Plant endophyte sorting device

Abstract

The invention discloses a plant endophyte sorting device which comprises a plant tissue grinding unit, a microfluidic sorting unit and an intelligent detection platform, the plant tissue grinding unit comprises a cutting mechanism, a grinding mechanism, a reagent bottle and a collecting bottle, and the cutting mechanism is used for cutting and crushing plant tissues; the grinding mechanism is used for grinding the crushed plant tissues; the micro-fluidic sorting unit is used for carrying out single bacterium sorting on the endophyte and collecting single bacterium cells; the intelligent detection platform comprises a microscope and a computer, the microscope is provided with a CCD image sensor and used for collecting images of the micro-droplets in real time, and the computer is used for analyzing whether the micro-droplets are single-bacterium liquid droplets or not according to the images of the micro-droplets and controlling the micro-fluidic sorting unit to perform single-bacterium sorting of endophytes. The device disclosed by the invention can be used for grinding various plant tissues, is suitable for separating endophytes of various plant tissues, and adopts a microfluidic technology to realize intelligent and automatic collection of single endophytes.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue endophyte sorting, and in particular to a plant endophyte sorting device. Background Art

[0002] Endophytes are microorganisms that live inside the tissues and organs of plants at certain stages or all stages, and do not cause obvious disease symptoms to plant tissues and organs. Plant endophytes mainly include endophytic bacteria and endophytic fungi. Endophytes are obviously universal and diverse. There are differences in endophytic groups between different plant species. There are also significant differences between different varieties, tissues, organs, growth regions and environments, and growth periods of the same plant. Endophytes are a very important type of microbial resource. In order to explore and find these resources, the isolation of plant endophytes is a key link in the research and application of endophytes.

[0003] Endophyte isolation is usually done by bringing plant materials back to the laboratory, disinfecting and sterilizing them, and then inoculating them in a culture medium under a sterile environment for isolation and culture. Due to the cumbersome operation during the separation process, the complex external environment of separation, and the inconvenient and incomplete sterilization of the separation equipment, contamination is prone to occur during the separation of endophytes, and the success rate of separation is low. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the first purpose of the present invention is to propose a plant endophyte sorting device, which adopts an integrated closed space, uses rapid cutting and grinding to process various plant tissues, and achieves rapid separation of endophytes, is suitable for separation of endophytes from a variety of plant tissues, and uses microfluidic technology for endophyte sorting, which has the characteristics of strong applicability and high success rate, so as to realize intelligent and automatic collection of endophyte single bacteria, and can effectively solve the problem of plant endophyte sorting in the prior art.

[0005] To achieve the above-mentioned purpose, the first embodiment of the present invention proposes a plant endophyte sorting device, comprising: a plant tissue grinding unit, the plant tissue grinding unit comprising a cutting mechanism, a grinding mechanism, a reagent bottle, and a collecting bottle, the cutting mechanism being used to cut and crush the plant tissue; the grinding mechanism being used to grind the crushed plant tissue; the reagent in the reagent bottle being used to sterilize the grinding mechanism and separate the endophytes from the ground and crushed plant tissue; the collecting bottle being used to contain an endophyte bacterial solution; a microfluidic sorting unit, the microfluidic sorting unit being connected to the collecting bottle, the microfluidic sorting unit being used to perform single bacterial sorting on the endophytes and collect single bacterial cells; an intelligent detection platform, the intelligent detection platform comprising a microscope and a computer, the microscope being electrically connected to the computer, the computer being electrically connected to the microfluidic sorting unit, the microscope being equipped with a CCD image sensor for real-time acquisition of images of microdroplets, the computer being used to analyze whether the microdroplets are single bacterial droplets based on the images of the microdroplets, and controlling the microfluidic sorting unit to perform single bacterial sorting of the endophytes.

[0006] According to the plant endophyte sorting device of the embodiment of the present invention, before sorting the plant endophyte, firstly, the corresponding reagent is added to the reagent bottle, and then the corresponding disinfection reagent is first introduced into the cutting mechanism and the grinding mechanism to disinfect the cutting mechanism and the grinding mechanism, and the collecting bottle is replaced after the disinfection and cleaning are completed. Then the plant tissue is put into the cutting mechanism, which cuts and crushes the plant tissue, and transports the crushed plant tissue to the grinding mechanism, which grinds the crushed plant tissue; while grinding, the reagent corresponding to the extraction of endophytes is introduced into the grinding mechanism, the endophytes are separated from the ground and crushed plant tissue, and an endophytic bacterial solution is obtained, and the endophytic bacterial solution is transported to a collection bottle; the microfluidic sorting unit extracts the endophytic bacterial solution from the collection bottle, the CCD image sensor in the microscope collects the image of the microdroplets in the microfluidic sorting unit in real time, and transmits the image signal to the computer, the computer analyzes whether the microdroplets are single bacterial droplets according to the image of the microdroplets, and when the microdroplets are single bacterial droplets, generates corresponding control signals and transmits the control signals to the microfluidic sorting unit to control the microfluidic sorting unit to perform single bacterial sorting of endophytes; the microfluidic sorting unit performs single-even sorting of the endophytes according to the control signal, and collects the single bacterial cells at the sorting location. Therefore, the device adopts an integrated closed space and utilizes rapid cutting and grinding to process various plant tissues to achieve rapid separation of endophytes. It is suitable for the separation of endophytes from a variety of plant tissues, and uses microfluidic technology for endophyte sorting. It has the characteristics of strong applicability and high success rate, thereby enabling intelligent and automated collection of endophyte single bacteria, and can effectively solve the problems of plant endophyte sorting in existing technologies.

[0007] In addition, the plant endophyte sorting device according to the above embodiment of the present invention may also have the following additional technical features:

[0008] According to one embodiment of the present invention, the cutting mechanism includes: a cutting chamber; an injection port, which is arranged at the upper part of the cutting chamber; a first motor, which is installed on the outer wall of one side of the cutting chamber, and the first motor is electrically connected to the computer; a first rotating shaft, which is connected to the first motor, and the first rotating shaft is located in the middle position of the cutting chamber; a first spiral blade, a cutting plate, and a cutting knife are installed on the first rotating shaft in sequence, and the cutting plate is located between the first spiral blade and the cutting knife; the cutting plate is provided with a plurality of circular holes; the first motor drives the first rotating shaft to drive the first spiral blade and the cutting knife to rotate, and the plant tissue is squeezed through the holes on the cutting plate and is cut and crushed by the cutting knife.

