagent. Pulling out the piston rod 302 from the piston cylinder 302, making the cracking binding reagent enter the piston cylinder 302 of the piston pump 300, moving the conduction valve 800, driving the conduction valve interface 802 to align with the liquid flowing pipeline 101g of the waste liquid cavity 102, making the waste liquid cavity 102 communicate with the piston cylinder 302 in the piston pump 300 after passing through the liquid flowing pipeline 101g and the conduction valve interface 802, pushing in the piston rod 302, making the cracking binding reagent discharge into the waste liquid cavity 102;

3) moving the conduction valve 800 to bring the conduction valve interface 802 into alignment with the liquid flow pipeline 101b of the storage tube 500b, so that the storage tube 500b is communicated with the piston cylinder 302 in the piston pump 300 after passing through the liquid flow pipeline 101b and the conduction valve interface 802, and pulling out the piston rod 302 to make a part of the magnetic ball cleaning reagent in the storage tube 500b enter the piston cylinder 302 of the piston pump 300;

4) and moving the conduction valve 800 to 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 communicated with the piston cylinder 302 in the piston pump 300 again after passing through the liquid flow pipeline 101b and the conduction valve interface 802, moving away the magnet outside the storage tube 500a, pushing the piston rod 302 in a reciprocating manner to enable the magnetic ball cleaning reagent to enter the storage tube 500a and be fully and uniformly mixed with the micro-nano magnetic ball, and then enabling the micro-nano magnetic ball to be adsorbed on the tube wall of the storage tube 500a and be separated from the magnetic ball cleaning reagent by utilizing the external magnet to be close to the storage tube 500 a. Then the piston rod 302 is pulled out, so that the magnetic ball cleaning reagent enters the piston cylinder 302 of the piston pump 300;

5) moving the conduction valve 800 to drive the conduction valve interface 802 to align with the liquid flowing pipeline 101g of the waste liquid cavity 102, so that the waste liquid cavity 102 is communicated with the piston cylinder 302 in the piston pump 300 again after passing through the liquid flowing pipeline 101g and the conduction valve interface 802, and pulling out the piston rod 302 to discharge the magnetic ball cleaning reagent into the waste liquid cavity 102;

6) repeating the operations 3), 4) and 5), and cleaning the micro-nano magnetic spheres again by using the residual magnetic sphere cleaning reagent in the storage tube 500 b; enabling the magnetic ball cleaning reagent to repeatedly enter and flow out of the storage tube 500a, and then cleaning and discharging after the magnetic ball cleaning reagent and the micro-nano magnetic balls are fully and uniformly mixed;

7) moving the conduction valve 800 to bring the conduction valve interface 802 into alignment with the liquid flow pipeline 101c of the storage tube 500c, so that the storage tube 500c is communicated with the piston cylinder 302 in the piston pump 300 after passing through the liquid flow pipeline 101c and the conduction valve interface 802, and pulling out the piston rod 302 to make a part of the chip washing reagent in the storage tube 500c enter the piston cylinder 302 of the piston pump 300;

8) moving the conduction valve 800 to drive the conduction valve interface 802 to align with the liquid flow pipeline 101b of the storage tube 500b, so that the storage tube 500b is communicated with the piston cylinder 302 in the piston pump 300 again after passing through the liquid flow pipeline 101b and the conduction valve interface 802, and pushing in and pulling out the piston rod 302 to discharge the chip washing reagent into the storage tube 500 b; the plunger rod 302 is then pulled out again, allowing the chip wash reagent to enter the plunger barrel 302 of the plunger pump 300.

The piston rod 302 is rapidly pushed to and fro, so that the chip washing reagent cleans the piston pump 300, the conduit 400, the storage tube 500b and the liquid flow pipeline 101b, and the influence of components (such as alcohol) possibly contained in the residual magnetic ball washing reagent on the subsequent nucleic acid amplification is eliminated.

