Long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms and detection method

Abstract

The invention relates to the technical field of deep-sea microorganism nucleic acid detection, in particular to a long-term experimental platform for deep-sea microorganism in-situ nucleic acid detection and a detection method. According to the invention, a water sample is subjected to tangential flow filtration and concentration through the filtration and sampling module, so that a concentrated microorganism sample is obtained. The method comprises the following steps of: transferring a sample into an amplification detection module under reduced pressure, transferring into a lysis purification module for cell lysis, injecting the cracked sample into a chromatographic column, filtering impurities and intercepting DNA by the chromatographic column, then washing by using an eluent to obtain a high-purity DNA sample, loading a suction head through a transfer washing liquid gun, mixing the high-purity DNA sample with an enzyme solution, adding into the amplification detection module for reaction, and detecting the DNA in the amplification detection module. And carrying out fluorescence labeling on the amplified gene, obtaining an amplification curve by using a fluorescence detector, and detecting the in-situ nucleic acid of the deep-sea microorganisms according to the change of the amplification curve. An automatic cleaning assembly is designed in the filtering and sampling module, and the cracking and purifying module is used for sampling and cleaning a cracking chamber and switching pipelines, so that cross contamination caused by deep sea in-situ multiple detection is prevented.

Description

Technical Field

[0001] The present invention relates to the technical field of deep-sea microbial nucleic acid detection, and specifically refers to a long-term experimental platform and detection method for in-situ nucleic acid detection of deep-sea microorganisms. Background Art

[0002] The species diversity and metabolic mechanism diversity of deep-sea functional microorganisms are very high, and the material and energy conversion and energy transfer networks they participate in are very complex. At the same time, environmental changes will significantly change the functional microbial community, and conversely, the changes in the functional microbial community can also indicate environmental changes. Long-term continuous monitoring of specific functional microbial groups can analyze the environmental change laws of typical deep-sea ecosystems, reveal the ecological functions of microorganisms, and provide a theoretical basis for evaluating the disturbance effects of human activities on typical deep-sea ecosystems and formulating effective deep-sea ecological security guarantee mechanisms.

[0003] For a long time, the monitoring of deep-sea environmental microorganisms has usually adopted the detection method of sampling from the deep sea to the laboratory for analysis. On the one hand, due to environmental changes and long-term transportation, the microorganisms in the samples will change, especially the low-abundance microorganisms may die and be difficult to detect; on the other hand, it is difficult to obtain time-series biological data by this method. In addition, it is difficult to obtain microbial data that cannot be isolated and cultured in the laboratory, especially microorganisms with low abundance, and the abundance changes of microorganisms during the process of environmental change. Therefore, there is an urgent need to develop instruments and methods for in-situ, instant detection and time-series analysis, especially microbial detection instruments that can be placed on the seabed for regular multiple detections, to provide data support for deep-sea biological research. To achieve long-term continuous in-situ detection of deep-sea functional microorganisms, it is necessary to solve technical problems such as fully automatic integrated low-abundance microbial concentration sampling, cell lysis and DNA purification treatment, real-time gene detection analysis, and prevention of cross-contamination. Summary of the Invention

[0004] The object of the present invention is to solve the problems existing in the prior art, that is, it is difficult to obtain high-purity DNA samples from low-abundance microbial samples in the sea, and the retrieval steps are numerous and the corresponding system is relatively complex. The present invention provides a long-term experimental platform and detection method for in-situ nucleic acid detection of deep-sea microorganisms.

[0005] To solve the problems existing in the prior art, the present invention adopts the following technical solutions:

[0006] A long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms includes a platform frame, a filtration sampling module, and a pressure-resistant instrument cabin. A lysis and purification module and an amplification and detection module are arranged in the pressure-resistant instrument cabin;

[0007] The filtration and detection module includes an in-situ deep-sea tangential flow filtration and circulation component. The filtration and detection module is used to filter and enrich microorganisms in seawater, discharge seawater, and intercept microorganisms in the water body.