[0009] According to one embodiment of the present invention, the grinding mechanism includes: a grinding chamber, which is connected to the bottom of the cutting chamber near the cutting knife through a connecting pipe; a second motor, which is installed above the grinding chamber; a second rotating shaft, which is connected to the second motor, and the second rotating shaft is located in the middle of the cutting chamber; a second spiral blade and a grinding rod are installed on the second rotating shaft in sequence, and the side walls of the grinding chamber and the grinding rod are provided with grinding grooves corresponding to each other; a sample outlet is provided at the bottom of the grinding chamber, and a filter is provided at the sample outlet; a liquid outlet pipe, one end of the liquid outlet pipe is connected to the sample outlet, the other end of the liquid outlet pipe is connected to a collecting bottle, and a first solenoid valve is provided on the liquid outlet pipe; a nozzle, which is provided at the top of the grinding chamber, the nozzle is connected to the reagent bottle through a liquid inlet pipe, and an infusion pump is provided on the liquid inlet pipe; the second motor, the first solenoid valve, and the infusion pump are all electrically connected to the computer.

[0010] According to one embodiment of the present invention, the reagent bottles include a 75% ethanol solution bottle, a 2% sodium hypochlorite solution bottle, a sterile water bottle, and a culture medium solution bottle; a second solenoid valve is arranged between the 75% ethanol solution bottle and the liquid inlet pipe; a third solenoid valve is arranged between the 2% sodium hypochlorite solution bottle and the liquid inlet pipe; a fourth solenoid valve is arranged between the sterile water bottle and the liquid inlet pipe; a fifth solenoid valve is arranged between the culture medium solution bottle and the liquid inlet pipe; the second solenoid valve, the third solenoid valve, the fourth solenoid valve, and the fifth solenoid valve are all electrically connected to the computer.

[0011] According to one embodiment of the present invention, the microfluidic sorting unit includes: a chip fixing table; a microfluidic chip, the microfluidic chip is fixedly connected to the top of the chip fixing table, and the microfluidic chip is made of glass; a manifold, the manifold is connected to the microfluidic chip, the manifold is respectively connected to the collection bottle, the mineral oil reagent bottle, the culture bottle, and the waste liquid bottle through a liquid pipeline and a constant pressure vacuum pump, the manifold is used to transport the endophytic bacterial solution and mineral oil to the microfluidic chip; the microfluidic chip is used to perform single bacterial sorting on the endophytic bacteria and collect single bacterial cells; the manifold is also used to collect the single bacterial solution flowing out of the microfluidic chip, and collect the waste liquid flowing out of the microfluidic chip.

[0012] According to one embodiment of the present invention, the manifold is provided with a first probe, a second probe, a third probe, and a fourth probe for docking with the inlet and outlet structures on the microfluidic chip.

[0013] According to one embodiment of the present invention, the liquid pipeline includes a first liquid inlet pipeline, a second liquid inlet pipeline, a first liquid outlet pipeline, and a second liquid outlet pipeline, wherein: one end of the first liquid inlet pipeline is inserted into the collecting bottle, and the other end of the first liquid inlet pipeline is connected to the first probe; the first liquid inlet pipeline is provided with a sixth solenoid valve and runs through the constant pressure vacuum pump, and the first liquid inlet pipeline is used to input endophyte solution into the microfluidic chip; one end of the second liquid inlet pipeline is inserted into the mineral oil reagent bottle, and the other end of the second liquid inlet pipeline is connected to the second probe; the second liquid inlet pipeline is provided with a sixth solenoid valve and runs through the constant pressure vacuum pump, and the first liquid inlet pipeline is used to input endophyte solution into the microfluidic chip; one end of the second liquid inlet pipeline is inserted into the mineral oil reagent bottle, and the other end of the second liquid inlet pipeline is connected to the second probe; Seven solenoid valves are connected through the constant-pressure vacuum pump, the second liquid inlet pipe is used to input mineral oil solution into the microfluidic chip; one end of the first liquid outlet pipe is inserted into the culture bottle, and the other end of the first liquid outlet pipe is connected to the third probe; the first liquid outlet pipe is used to collect the single bacterial solution flowing out of the microfluidic chip; one end of the second liquid outlet pipe is inserted into the waste liquid bottle, and the other end of the second liquid outlet pipe is connected to the fourth probe; the second liquid outlet pipe is used to collect the waste liquid flowing out of the microfluidic chip; the sixth solenoid valve, the seventh solenoid valve and the constant-pressure vacuum pump are all electrically connected to the computer.

[0014] According to one embodiment of the present invention, the inlet and outlet structure of the microfluidic chip includes: a bacterial solution inlet, which is connected to the first probe and is used to inject a culture solution containing endophytic bacteria as a dispersed phase for droplet generation; a mineral oil inlet, which is connected to the second probe and is used to inject mineral oil as a continuous phase for droplet generation; a first solution outlet, which is connected to the third probe; and a second solution outlet, which is connected to the fourth probe.

[0015] According to one embodiment of the present invention, the microfluidic chip also includes a channel structure, which includes: a mixing channel, a first splitting channel, a second splitting channel, a detection channel and a sorting channel, wherein the first input end of the mixing channel is connected to the bacterial solution inlet, and the second input end of the mixing channel is connected to the mineral oil inlet. After the endogenous bacterial solution and the mineral oil enter the chip pipeline, they are mixed at the trifurcated structure to produce a water-oil mixed phase; the input end of the first splitting channel is connected to the output end of the mixing channel, and a first micro-droplet generation port and a storage chamber are provided in the first splitting channel. The water-oil mixed phase flows through the first micro-droplet generation port and splits into micro-droplets. The split droplets enter the storage chamber for temporary storage; the input end of the detection channel is connected to the storage chamber, and the detection channel is used to observe and capture the image of the droplets passing through the detection point in real time through the microscope, and perform real-time target detection on the bacteria in the droplets; the sorting channel includes a first sorting branch and a second sorting branch, One end of the first sorting branch is connected to the output end of the detection channel and is provided with a first control valve, and the other end of the first sorting branch is connected to the second solution outlet; one end of the second sorting branch is connected to the output end of the detection channel and is provided with a second control valve, and the other end of the second sorting branch is connected to the first solution outlet; the sorting channel is used to sort single bacterial droplets, and single bacterial droplets flow out from the first solution outlet through the second sorting branch; sterile droplets flow out from the second solution outlet through the first sorting branch; droplets containing more than two bacteria flow into the second splitting channel; one end of the second splitting channel is connected to the output end of the detection channel and is provided with a third control valve, the other end of the second splitting channel is connected to the storage chamber, and a second micro-droplet generation port is provided on the second splitting channel, which is used to split droplets containing more than two bacteria to generate single bacterial droplets; the first control valve, the second control valve and the third control valve are all electrically connected to the computer.