9) After the piston pump 300, the conduit 400, the storage tube 500b and the liquid flow tube 101b are cleaned, the conduction valve 800 is moved to bring the conduction valve interface 802 into alignment with the liquid flow tube 101a of the storage tube 500a, so that the storage tube 500a is communicated with the piston cylinder 302 of the piston pump 300 again after passing through the liquid flow tube 101a and the conduction valve interface 802, and the piston rod 302 is pushed in, so that the chip washing reagent is discharged into the bottom of the storage tube 500a (where the liquid cannot enter the tube, and the liquid can contact the magnetic beads); the plunger rod 302 is then pulled out again, allowing the chip wash reagent to enter the plunger barrel 302 of the plunger pump 300.

The piston rod 302 is rapidly pushed back and forth, so that the chip washing reagent cleans the liquid flow pipe 101 a. The washing times can be determined according to actual conditions in the whole washing process, and meanwhile, the chip washing reagent is prevented from contacting the micro-nano magnetic ball.

In the examples of the present invention, the number of washing was repeated three times.

10) After the washing process is finished, the conduction valve 800 is moved to drive the conduction valve interface 802 to be communicated with the exhaust pipeline 108, so that external clean gas is communicated with the piston cylinder 302 in the piston pump 300 after passing through the air holes 103 and the conduction valve interface 802, the piston rod 302 is pulled out, and the external clean gas is sucked into the piston cylinder 302 of the piston pump 300; the conductance valve 800 is moved to bring the conductance valve port 802 into alignment with the fluid flow channel 101a of the storage tube 500a, and then the piston rod 302 is pushed in to discharge the external cleaning gas out of the piston cylinder 302 of the piston pump 300.

Whole process is that the clean air of inhaling is from ventilative mouthful, squeezes into storage tube 500a again, then goes to inhale clean air again and from ventilative mouthful, squeezes into storage tube 500a again, and is reciprocal to be gone on, realizes that it washes to swing to chip pipeline, storage tube to inhale clean gas from the external world, blows and beats, further cleans.

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

12) and moving the conduction valve 800 to 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 communicated with the piston cylinder 302 in the piston pump 300 again after passing through the liquid flow pipeline 101a and the conduction valve interface 802, moving the external magnet close to the storage tube 500a, rapidly and reciprocally pushing in and pulling out the piston rod 302, and enabling the elution reagent in the piston cylinder 302 to repeatedly enter and flow out of the storage tube 500a to be fully and uniformly mixed with the micro-nano magnetic ball. Then, an external magnet is close to the storage tube 500a, so that the micro-nano magnetic ball is adsorbed on the tube wall of the storage tube 500a and is separated from the elution reagent, and the nucleic acid molecules are eluted by the elution reagent and enter the elution reagent; pulling out the piston rod 302 to make the elution reagent containing nucleic acid molecules enter the piston cylinder 302 of the piston pump 300;

13) the conduction valve 800 is moved to drive the conduction valve interface 802 to align with the liquid flow pipeline 101f of the reaction chamber 104, so that the reaction chamber 104 is communicated with the piston cylinder 302 in the piston pump 300 through the liquid flow pipeline 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 reaction chamber 104 to dissolve the solidified nucleic acid amplification reagent. The reaction chamber 104 is used for nucleic acid amplification with the help of an external temperature control device. After the amplification reaction is finished, the piston rod 302 is pulled out, so that the amplification product in the reaction chamber 104 enters the piston cylinder 302 of the piston pump 300.

14) Moving the conduction valve 800 to drive the conduction valve interface 802 to align with the liquid flow pipeline 101e of the storage tube 500e, so that the storage tube 500e is communicated with the piston cylinder 302 in the piston pump 300 after passing through the liquid flow pipeline 101e and the conduction valve interface 802, and the piston rod 302 is pushed in and pulled out rapidly and reciprocally, so that the amplification product in the piston cylinder 302 repeatedly enters and flows out of the storage tube 500e and is uniformly mixed with the diluent in the storage tube 500 e.