[0008] The lysis and purification module includes a lysis chamber and a chromatography column. The lysis and purification module is used to extract DNA from the microorganisms and obtain a high-purity DNA sample through elution with the chromatography column and eluent.

[0009] The amplification and detection module includes a microfluidic chip and a fluorescence detector. The amplification and detection module is used to inject the high-purity DNA sample and reagents into the microfluidic chip for fluorescence labeling amplification of microbial genes.

[0010] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the in-situ deep-sea tangential flow filtration and circulation component includes a tangential flow filtration membrane package, a metering chamber, a deep-sea circulation pump, a deep-sea sampling pump, a three-way valve, and a circulation pipeline.

[0011] The water inlet of the tangential flow filtration membrane package is connected to the deep-sea circulation pump through the circulation pipeline. The water return port of the tangential flow filtration membrane package is connected to the metering chamber and the pressure-resistant instrument chamber through the circulation pipeline and one inlet and outlet of the three-way valve.

[0012] The metering chamber, the deep-sea circulation pump, and the tangential flow filtration membrane package form a deep-sea tangential flow concentration and circulation.

[0013] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the filtration and sampling module further includes an in-situ deep-sea automatic cleaning component, which uses the filtered seawater to clean and protect the tangential flow filtration and circulation component to prevent cross-contamination during multiple samplings in the deep-sea environment.

[0014] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the in-situ deep-sea automatic cleaning component includes a filtered water collection chamber, a cleaning agent chamber, a deep-sea three-in-one-out reversing valve, and a deep-sea stop valve.

[0015] The filtered water collection chamber is connected to the water outlet of the tangential flow filtration membrane package.

[0016] The deep-sea three-in-one-out reversing valve is arranged on the pipeline between the deep-sea experience pump and the metering chamber, and the first interface of the deep-sea three-in-one-out reversing valve is connected to the filtered water collection chamber, and the second interface is connected to the cleaning agent chamber.

[0017] The deep-sea stop valve is connected to the end cover of the metering chamber through a pipeline.

[0018] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the deep-sea in-situ tangential flow filtration and circulation module further includes a flow meter and a coarse filter;

[0019] Both the flow meter and the coarse filter are arranged at the front end of the sampling port of the metering chamber. The flow meter is used to record the water volume sampled into the metering chamber and output data in real time; the coarse filter is used to prevent larger particles from entering the deep-sea tangential flow concentration and circulation.

[0020] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the lysis and purification module includes a sample chamber, the lysis chamber, the chromatography column, an air pump, an alcohol reagent bag, an eluent reagent bag, a multi-channel rotary valve, a one-way valve, a reversing valve, a waste liquid chamber, an alcohol adsorption chamber, a DNA collection tube, a plurality of plunger pumps and a circulation pipeline, and among them, the plurality of plunger pumps include a first plunger pump, a second plunger pump and a third plunger pump;

[0021] The sample chamber is connected to the lysis chamber through the first plunger pump, and a semiconductor heating sheet is arranged on the wall surface of the lysis chamber;

[0022] The alcohol reagent bag is connected to the lysis chamber through the second plunger pump. A one-way valve is arranged at the discharge port of the lysis chamber and is connected to the multi-channel rotary valve;

[0023] The front end of the chromatography column is connected to the multi-channel rotary valve, and the rear end is respectively connected to the DNA collection tube and the waste liquid chamber through the reversing valve;

[0024] The sampling port of the lysis chamber is connected to the chromatography column and the waste liquid chamber, and the eluent reagent bag is connected to the multi-channel rotary valve through the third plunger pump.

[0025] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the amplification and detection module includes a manipulator, an electric pipette, a constant-temperature amplification chamber, a fluorescence detector, a plurality of tips, the microfluidic chip and a chip chamber, and among them, the chip chamber includes two chip storage chambers and one chip waste chamber; the manipulator includes a support frame, a Z-axis motor, a radial motor, a rotating main shaft and an electric claw clamp.

[0026] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, the pressure-resistant instrument chamber is a cylindrical pressure-resistant instrument chamber, and the lysis and purification module and the amplification and detection module are jointly encapsulated in the pressure-resistant instrument chamber;

[0027] A sample high and low pressure transfer module is provided on the end cover of the pressure-resistant instrument cabin for transferring the concentrated microbial sample after lysis into the pressure-resistant instrument cabin.