[0016] According to one embodiment of the present invention, the computer is installed with software with the functions of real-time observation, detection and sorting of micro-droplets, and the image of the micro-droplets is observed in real time through the software; the micro-droplets are intelligently detected and automatically sorted by setting detection parameters, and the detection results and sorting results are output to a display for display.

[0017] Compared with the existing technology, the present invention has the following beneficial effects:

[0018] (1) The plant endophyte separation device of the embodiment of the present invention provides a closed plant tissue cutting and grinding space, which is convenient for disinfection and pretreatment, and easily achieves a sterile environment, thereby avoiding the introduction of contaminating microorganisms during the process of plant tissue crushing and endophyte separation.

[0019] (2) In the plant endophyte sorting device of the embodiment of the present invention, the first rotating shaft drives the first spiral blade to squeeze the plant tissue through the mesh cutting plate, which is then cut by the cutting knife and enters the grinding groove. The endophytes in the plant tissue are fully released through rapid grinding between the grinding rod and the raised part of the inner wall of the grinding groove, thereby achieving rapid and efficient separation.

[0020] (3) The plant endophyte sorting device of the embodiment of the present invention uses a microfluidic chip to sort endophytes, achieving fast and efficient single bacterial screening. Compared with other endophyte separation technologies, it avoids the need for secondary separation after endophyte propagation and cultivation.

[0021] (4) In the plant endophyte sorting device of the embodiment of the present invention, a secondary splitting channel is designed in the droplet splitting part of the microfluidic chip. When it is detected that the droplet contains non-single bacteria, the second splitting channel is used to perform secondary splitting, thereby improving the accuracy and success rate of sorting single bacteria.

[0022] (5) The plant endophyte sorting device of the embodiment of the present invention has an intelligent detection platform and sorting control software that establishes communication between the computer and the constant pressure vacuum pump based on the droplet target detection algorithm, thereby realizing intelligent and automated sorting.

[0023] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the overall structure of a plant endophyte sorting device according to an embodiment of the present invention;

[0025] Figure 2 It is a schematic structural diagram of a cutting plate of a plant endophyte sorting device according to an embodiment of the present invention;

[0026] Figure 3 This is a schematic structural diagram of a manifold plate of a plant endophyte sorting device according to an embodiment of the present invention;

[0027] Figure 4 A schematic diagram of the internal channel structure of a microfluidic chip of a plant endophyte sorting device according to an embodiment of the present invention;

[0028] Figure 5 This is a schematic structural diagram of a mixing channel and a first splitting channel of a plant endophyte sorting device according to an embodiment of the present invention.

[0029] Reference numerals:

[0030] 100, plant tissue grinding unit; 101, first motor; 102, sample inlet; 103, first rotating shaft; 104, first spiral blade; 105, cutting chamber; 106, cutting plate; 107, hole; 108, cutting knife; 109, connecting pipe; 110, grinding chamber; 111, grinding rod; 112, grinding groove; 113, sample outlet; 114, filter screen; 115, liquid outlet pipe; 116, collecting bottle; 11 7. Nozzle; 118. Infusion pump; 119. Liquid inlet tube; 120. Second solenoid valve; 121. 75% ethanol solution bottle; 122. 2% sodium hypochlorite solution bottle; 123. Sterile water bottle; 124. Culture medium solution bottle; 125. Second motor; 126. Second rotating shaft; 127. Second spiral blade; 128. First solenoid valve; 129. Third solenoid valve; 130. Fourth solenoid valve; 131. Fifth solenoid valve;

[0031] 200, microfluidic sorting unit; 201, constant pressure vacuum pump; 202, first liquid inlet pipeline; 203, second liquid inlet pipeline; 204, first liquid outlet pipeline; 205, second liquid outlet pipeline; 206, manifold; 207, microfluidic chip; 208, chip fixing table; 209, mineral oil reagent bottle; 210, waste liquid bottle; 211, culture bottle; 212, first probe; 213, second probe; 214, third probe; 215, fourth probe; 216, bacterial solution inlet; 217, mineral oil inlet; 218 , mixing channel; 219, first splitting channel; 220, first micro-droplet generation port; 221, storage chamber; 222, detection channel; 223, second splitting channel; 224, detection point; 225, first control valve; 226, second control valve; 227, third control valve; 228, first sorting branch; 229, first solution outlet; 230, second solution outlet; 231, micro-droplet; 232, second sorting branch; 233, second micro-droplet generation port; 234, sixth solenoid valve; 235, seventh solenoid valve;

[0032] 300. Intelligent detection platform; 301. Computer; 302. Microscope. DETAILED DESCRIPTION

[0033] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0034] The following describes a plant endophyte sorting device according to an embodiment of the present invention with reference to the accompanying drawings.

[0035] like Figure 1As shown, the plant endophyte sorting device according to the embodiment of the present invention may include: a plant tissue grinding unit 100 , a microfluidic sorting unit 200 and an intelligent detection platform 300 .

[0036] Among them, the plant tissue grinding unit 100 includes a cutting mechanism, a grinding mechanism, a reagent bottle, and a collecting bottle 116. The cutting mechanism is used to cut and crush the plant tissue; the grinding mechanism is used to grind the crushed plant tissue; the reagent in the reagent bottle is used to disinfect the grinding mechanism and separate the endophytes from the ground and crushed plant tissue; the collecting bottle 116 is used to contain the endophytic bacterial solution. The microfluidic sorting unit 200 is connected to the collecting bottle 116, and the microfluidic sorting unit 200 is used to perform single bacterial sorting on endophytes and collect single bacterial cells; the intelligent detection platform 300 includes a microscope 302 and a computer 301, the microscope 302 is electrically connected to the computer 301, and the computer 301 is electrically connected to the microfluidic sorting unit 200, the microscope 302 is equipped with a CCD image sensor for real-time acquisition of images of microdroplets 231, the computer 301 is used to analyze whether the microdroplet 231 is a single bacterial droplet based on the image of the microdroplet 231, and control the microfluidic sorting unit 200 to perform single bacterial sorting of endophytes.