15) And moving the conduction valve 800 to drive the conduction valve interface 802 to be sequentially aligned with the

liquid flow pipelines

101h and 101i of the

storage tubes

500f and 500g, and uniformly distributing the diluted amplification products into the storage tube 500f and the storage tube 500g respectively to dissolve the solidified CRISPR reagent for product detection. Although the amplification product is diluted to some extent by the diluting reagent, the amount of the amplification product after nucleic acid amplification is in the order of millions, and CRISPR detection is very sensitive, so that the overall detection sensitivity is not affected. Meanwhile, under the condition of high requirement on detection sensitivity, a buffer solution required by CRISPR detection can be stored in the buffer chamber and is used as a diluting reagent. Although the target nucleic acid is diluted by the CRISPR detection buffer, the concentrations of each active component and the target nucleic acid to be detected in the CRISPR system are kept unchanged by using a concentration reagent or a freeze-drying reagent in a subsequent CRISPR detection cavity. Therefore, the detection sensitivity is ensured, and the extraction and the transfer of the volume of the trace liquid can be avoided. The buffer solution specific components of the CRISPR detection system have been reported (for example, Science 360(2018)436-439), and can be further optimized according to the reagent conditions.

In operation, if there is liquid remaining in the conduit or it is desired to facilitate the volatilization of the organic reagent, the conductance valve 800 can be moved to align the port 802 with the exhaust conduit 108, pulling the piston rod 302 outward, causing the piston pump 300 to create a column of clean air. The piston rod 302 is then pushed rapidly inward to drain the channel of residual liquid or to volatilize the organic reagent.

When the amplification product is detected, the end point detection can be carried out, and the result judgment can be carried out by observing whether a fluorescence signal exists or not; real-time fluorescence signal detection is also possible, but the presence of bubbles in the reaction solution will interfere with the reading of the fluorescence signal. The image sensor is used for identifying the bubbles in the reaction liquid, so that the fluorescent signal acquisition of the bubble position is avoided, the spatial distribution detection of the fluorescent signal of the reaction liquid is realized, and the interference of the possibly existing bubbles on the fluorescent signal detection is avoided. If the existence of the bubbles is detected, liquid can be further extracted from the detection chamber by using a pump valve of the chip so as to eliminate the bubbles in the chamber, thereby eliminating the interference of the factors such as the bubbles on the detection of the fluorescence signal.

The image sensor can adopt a common CMOS or CCD type product on the market, and the selected pixel specification can be determined according to the requirement on the detection precision of bubbles, impurities and the like. Typically, a mega pixel image sensor can meet the detection requirement.

Example 3

Taking the double detection of the CaMV35S sequence and the Lectin sequence in the transgenic soybean leaves as an example.

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

The nucleic acid amplification adopts an isothermal amplification method, and the nucleic acid amplification reagents are as follows: bst DNA polymerase 16 units, 10x Thermopol reaction buffer 5. mu.l, betaine 0.8. mu. per liter, dNTP 0.35. mu. per liter, magnesium sulfate 2. mu. per liter, primer mix (containing 4 inner primers, 1.6. mu. mol each per liter of 2 inner primers, 0.8. mu. mol each per liter of 2 inner primers, 4 outer primers, 0.2. mu. mol each per liter of 2 outer primers, 0.1. mu. mol each liter of 2 outer primers, 4 loop primers, 0.4. mu. mol each liter of 2 loop primers, 0.2. mu. mol each of 2 loop primers). These reagents are freeze-dried to form a solid, which is placed in the reaction chamber 104 in advance.

The CRISPR detection reagent is used for a CRISPR/Cas12a detection reagent, and comprises the following main components: cas12a protein 200 nmol per liter, 10. times. NEBuffer 2.1 buffer 10. mu.l, single stranded DNA probe 2.5. mu.l per liter, guide RNA600 nmol per liter, RNase inhibitor 20 units. These reagents were freeze-dried to form solids and placed in

storage tubes

500f and 500g in advance. The storage tube 500f detects the CaMV35S sequence, and the storage tube 500g detects the Lectin sequence.

The nano magnetic ball adopts SeraSil-MagTMSpeedBeads carboxyl magnetic ball of GE company, and can also adopt other commercialized magnetic balls or be prepared by the nano magnetic ball.

Before use, 200. mu.l of lysis reagent (4. mu.l of guanidinium isothiocyanate, 50. mu.l of tris hydrochloride, 20. mu.l of ethylenediaminetetraacetic acid, pH 7.6-8.0), 200. mu.l of isopropanol and 10. mu.l of nanospheres) were placed in

reservoir

500a, 800. mu.l of magnetic bead washing reagent (80% ethanol) was placed in reservoir 500b, 1.2 ml of chip washing reagent (sterile water) was placed in reservoir 500c, 50. mu.l of elution reagent (sterile water) was placed in reservoir 500d, and 50. mu.l of diluting reagent (sterile water or buffer for CRISPR detection, such as NEBuffer 2.1 buffer) was placed in reservoir 500 e.