[0028] A method for in-situ nucleic acid detection of deep-sea microorganisms uses the long-term experimental platform for in-situ detection of deep-sea microorganisms as described above, and includes the following steps:

[0029] Use the filtration sampling module to perform negative pressure vertical filtration to filter and enrich microorganisms in-situ underwater, and intercept the microorganisms remaining in the water body;

[0030] Lyse the microorganisms through the lysis and purification module, and obtain a high-purity DNA sample through a chromatography column and eluent;

[0031] Inject the high-purity DNA sample into the microfluidic chip, and synchronously detect the fluorescence signal to obtain an amplification curve for detecting specific microorganisms.

[0032] As an improvement to the technical solution of the method for in-situ nucleic acid detection of deep-sea microorganisms of the present invention, it includes the following steps:

[0033] S1. Concentrate microorganisms in-situ underwater by negative pressure vertical filtration, and obtain a sample with concentrated microorganisms through the filtration sampling module;

[0034] S2. The sample of concentrated microorganisms enters the lysis and purification module in the pressure-resistant cabin through the high and low pressure conversion valve;

[0035] S3. Flush the pipeline and membrane package of the filtration sampling module and perform nucleic acid digestion protection;

[0036] S4. Lyse the cells in the sample through the lysis and purification module;

[0037] S5. Use an air pump to blow air to make the sample of lysed cells flow through the chromatography column, wash the chromatography column with alcohol, and dry the alcohol with an air pump;

[0038] S6. Wash with eluent to obtain a high-purity DNA sample, and send the DNA sample into the sample pool;

[0039] S7. Use an electric pipette to load the tip, suck the DNA sample from the sample pool, inject it into the sample pool containing nucleic acid amplification reagents for mixing, and then use the pipette to suck the mixed solution and inject it into the microfluidic chip;

[0040] S8. The microfluidic chip is centrifuged twice to evenly distribute and quantify the mixed solution into the detection chamber of the microfluidic chip;

[0041] S9. Place the microfluidic chip in a constant temperature chamber to amplify the DNA and synchronously detect the fluorescence signal.

[0042] S10. Clean the flow path of the lysis and purification module.

[0043] Advantages of the present invention:

[0044] In the present invention, by using the tangential flow filtration sampling assembly in-situ in the deep sea to enrich microorganisms, on the one hand, the filtered water sample volume is increased, which is more conducive to collecting microorganisms with very low abundance. On the other hand, the problem that the existing in-situ concentration and collection of deep-sea microorganisms cannot be quantified is solved.

[0045] In the present invention, by using the deep-sea in-situ automatic cleaning assembly, the pipelines in the tangential flow filtration sampling assembly are cleaned after each operation, avoiding cross-contamination of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms during multiple enrichments of microorganisms in the deep sea.

[0046] In the present invention, a quantitative amount of the concentrated solution is used for cell lysis in the lysis and purification module. The lysed sample is injected into the chromatography column, impurities are filtered out and DNA is retained by the chromatography column, and then an eluent is used to wash it to obtain a high-purity DNA sample, ensuring the reliability and stability of the detection; multiple high-purity DNA samples are independently collected by switching the multi-channel valve, effectively preventing cross-contamination; then a manipulator is used to drive a high-precision pipette for accurate positioning, and in the way of automatically loading the pipette tip, the high-purity DNA sample is accurately and quantitatively added to the microfluidic chip in the amplification detection module for amplification reaction, the fluorescence value is detected and an amplification curve is obtained, and the in-situ nucleic acid of deep-sea microorganisms is detected through the change of the amplification curve, ensuring the success rate of the detection.