[0037] Specifically, before sorting the plant endophytes, first add the corresponding reagents to the reagent bottle, and then pass the corresponding reagents for disinfection into the cutting mechanism and the grinding mechanism to disinfect the cutting mechanism and the grinding mechanism. After disinfection and cleaning, replace the collection bottle 116. Then put the plant tissue into the cutting mechanism, the cutting mechanism cuts and crushes the plant tissue, and the crushed plant tissue is transported to the grinding mechanism, and the grinding mechanism grinds the crushed plant tissue. While grinding, the reagent corresponding to the extraction of endophytes is passed into the grinding mechanism, and the endophytes are separated from the ground and crushed plant tissue to obtain an endophytic bacterial body solution, which is transported to the collection bottle 116. The microfluidic sorting unit 200 extracts the endophytic bacterial solution from the collection bottle 116, and the CCD image sensor in the microscope 302 collects the image of the micro-droplet 231 in the microfluidic sorting unit 200 in real time, and transmits the image signal to the computer 301. The computer 301 analyzes whether the micro-droplet 231 is a single bacterial droplet based on the image of the micro-droplet 231, and generates a corresponding control signal when the micro-droplet 231 is a single bacterial droplet and transmits the control signal to the microfluidic sorting unit 200 to control the microfluidic sorting unit 200 to perform single bacterial sorting of endophytes. The microfluidic sorting unit 200 performs single-even sorting of endophytes according to the control signal, and collects the single bacterial cells at the sorting location.

[0038] According to one embodiment of the present invention, Figure 1 and Figure 2As shown, the cutting mechanism includes: a cutting chamber 105; an injection port 102, which is arranged at the upper part of the cutting chamber 105; a first motor 101, which is installed on the outer wall of one side of the cutting chamber 105, and the first motor 101 is electrically connected to the computer 301; a first rotating shaft 103, which is connected to the first motor 101, and the first rotating shaft 103 is located in the middle position of the cutting chamber 105; a first spiral blade 104, a cutting plate 106, and a cutting knife 108 are installed on the first rotating shaft 103 in sequence, and the cutting plate 106 is located between the first spiral blade 104 and the cutting knife 108; the cutting plate 106 is provided with a plurality of circular holes 107; the first motor 101 drives the first rotating shaft 103 to drive the first spiral blade 104 and the cutting knife 108 to rotate, and the plant tissue is squeezed through the holes 107 on the cutting plate 106 and then cut and crushed by the cutting knife 108.

[0039] Specifically, when sorting plant endophytes, the first motor 101 is started first, and then the plant tissue is placed in the cutting chamber 105 through the sampling port 102. The first motor 101 drives the first rotating shaft 103 to drive the first spiral blade 104 and the cutting knife 108 to rotate. The first spiral blade 104 squeezes the plant tissue through the hole 107 on the cutting plate 106, and is cut and broken by the rotating cutting knife 108.

[0040] According to one embodiment of the present invention, Figure 1 As shown, the grinding mechanism includes: a grinding chamber 110, which is connected to the bottom of the cutting chamber 105 near the cutting blade 108 through a connecting pipe 109; a second motor 125, which is installed above the grinding chamber 110; a second rotating shaft 126, which is connected to the second motor 125, and the second rotating shaft 126 is located in the middle of the cutting chamber 105; a second spiral blade 127 and a grinding rod 111 are sequentially installed on the second rotating shaft 126, and a grinding groove 112 is provided on the side wall of the grinding chamber 110 and the grinding rod 111 correspondingly; the grinding chamber A sample outlet 113 is provided at the bottom of 110, and a filter screen 114 is provided at the sample outlet 113; a liquid outlet pipe 115, one end of which is connected to the sample outlet 113, and the other end of which is connected to a collecting bottle 116, and a first solenoid valve 128 is provided on the liquid outlet pipe 115; a nozzle 117, which is provided at the top of the grinding chamber 110, and is connected to the reagent bottle through a liquid inlet pipe 119, and an infusion pump 118 is provided on the liquid inlet pipe 119; the second motor 125, the first solenoid valve 128, and the infusion pump 118 are all electrically connected to the computer 301.

[0041] Further, according to one embodiment of the present invention, Figure 1As shown, the reagent bottles include a 75% ethanol solution bottle 121, a 2% sodium hypochlorite solution bottle 122, a sterile water bottle 123, and a culture medium solution bottle 124; a second solenoid valve 120 is arranged between the 75% ethanol solution bottle 121 and the liquid inlet pipe 119; a third solenoid valve 129 is arranged between the 2% sodium hypochlorite solution bottle 122 and the liquid inlet pipe 119; a fourth solenoid valve 130 is arranged between the sterile water bottle 123 and the liquid inlet pipe 119; a fifth solenoid valve 131 is arranged between the culture medium solution bottle 124 and the liquid inlet pipe 119; the second solenoid valve 120, the third solenoid valve 129, the fourth solenoid valve 130, and the fifth solenoid valve 131 are all electrically connected to the computer 301.

[0042] Specifically, before the formal operation of separating endophytes, the computer 301 controls the opening of the second solenoid valve 120 and the third solenoid valve 129, closes the fourth solenoid valve 130 and the fifth solenoid valve 131, and turns on the infusion pump 118, the first motor 101 and the second motor 125, and pumps a sufficient amount of 75% ethanol solution and 2% sodium hypochlorite solution into the grinding chamber 110 and the cutting chamber 105 through the nozzle 117 to disinfect the grinding chamber 110 and the cutting chamber 105; then close the second solenoid valve 120, the third solenoid valve 129, the infusion pump 118, and turn on the third solenoid valve 129. A solenoid valve 128 is used to discharge 75% ethanol and 2% sodium hypochlorite into the collecting bottle 116; the fourth solenoid valve 130 is opened, the first solenoid valve 128 is closed, and the infusion pump 118 is turned on to pump a sufficient amount of sterile water into the grinding chamber 110 and the cutting chamber 105 through the nozzle 117 for thorough cleaning; after the cleaning is completed, the first solenoid valve 128 is opened, the fourth solenoid valve 130 and the infusion pump 118 are closed, and the sterile water is discharged into the collecting bottle 116; a new collecting bottle 116 is replaced, and the first solenoid valve 128, the first motor 101 and the second motor 125 are closed.