Mashing soybean leaves, adding 100 microliters of supernatant into the storage tube 500a, covering the tube cover, then pushing the piston pump to realize the full and uniform mixing of the sample to be detected and the nano magnetic ball in a reciprocating manner, and incubating for 10 minutes.

The procedure is as in example 2, wherein the number of reciprocations of the syringe is at least ten in order to ensure a homogeneous mixing of the liquids. When the magnetic ball cleaning reagent is used for cleaning the micro-nano magnetic ball, 400 microliter of the cleaning reagent is absorbed each time for cleaning, and the cleaning is performed twice in total. When the piston pump 300 and the tube 400 are washed with the chip washing reagent, 300. mu.l of the reagent is sucked and washed three times in total. In washing the liquid flow line 101a, washing was performed by sucking 100. mu.l each time, and washing was performed three times in total.

In the CRISPR detection, the

storage tubes

500f and 500g can be observed with a portable fluorescence observation device. Positive amplification will produce a fluorescent signal, while negative amplification will not.

For experimental information such as design of primer sequences and design of guide RNA sequences to be detected, reference is made to the literature (Biosensors and Bioelectronics 157(2020) 112153).

The above has described in detail the specific embodiment of the nucleic acid analysis chip of the present invention for nucleic acid analysis, but the above examples are illustrative, 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 is not to be construed as limiting the present invention. In the design of the present invention, similar modifications, substitutions, variations, etc. may be made to the technical solution of the present invention, and these are all within the scope of the present invention.

Claims

1. An integrated nucleic acid analysis chip, said chip being provided with a plurality of chambers, characterized in that:

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

and after the nucleic acid amplification reaction is finished, the amplification products are respectively pumped into the CRISPR detection cavities, so that the end-point multiplex detection is realized.

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

the chip is transparent at the cavity and is provided with an image sensor, and the image sensor is used for detecting bubbles of fluorescent signals in the cavity; when the existence of air bubbles is found, the liquid in the chamber is further extracted by a pump valve on the chip to eliminate the air bubbles in the chamber.

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

the chip is provided with a washing chamber, a chip washing reagent is arranged in the washing chamber, a pipeline and other chambers in the chip are washed, and the reagent which has an influence on subsequent nucleic acid amplification and is used in the nucleic acid experiment process is removed completely, so that the influence on the nucleic acid amplification is avoided.

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

the chip is equipped with the exhaust duct who takes the diaphragm and connects exhaust duct's bleeder vent, does not have the nucleic acid molecule exchange with the external world at the chip under the prerequisite, inhales external clean gas to the chip inside from the bleeder vent via exhaust duct, and then washes to the inside pipeline of chip and cavity and swing and blow and beat.

6. An integrated nucleic acid analysis chip, comprising:

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

the slide rail is tightly fixed with the chip through a slide rail interface and used for moving the conduction valve, so that the air holes, the reaction cavity, the waste liquid cavity and different storage tubes are selectively conducted with the conduction valve through a liquid flow pipeline/an exhaust pipeline of the chip;

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

and the piston pump is connected with the conduction valve in a sealing way through a guide pipe and is used for pumping or discharging liquid.

7. An integrated nucleic acid analysis chip according to claim 6, wherein:

8. An integrated nucleic acid analysis chip according to claim 6, wherein:

the chip is provided with a washing chamber, a chip washing reagent is arranged in the washing chamber, the washing chamber is communicated with a pipeline port through a respective liquid flowing pipeline and then selectively communicated with a conduction valve, and the pipeline and other chambers in the chip are washed.

9. An integrated nucleic acid analysis chip according to claim 6, 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 of fluorescent signals in the cavities; when the existence of air bubbles is found, the liquid in the chamber is further extracted by a pump valve on the chip to eliminate the air bubbles in the chamber.

10. An integrated nucleic acid analysis chip according to claim 6, wherein:

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