[0047] In the present invention, a fully automated process from sample collection to result output is realized under the condition of in-situ unmanned operation in the deep sea. Moreover, automatic cleaning and maintenance are realized during the microorganism sampling process, and independent pipelines are used for different samples during the DNA extraction process, so that the instrument can perform multiple in-situ detections in the deep sea. After a set interval time, it can be put into the deep sea for long-term time-series microorganism detection, providing data on the dynamic changes of deep-sea microorganisms for scientists. Description of the Drawings

[0048] Figure 1 Schematic diagram of the long-term experimental platform for deep-sea nucleic acid detection of the present invention;

[0049] Figure 2 Schematic diagram of the structure of the filtration sampling module in the present invention;

[0050] Figure 3 Schematic diagram of the structure of the lysis and purification module in the present invention;

[0051] Figure 4 For Figure 3 Split structure schematic diagram;

[0052] Figure 5 It is a schematic structural diagram of the amplification detection module in the present invention;

[0053] Figure 6 It is a flow chart of the in-situ nucleic acid detection method for deep-sea microorganisms in the present invention.

[0054] Explanation of the reference numerals in the drawings: 1 - platform frame; 2 - power supply; 3 - filtration sampling module; 4 - lysis and purification module; 5 - amplification detection module; 6 - pressure-resistant instrument cabin; 31 - deep-sea in-situ tangential flow filtration circulation assembly; 311 - tangential flow filtration membrane package; 312 - quantitative cabin; 313 - deep-sea circulation pump; 314 - deep-sea injection pump; 315 - flow meter; 316 - coarse filter; 317 - three-way valve; 321 - filtered water collection cabin; 322 - cleaning agent cabin; 323 - deep-sea three-in-one-out reversing valve; 324 - deep-sea stop valve; 411 - sample chamber; 412 - lysis chamber; 413 - chromatography column; 414 - air pump; 415 - alcohol reagent bag; 416 - eluent reagent bag; 417 - multi-channel rotary valve; 418 - reversing valve (one-way valve); 419 - waste liquid chamber; 420 - alcohol adsorption chamber; 421 - DNA collection tube; 422 - first plunger pump; 423 - second plunger pump; 424 - third plunger pump; 51 - manipulator; 511 - support frame; 512 - Z-axis motor; 513 - radial motor; 514 - rotating main shaft; 515 - electric claw gripper; 52 - electric pipette; 53 - constant-temperature amplification chamber; 54 - fluorescence detector; 55 - tip; 56 - microfluidic chip; 571 - chip storage cabin; 572 - chip waste cabin. Detailed implementation manners

[0055] To make the invention purpose, technical solutions and beneficial effects of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0056] As Figures 1 to 5 shown, on the one hand, the present invention provides a long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms, including: a platform frame 1 to realize the position setting or optimization of each component.

[0057] A filtration sampling module 3, which is used to filter seawater. The filtration sampling module 3 uses the tangential flow filtration method to concentrate microorganisms in-situ in water to obtain a sample with concentrated microorganisms;

[0058] A lysis and purification module 4, which includes a chromatography column. The lysis and purification module 4 is used to lyse the cells in the sample and obtain a high-purity DNA sample through the chromatography column;

[0059] The amplification detection module 5 is used to amplify high-purity DNA samples to increase the number of microorganisms and perform detection.

[0060] In the present invention, the filtration sampling module 3 uses a negative pressure vertical filtration method to perform in-situ microbial filtration and enrichment underwater and intercept the microorganisms remaining in the water body.

[0061] The lysis and purification module 4 lyses the microorganisms, and a high-purity DNA sample is obtained through a chromatography column and an eluent.

[0062] The high-purity DNA sample is injected into the microfluidic chip 56, and the fluorescence signal is synchronously detected to obtain an amplification curve for detecting specific microorganisms.

[0063] Specifically, in the present invention, a certain amount of environmental water sample is collected by the filtration sampling module 3 for filtration and concentration to obtain a concentrated solution; a quantitative concentrated solution enters the sample chamber 411 of the pressure-resistant instrument cabin 6 through the high-low pressure conversion module and is then introduced into the lysis and purification module 4 for cell lysis. The lysed sample flows through the chromatography column, impurities are filtered out, and DNA is intercepted by the chromatography column. Then, an eluent is used to wash it to obtain a high-purity DNA sample. Then, the high-purity DNA sample is mixed with an enzyme solution, quantitatively added to the amplification detection module 5 for isothermal amplification to increase the number of DNA with fluorescent labels, and an amplification curve is obtained through a fluorescence detector to realize the in-situ nucleic acid detection of deep-sea microorganisms.