[0043] When sorting plant endophytes, the first motor 101 and the second motor 125 are first started, and then the plant tissue is placed in the cutting chamber 105 through the sample inlet 102. The first motor 101 drives the first rotating shaft 103 to drive the first spiral blade 104 and the cutting knife 108 to rotate. The first spiral blade 104 squeezes the plant tissue through the hole 107 on the cutting plate 106, and is cut and broken by the rotating cutting knife 108. The cut and broken plant tissue is transported to the grinding chamber 110 through the connecting pipe 109, and the second motor 125 drives the second rotating shaft 126 to drive the second spiral blade 127 and the grinding rod 111 to rotate. The grinding rod 111 and the raised part of the inner wall of the grinding groove 112 perform secondary grinding on the cut and broken plant tissue, so that the endophytes in the plant tissue are fully released. At the same time, the infusion pump 118 is turned on, the fifth solenoid valve 131 is opened, and the culture medium solution is pumped into the grinding chamber 110 through the nozzle 117. The culture medium solution can elute the endophytes contained in the ground plant tissue. The culture medium solution can fully elute the endophytes and protect the endophytes to avoid the death of the endophytes. After the elution is completed, the infusion pump 118 and the fifth solenoid valve 131 are turned off, the first solenoid valve 128 is opened, and the endophytic bacterial solution filtered by the filter 114 is transported to the collection bottle 116 through the liquid outlet pipe 115.

[0044] According to one embodiment of the present invention, Figure 1 As shown, the microfluidic sorting unit 200 includes: a chip fixing table 208; a microfluidic chip 207, which is fixedly connected to the top of the chip fixing table 208 and is made of glass; a manifold 206, which is connected to the microfluidic chip 207, and the manifold 206 is respectively connected to the collection bottle 116, the mineral oil reagent bottle 209, the culture bottle 211, and the waste liquid bottle 210 through a liquid pipeline and a constant pressure vacuum pump 201, and the manifold 206 is used to transport the endophytic bacterial solution and mineral oil to the microfluidic chip 207; the microfluidic chip 207 is used to perform single bacterial sorting on the endophytic bacteria and collect single bacterial cells; the manifold 206 is also used to collect the single bacterial solution flowing out of the microfluidic chip 207, and collect the waste liquid flowing out of the microfluidic chip 207.

[0045] According to one embodiment of the present invention, Figure 3 As shown, the manifold 206 is provided with a first probe 212 , a second probe 213 , a third probe 214 , and a fourth probe 215 for docking with the inlet and outlet structures on the microfluidic chip 207 .

[0046] According to one embodiment of the present invention, Figure 1 and Figure 3As shown, the liquid pipeline includes a first liquid inlet pipeline 203, a second liquid inlet pipeline 204, a first liquid outlet pipeline 205, and a second liquid outlet pipeline 206, wherein: one end of the first liquid inlet pipeline 203 is inserted into the collecting bottle 116, and the other end of the first liquid inlet pipeline 203 is connected to the first probe 212; the first liquid inlet pipeline 203 is provided with a sixth solenoid valve 234, and runs through the constant pressure vacuum pump 201, and the first liquid inlet pipeline 203 is used to input the endophytic bacterial solution into the microfluidic chip 207; one end of the second liquid inlet pipeline 204 is inserted into the mineral oil reagent bottle 209, and the other end of the second liquid inlet pipeline 204 is connected to the second probe 213; the second liquid inlet pipeline 204 is provided with a seventh solenoid valve 235 , and runs through the constant pressure vacuum pump 201, the second liquid inlet pipe 204 is used to input mineral oil solution into the microfluidic chip 207; one end of the first liquid outlet pipe 205 is inserted into the culture bottle 211, and the other end of the first liquid outlet pipe 205 is connected to the third probe 214; the first liquid outlet pipe 205 is used to collect the single bacterial solution flowing out of the microfluidic chip 207; one end of the second liquid outlet pipe 206 is inserted into the waste liquid bottle 210, and the other end of the second liquid outlet pipe 206 is connected to the fourth probe 215; the second liquid outlet pipe 206 is used to collect the waste liquid flowing out of the microfluidic chip 207; the sixth solenoid valve 234, the seventh solenoid valve 235 and the constant pressure vacuum pump 201 are all electrically connected to the computer 301.

[0047] According to one embodiment of the present invention, Figure 3 and Figure 4 As shown, the inlet and outlet structure of the microfluidic chip 207 includes: a bacterial solution inlet 216, which is connected to the first probe 212 and is used to inject a culture solution containing endophytic bacteria as a dispersed phase for droplet generation; a mineral oil inlet 217, which is connected to the second probe 213 and is used to inject mineral oil as a continuous phase for droplet generation; a first solution outlet 229, which is connected to the third probe 214; and a second solution outlet 230, which is connected to the fourth probe 215.

[0048] According to one embodiment of the present invention, Figure 4 and Figure 5As shown, the microfluidic chip 207 also includes a channel structure, which includes: a mixing channel 218, a first splitting channel 219, a second splitting channel 223, a detection channel 222 and a sorting channel, wherein the first input end of the mixing channel 218 is connected to the bacterial solution inlet 216, and the second input end of the mixing channel 218 is connected to the mineral oil inlet 217. After the endophytic bacterial solution and the mineral oil enter the chip pipeline, they are mixed at the trifurcated structure to generate a water-oil mixed phase; the input end of the first splitting channel 219 is connected to the output end of the mixing channel 218 The first splitting channel 219 is provided with a first micro-droplet generation port 220 and a storage chamber 221. The water-oil mixed phase flows through the first micro-droplet generation port 220 and splits into micro-droplets 231. The split droplets enter the storage chamber 221 for temporary storage. The input end of the detection channel 222 is connected to the storage chamber 221. The detection channel 222 is used to observe and take images of droplets passing through the detection point 224 in real time through a microscope 302, and perform real-time target detection of bacteria in the droplets. The sorting channel includes a first sorting branch 228 and a second sorting branch 232. The first sorting branch One end of the first sorting branch 228 is connected to the output end of the detection channel 222, and is provided with a first control valve 225, and the other end of the first sorting branch 228 is connected to the second solution outlet 230; one end of the second sorting branch 232 is connected to the output end of the detection channel 222, and is provided with a second control valve 226, and the other end of the second sorting branch 232 is connected to the first solution outlet 229; the sorting channel is used to sort single bacterial droplets, and a single bacterial droplet flows out from the first solution outlet 229 through the second sorting branch 232; sterile body droplets flow out from the first solution outlet 229 through the first sorting branch 228 The second solution flows out from the second solution outlet 230; the droplets containing more than two bacteria flow into the second split channel 223; one end of the second split channel 223 is connected to the output end of the detection channel 222, and is provided with a third control valve 227, the other end of the second split channel 223 is connected to the storage chamber 221, and the second split channel 223 is provided with a second micro-droplet generation port 233, which is used for the droplets containing more than two bacteria to split and generate single bacterial droplets; the first control valve 225, the second control valve 226 and the third control valve 227 are all electrically connected to the computer 301.

[0049] Specifically, after the endophytic bacteria cell solution is collected, the computer 301 controls the opening of the constant pressure vacuum pump 201, the sixth solenoid valve 234, the seventh solenoid valve 235, and the microscope 302, so that the endophytic bacteria cells in the collection bottle 116 are transported to the cell solution inlet 216 at a relatively slow speed through the first liquid inlet pipe 203 and the first probe 212, and the mineral oil in the mineral oil reagent bottle 209 is transported to the mineral oil inlet 217 at a relatively slow speed through the second liquid inlet pipe 204 and the second probe 213. After the endophytic bacteria solution and the mineral oil are mixed in the mixing channel 218, they enter the first splitting channel 219 and are split into micro-droplets 231 by the first micro-droplet generation port 220, and the split droplets enter the storage chamber 221 for temporary storage. Then the micro-droplets 231 enter the detection channel 222, and the microscope 302 observes and captures the droplet images passing through the detection point 224 in real time, and performs real-time target detection on the bacteria in the droplets. When a sterile body droplet is detected, the second control valve 226 and the third control valve 227 are closed, and the first control valve 225 is opened, and the sterile body droplet is transported to the second solution outlet 230 through the first sorting branch 228, and is transported to the waste liquid bottle 210 through the fourth probe 215 and the second liquid outlet pipe 206; when a single bacterial body droplet is detected, the first control valve 225 and the third control valve 227 are closed, and the second control valve 226 is opened, and the single bacterial body droplet is transported to the first solution outlet 230 through the second sorting branch 232. The solution outlet 229 is transported to the culture bottle 211 through the third probe 214 and the first liquid outlet pipe 115 for subsequent culture and observation; when droplets containing more than two bacteria are detected, the first control valve 225 and the third control valve 227 are closed, and the second control valve 226 is opened, and the multi-bacterial droplets enter the second splitting channel 223, and the second micro-droplet generation port 233 on the second splitting channel 223 splits the multi-bacterial droplets into single bacterial droplets, which flow into the storage chamber 221 again for temporary storage.

[0050] According to one embodiment of the present invention, the computer 301 is installed with software with the functions of real-time observation, detection and sorting of the micro-droplets 231, and the image of the micro-droplets 231 is observed in real time through the software; the micro-droplets 231 are intelligently detected and automatically sorted by setting detection parameters, and the detection results and sorting results are output to the display for display.

[0051] Combine the following Figures 1 to 5 The working principle of the plant endophyte sorting device according to the embodiment of the present invention is described in detail, which may specifically include the following steps:

[0052] S1, refill the reagent bottle with the corresponding reagent; place the microfluidic chip 207 on the chip fixing table 208;

[0053] S2; Open the software on the computer 301, and set the detection and sorting parameters in the software according to the requirements;

[0054] S3, the computer 301 controls to open the second solenoid valve 120 and the third solenoid valve 129, close the fourth solenoid valve 130 and the fifth solenoid valve 131, and turn on the infusion pump 118, the first motor 101 and the second motor 125, and pumps a sufficient amount of 75% ethanol solution and 2% sodium hypochlorite solution into the grinding chamber 110 and the cutting chamber 105 through the nozzle 117 to disinfect the grinding chamber 110 and the cutting chamber 105; then close the second solenoid valve 120, the third solenoid valve 129, and the infusion pump 118, open the first solenoid valve 128, and discharge 75% ethanol and 2% sodium hypochlorite into the collection bottle 116;

[0055] S4, open the fourth solenoid valve 130, close the first solenoid valve 128, and start the infusion pump 118, pump a sufficient amount of sterile water into the grinding chamber 110 and the cutting chamber 105 through the nozzle 117 for thorough cleaning; after the cleaning is completed, open the first solenoid valve 128, close the fourth solenoid valve 130 and the infusion pump 118, and discharge the sterile water into the collection bottle 116; replace the new collection bottle 116, and close the first solenoid valve 128, the first motor 101 and the second motor 125;

[0056] S5, start the first motor 101 and the second motor 125, then put the plant tissue into the cutting chamber 105 through the sample inlet 102, the first motor 101 drives the first rotating shaft 103 to drive the first spiral blade 104 and the cutting knife 108 to rotate, the first spiral blade 104 squeezes the plant tissue through the hole 107 on the cutting plate 106, and is cut and broken by the rotating cutting knife 108; the cut and broken plant tissue is transported to the grinding chamber 110 through the connecting tube 109, the second motor 125 drives the second rotating shaft 126 to drive the second spiral blade 127 and the grinding rod 111 to rotate, so as to perform secondary grinding on the cut and broken plant tissue, so that the endophytes in the plant tissue are fully released; at the same time, start the infusion pump 118, open the fifth solenoid valve 131, and pump the culture medium solution into the grinding chamber 110 through the nozzle 117, and the culture medium solution can elute the endophytes contained in the ground plant tissue;

[0057] S6, after the elution is completed, the infusion pump 118 and the fifth solenoid valve 131 are closed, the first solenoid valve 128 is opened, and the endophytic bacterial cell solution filtered by the filter screen 114 is transported to the collection bottle 116 through the liquid outlet pipe 115;