[0064] In the prior art, generally, a negative pressure vertical filtration method is used to concentrate microorganisms in-situ underwater. The amounts of microorganisms in the filter membrane and the concentrated solution are different. Whether flushing the filter membrane or collecting the concentrated solution, the concentration of microorganisms in the sample cannot be accurately quantified.

[0065] In the present invention, a tangential flow filtration method is used to concentrate microorganisms in-situ in water. During the filtration process, the flow direction of the sample is parallel to the surface of the filter membrane, which can scour the surface of the filter membrane, effectively alleviating the accumulation of cells or particles on the filter membrane. On the one hand, it avoids insufficient water filtration caused by filter membrane blockage. On the other hand, the collected microorganisms can be evenly retained in the concentrated solution, facilitating post-treatment.

[0066] Among them, the present invention further includes a power supply 2, and the power supply 2 is electrically connected to the filtration sampling module 3, the pressure-resistant instrument cabin 6, the amplification detection module 5, and the lysis and purification module 4 respectively to supply power to them.

[0067] In some embodiments of the present invention, the concentrated microbial sample is automatically obtained through the deep-sea in-situ tangential flow filtration circulation assembly 31.

[0068] Among them, the in-situ tangential flow filtration and circulation component 31 in the deep sea includes a tangential flow filtration membrane module 311, a metering chamber 312, a deep-sea circulation pump 313, a deep-sea sampling pump 314, a deep-sea pressure reducing valve, a flowmeter 315, and a coarse filter 316; the water inlet of the tangential flow filter membrane module is connected to the deep-sea circulation pump 313 through pipes respectively, and the water return port of the tangential flow filter membrane module is connected to the metering chamber 312 and the pressure-resistant instrument chamber 6 through pipes and a three-way valve 317; the metering chamber 312, the deep-sea circulation pump 313, and the tangential flow filtration membrane module 311 form a tangential flow filtration and circulation.

[0069] Among them, a flowmeter 315 and a coarse filter 316 are provided at the front end of the sampling port of the metering chamber 312. The flowmeter 315 is used to record the sampling water volume and output data in real time; the coarse filter 316 is used to prevent larger particles from entering the tangential flow filtration and circulation and blocking the pipeline and the tangential flow membrane module. Preferably, the tangential flow filter membrane module adopts Millipore P2GVPPV05 to achieve a seawater filtration volume of more than 30 L and meet a concentration ratio of ≥100:1.

[0070] In some embodiments of the present invention, multiple non-cross-contamination samplings are realized through the in-situ automatic cleaning component in the deep sea; the in-situ automatic cleaning component in the deep sea includes a filtered water collection chamber 321, a cleaning agent chamber 321, a deep-sea three-in-one-out reversing valve 323, and a deep-sea stop valve 324.

[0071] Furthermore, the filtered water collection chamber 321 is provided with an interface connected to the water outlet of the tangential flow filtration membrane module 311; the deep-sea three-in-one-out reversing valve 323 is arranged on the connecting pipeline between the deep-sea sampling pump 314 and the metering chamber 312, one of the interfaces is connected to the filtered water collection chamber 321, and one interface is connected to the cleaning agent chamber 321; the deep-sea stop valve 324 is installed on the end cover of the metering chamber 312; the cleaning agent chamber 321 is filled with NaOH solution.

[0072] In some embodiments of the present invention, the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms further includes a cylindrical pressure-resistant instrument chamber 6, and the lysis and purification module 4 and the amplification and detection module 5 are jointly encapsulated in the pressure-resistant instrument chamber 6; the pressure environment in the pressure-resistant instrument chamber 6 is an atmospheric pressure environment; a sample high-low pressure transfer module is provided on the end cover of the pressure-resistant instrument chamber 6 for transferring the concentrated microorganism sample to the atmospheric pressure environment in the pressure-resistant instrument chamber 6.