[0058] S7, the computer 301 controls the opening of the constant pressure vacuum pump 201, the sixth solenoid valve 234, the seventh solenoid valve 235, and the microscope 302, so that the endophytic bacteria in the collection bottle 116 are transported to the bacterial solution inlet 216 at a relatively slow speed through the first liquid inlet pipe 203 and the first probe 212, and the mineral oil in the mineral oil reagent bottle 209 is transported to the mineral oil inlet 217 at a relatively slow speed through the second liquid inlet pipe 204 and the second probe 213, and the endophytic bacteria solution and the mineral oil are mixed in the mixing channel 218, enter the first splitting channel 219, and then split into micro-droplets 231 by the first micro-droplet generating port 220, and the split droplets enter the storage chamber 221 for temporary storage;

[0059] S8, the micro-droplet 231 enters the detection channel 222, and the microscope 302 observes and captures the image of the droplet passing through the detection point 224 in real time, and performs real-time target detection on the bacteria in the droplet; when a sterile droplet is detected, the second control valve 226 and the third control valve 227 are closed, and the first control valve 225 is opened, and the sterile droplet is transported to the second solution outlet 230 through the first sorting branch 228, and is transported to the waste liquid bottle 210 through the fourth probe 215 and the second liquid outlet pipe 206; when a single bacterial droplet is detected, the first control valve 225 and the third control valve 227 are closed, and the first control valve 225 is opened. The second control valve 226 is opened, and the single bacterial droplet is transported to the first solution outlet 229 through the second sorting branch 232, and is transported to the culture bottle 211 through the third probe 214 and the first liquid outlet pipe 115 for subsequent culture and observation; when droplets containing more than two bacteria are detected, the first control valve 225 and the third control valve 227 are closed, and the second control valve 226 is opened, and the multi-bacterial droplets enter the second splitting channel 223, and the second micro-droplet generation port 233 on the second splitting channel 223 splits the multi-bacterial droplets into single bacterial droplets, which flow into the storage chamber 221 again for temporary storage.

[0060] According to the plant endophyte sorting device of the embodiment of the present invention, before sorting the plant endophyte, firstly, the corresponding reagent is added to the reagent bottle, and then the corresponding disinfection reagent is first introduced into the cutting mechanism and the grinding mechanism to disinfect the cutting mechanism and the grinding mechanism, and the collecting bottle is replaced after the disinfection and cleaning are completed. Then the plant tissue is put into the cutting mechanism, which cuts and crushes the plant tissue, and transports the crushed plant tissue to the grinding mechanism, which grinds the crushed plant tissue; while grinding, the reagent corresponding to the extraction of endophytes is introduced into the grinding mechanism, the endophytes are separated from the ground and crushed plant tissue, and an endophytic bacterial solution is obtained, and the endophytic bacterial solution is transported to a collection bottle; the microfluidic sorting unit extracts the endophytic bacterial solution from the collection bottle, the CCD image sensor in the microscope collects the image of the microdroplets in the microfluidic sorting unit in real time, and transmits the image signal to the computer, the computer analyzes whether the microdroplets are single bacterial droplets according to the image of the microdroplets, and when the microdroplets are single bacterial droplets, generates corresponding control signals and transmits the control signals to the microfluidic sorting unit to control the microfluidic sorting unit to perform single bacterial sorting of endophytes; the microfluidic sorting unit performs single-even sorting of the endophytes according to the control signal, and collects the single bacterial cells at the sorting location. Therefore, the device adopts an integrated closed space and utilizes rapid cutting and grinding to process various plant tissues to achieve rapid separation of endophytes. It is suitable for the separation of endophytes from a variety of plant tissues, and uses microfluidic technology for endophyte sorting. It has the characteristics of strong applicability and high success rate, thereby enabling intelligent and automated collection of endophyte single bacteria, and can effectively solve the problems of plant endophyte sorting in existing technologies.

[0061] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0062] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0063] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0064] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A plant endophyte sorting device, characterized in that: include: A plant tissue grinding unit, comprising a cutting mechanism, a grinding mechanism, a reagent bottle, and a collection bottle, wherein the cutting mechanism is used to cut and crush the plant tissue; The grinding mechanism is used to grind the crushed plant tissue; the reagent in the reagent bottle is used to sterilize the grinding mechanism and separate the endophytic bacteria from the crushed plant tissue; the collecting bottle is used to contain the endophytic bacteria solution; A microfluidic sorting unit, the microfluidic sorting unit is connected to the collection bottle, and the microfluidic sorting unit is used to sort the endophytes by single bacteria and collect single bacterial cells; An intelligent detection platform, the intelligent detection platform comprises a microscope and a computer, the microscope is electrically connected to the computer, the computer is electrically connected to the microfluidic sorting unit, the microscope is equipped with a CCD image sensor for real-time acquisition of images of microdroplets, the computer is used to analyze whether the microdroplets are single bacterial droplets based on the images of the microdroplets, and control the microfluidic sorting unit to perform single bacterial sorting of endophytes.

2. The plant endophyte sorting device according to claim 1, characterized in that: The cutting mechanism comprises: Cutting chamber; An injection port, the injection port is arranged at the upper part of the cutting chamber; A first motor, the first motor is installed on an outer wall of one side of the cutting chamber, and the first motor is electrically connected to the computer; A first rotating shaft, the first rotating shaft is connected to the first motor, and the first rotating shaft is located in the middle of the cutting chamber; The first spiral blade, the cutting plate and the cutting knife are sequentially mounted on the first rotating shaft, and the cutting plate is located between the first spiral blade and the cutting knife; the cutting plate is provided with a plurality of circular holes; The first motor drives the first rotating shaft to drive the first spiral blade and the cutting knife to rotate, so that the plant tissue is squeezed through the holes on the cutting plate and then cut and crushed by the cutting knife.

3. The plant endophyte sorting device according to claim 2, characterized in that: The grinding mechanism comprises: A grinding chamber, the grinding chamber is connected to the bottom of one side of the cutting chamber close to the cutting blade through a connecting pipe; a second motor, wherein the second motor is installed above the grinding bin; A second rotating shaft, the second rotating shaft is connected to the second motor, and the second rotating shaft is located in the middle of the cutting chamber; The second spiral blade and the grinding rod are sequentially mounted on the second rotating shaft, and the side wall of the grinding chamber and the grinding rod are provided with grinding grooves corresponding to each other; A sample outlet is provided at the bottom of the grinding chamber, and a filter screen is provided at the sample outlet; a liquid outlet pipe, one end of which is connected to the sample outlet, the other end of which is connected to the collecting bottle, and a first solenoid valve is provided on the liquid outlet pipe; A nozzle, which is arranged on the top of the grinding chamber, and is connected to the reagent bottle through a liquid inlet pipe, and an infusion pump is arranged on the liquid inlet pipe; The second motor, the first solenoid valve, and the infusion pump are all electrically connected to the computer.

4. The plant endophyte sorting device according to claim 3, characterized in that: The reagent bottles include a 75% ethanol solution bottle, a 2% sodium hypochlorite solution bottle, a sterile water bottle, and a culture medium solution bottle; A second solenoid valve is provided between the 75% ethanol solution bottle and the liquid inlet pipe; A third solenoid valve is provided between the 2% sodium hypochlorite solution bottle and the liquid inlet pipe; A fourth solenoid valve is provided between the sterile water bottle and the liquid inlet pipe; A fifth solenoid valve is provided between the culture medium solution bottle and the liquid inlet pipe; The second solenoid valve, the third solenoid valve, the fourth solenoid valve, and the fifth solenoid valve are all electrically connected to the computer.

5. The plant endophyte sorting device according to claim 1, characterized in that: The microfluidic sorting unit comprises: Chip fixing platform; A microfluidic chip, wherein the microfluidic chip is fixedly connected to the top of the chip fixing platform, and the microfluidic chip is made of glass; A manifold, the manifold is connected to the microfluidic chip, the manifold is respectively connected to the collection bottle, the mineral oil reagent bottle, the culture bottle, and the waste liquid bottle through a liquid pipeline and a constant pressure vacuum pump, and the manifold is used to transport the endophytic bacteria solution and the mineral oil to the microfluidic chip; The microfluidic chip is used to sort endophytes by single bacteria and collect single bacterial cells; The confluence plate is also used to collect the single bacterial solution flowing out of the microfluidic chip, and to collect the waste liquid flowing out of the microfluidic chip.

6. The plant endophyte sorting device according to claim 5, characterized in that: The manifold is provided with a first probe, a second probe, a third probe, and a fourth probe for docking with the inlet and outlet structures on the microfluidic chip.

7. The plant endophyte sorting device according to claim 6, characterized in that: The liquid pipeline comprises a first liquid inlet pipeline, a second liquid inlet pipeline, a first liquid outlet pipeline, and a second liquid outlet pipeline, wherein: One end of the first liquid inlet pipe is inserted into the collecting bottle, and the other end of the first liquid inlet pipe is connected to the first probe; a sixth solenoid valve is provided on the first liquid inlet pipe and passes through the constant pressure vacuum pump, and the first liquid inlet pipe is used to input endophytic bacterial solution into the microfluidic chip; One end of the second liquid inlet pipe is inserted into the mineral oil reagent bottle, and the other end of the second liquid inlet pipe is connected to the second probe; a seventh solenoid valve is provided on the second liquid inlet pipe and passes through the constant pressure vacuum pump, and the second liquid inlet pipe is used to input the mineral oil solution into the microfluidic chip; One end of the first liquid outlet pipe is inserted into the culture bottle, and the other end of the first liquid outlet pipe is connected to the third probe; the first liquid outlet pipe is used to collect the single bacterial solution flowing out of the microfluidic chip; One end of the second liquid outlet pipe is inserted into the waste liquid bottle, and the other end of the second liquid outlet pipe is connected to the fourth probe; the second liquid outlet pipe is used to collect waste liquid flowing out of the microfluidic chip; The sixth solenoid valve, the seventh solenoid valve and the constant-pressure vacuum pump are all electrically connected to the computer.

8. The plant endophyte sorting device according to claim 7, characterized in that: The inlet and outlet structure of the microfluidic chip includes: a bacterial solution inlet, the bacterial solution inlet being connected to the first probe and used for injecting a culture solution containing endophytic bacteria as a dispersed phase for droplet generation; a mineral oil inlet, the mineral oil inlet being connected to the second probe and used for injecting mineral oil as a continuous phase for droplet generation; a first solution outlet, wherein the first solution outlet is connected to the third probe; The second solution outlet is connected to the fourth probe.

9. The plant endophyte sorting device according to claim 8, characterized in that: The microfluidic chip also includes a channel structure, which includes: a mixing channel, a first splitting channel, a second splitting channel, a detection channel and a sorting channel, wherein: The first input end of the mixing channel is connected to the bacterial solution inlet, and the second input end of the mixing channel is connected to the mineral oil inlet. After the endophytic bacterial solution and the mineral oil enter the chip pipeline, they are mixed at the trifurcated structure to generate a water-oil mixed phase; The input end of the first splitting channel is connected to the output end of the mixing channel. The first splitting channel is provided with a first micro-droplet generation port and a storage chamber. The water-oil mixed phase flows through the first micro-droplet generation port and splits into micro-droplets. The split droplets enter the storage chamber for temporary storage. The input end of the detection channel is connected to the storage chamber, and the detection channel is used to observe and take images of droplets passing through the detection point in real time through the microscope, and perform real-time target detection of bacteria in the droplets; The sorting channel comprises a first sorting branch and a second sorting branch, one end of the first sorting branch is connected to the output end of the detection channel and is provided with a first control valve, and the other end of the first sorting branch is connected to the second solution outlet; one end of the second sorting branch is connected to the output end of the detection channel and is provided with a second control valve, and the other end of the second sorting branch is connected to the first solution outlet; The sorting channel is used to sort a single bacterial droplet, and the single bacterial droplet flows out from the first solution outlet through the second sorting branch; a sterile droplet flows out from the second solution outlet through the first sorting branch; a droplet containing more than two bacterial cells flows into the second splitting channel; One end of the second split channel is connected to the output end of the detection channel and is provided with a third control valve, the other end of the second split channel is connected to the storage chamber, and the second split channel is provided with a second micro-droplet generation port for splitting droplets containing more than two bacterial cells to generate single bacterial droplets; The first control valve, the second control valve and the third control valve are all electrically connected to the computer.

10. The plant endophyte sorting device according to claim 1, characterized in that: The computer is installed with software with the functions of real-time observation, detection and sorting of micro-droplets, and the images of micro-droplets are observed in real time through the software; the micro-droplets are intelligently detected and automatically sorted by setting detection parameters, and the detection results and sorting results are output to a display for display.

Images