[0073] As a specific embodiment of this implementation manner, the deep-sea in-situ automatic cleaning process is as follows: When performing tangential flow filtration circulation, the filtered seawater discharged is collected in the filtered water collection tank 321; after completing the tangential flow filtration circulation, the deep-sea three-in-one-out reversing valve 323 is reversed to the interface of the filtered water collection tank 321, and the deep-sea circulation pump 313 is started to inject the seawater in the filtered water collection tank 321 into the pipeline of the deep-sea in-situ tangential flow filtration circulation assembly 31 and the tangential flow filtration membrane module 311. At the same time, the deep-sea stop valve 324 is opened to discharge the seawater and rinse the sample remaining in the deep-sea in-situ tangential flow filtration circulation assembly 31 clean; the deep-sea stop valve 324 is closed, and the deep-sea three-in-one-out reversing valve 323 is reversed to the cleaning agent tank 321 interface to inject a certain amount of NaOH solution to digest the remaining microorganisms or DNA; before the next sampling and concentration, the deep-sea three-in-one-out reversing valve 323 is reversed to the interface of the deep-sea sampling pump 314, and the deep-sea circulation pump 313 and the deep-sea stop valve 324 are delayed to be opened to rinse off the NaOH solution with in-situ seawater.

[0074] In some implementation manners of the present invention, the lysis and purification module 4 includes a sample chamber 411, a lysis chamber 412, a chromatography column 413, an air pump 414, an alcohol reagent bag 415, an eluent reagent bag 416, a multi-channel rotary valve 417, a solenoid valve, a waste liquid chamber 419, an alcohol adsorption chamber 420, a DNA collection tube 421, a plurality of plunger pumps and flow channels; the plurality of plunger pumps include a first plunger pump 422, a second plunger pump 423 and a third plunger pump 424.

[0075] Further, the sample chamber 411 is connected to the lysis chamber 412 through the first plunger pump 422, and a semiconductor heating sheet is provided on the wall surface of the lysis chamber 412 to achieve high-temperature lysis of cells; the alcohol liquid reagent bag is connected to the lysis chamber 412 through the second plunger pump 423 to flush the lysed sample into the chromatography column 413; a check valve is provided at the discharge port of the lysis chamber 412 and is connected to the multi-channel rotary valve 417; the front end of the chromatography column 413 is connected to one of the channels of the multi-channel rotary valve 417, and the rear end is connected to a reversing valve 418, and the reversing valve 418 is respectively connected to the DNA collection tube 421 and the waste liquid chamber 419.

[0076] The lysed sample flows through the chromatography column 413 and enters the waste liquid chamber 419, and the DNA is intercepted by the chromatography column 413 to achieve DNA purification; the eluent reagent bag 416 is connected to the multi-channel rotary valve 417 through the third plunger pump 424. After the reversing valve 418 is reversed, the eluent flows through the chromatography column 413 to elute the adsorbed DNA and send it into the DNA collection tube 421; the chromatography column 413, the DNA collection tube 421, the reversing valve 418 and the pipelines connected thereto are multiple sets of independent pipelines, and the multi-channel rotary valve 417 is used for switching each time a detection is performed to prevent cross-contamination.

[0077] Further, in the present invention, the microbial lysis is described by taking the high-temperature lysis method as an example; the DNA purification is described by taking the silica gel membrane chromatography column 413 method as an example.

[0078] As a specific embodiment of this embodiment, the automatic DNA extraction and purification process is as follows: The concentrated microbial sample in the sample chamber 411 enters the lysis chamber 412 through the first plunger pump 422. The temperature of the lysis chamber 412 is raised to 95°C by a semiconductor heating sheet and maintained for 10 minutes to destroy the microbial cells, and the DNA in the cells enters the liquid; anhydrous alcohol is injected into the lysis chamber 412 through the second injection pump, and a 70% ethanol environment mixture is formed in the lysis chamber 412. Then, the mixture is made to flow through the silica gel membrane chromatography column 413 by the air pump 414, and the flowing liquid enters the waste liquid chamber 419, and the DNA is intercepted by the chromatography column 413; then, 70% ethanol is injected by the second plunger pump 423 to wash the chromatography column 413 1-2 times to better remove impurities, and the air pump 414 runs continuously for 5 minutes to blow off the alcohol in the chromatography column 413 and the pipeline; the gas flows through the alcohol adsorption chamber 420 filled with activated carbon, and the alcohol is adsorbed to prevent the alcohol in the air in the instrument cabin from being too high and avoid danger. Finally, the eluent is added by the third plunger pump 424 to elute the DNA from the chromatography column 413 to achieve DNA purification; after the switching valve 418 is switched, the air pump 414 is started, and the air flow sends the DNA sample into the DNA collection tube 421.

[0079] In some embodiments of the present invention, the multi-channel rotary valve 417 is a 24-channel rotary valve, and the chromatography column 413, the DNA collection tube 421, the switching valve 418 and the pipelines connected thereto are 24 sets of independent components. Each set of components is connected to 1 channel of the multi-channel rotary valve 417, so that the sample after each sample lysis enters a new pipeline and chromatography column 413, and the DNA purification of 24 samples can be completed to avoid contamination in multiple detections.

[0080] In some embodiments of the present invention, the amplification detection module 5 includes a manipulator 51, an electric pipette 52, a constant temperature amplification chamber 53, a fluorescence detector 54, a plurality of pipette tips 55, a microfluidic chip 56 and a chip chamber; the manipulator 51 includes a support frame 511, a Z-axis motor 512, a radial motor 513, a rotating main shaft 514 and an electric claw 515; the chip chamber includes 2 chip storage chambers 571 and 1 chip waste chamber 572;

[0081] As a specific embodiment of this implementation manner, the automatic DNA amplification detection process is as follows: The radial motor 513 drives the rotating spindle 514 to drive the electronic pipette and the electric claw gripper 515 to achieve planar movement and positioning; the Z-axis motor 512 drives the electronic pipette and the electric claw gripper 515 to achieve vertical movement to reach the positions for performing actions such as grasping the chip, loading the pipette tip 55, and aspirating and adding liquid. The chip compartment includes two chip storage compartments 571 and one chip waste compartment 572; each chip storage compartment 571 houses multiple microfluidic chips 56; the multiple microfluidic chips 56 are transferred by the manipulator 51, and the electric claw gripper 515 is moved to the designated position through the moving device; the electric claw gripper 515 grabs the microfluidic chip 56 from the chip storage compartment 571 and places it into the constant-temperature amplification chamber 53; after the detection is completed, the electric claw gripper 515 grabs the microfluidic chip 56 and moves it to the chip waste compartment 572 to discard the microfluidic chip 56.

[0082] Specifically, the radial motor 513 and the rotating spindle 514 drive the electronic pipette and the electric claw gripper 515 to achieve planar movement and positioning; the Z-axis motor 512 drives the electronic pipette and the electric claw gripper 515 to achieve vertical movement to reach the positions for performing actions such as grasping the microfluidic chip 56, loading and unloading the pipette tip 55, and aspirating and adding liquid. The chip compartment includes two chip storage compartments 571 and one chip waste compartment 572; each chip storage compartment 571 houses multiple microfluidic chips 56; the multiple microfluidic chips 56 are transferred by the manipulator 51, and the electric claw gripper 515 is moved above the chip storage compartment 571 through the moving device; the electric claw gripper 515 grabs the microfluidic chip 56 and places it into the constant-temperature amplification chamber 53; the microfluidic chip 56 rotates in the constant-temperature amplification chamber 53, and the purified DNA sample is quantitatively distributed into the chambers of the microfluidic chip 56 by centrifugal force to be mixed with the preset reagent, and heated to maintain a specific temperature for the amplification reaction; the fluorescence detector 54 detects the fluorescence value of the reaction area, and the detected fluorescence value is plotted into a reaction curve after a certain time; the reaction curve exceeding a certain threshold is considered to detect microorganisms, and the time to reach the threshold is converted into the DNA concentration; after the detection is completed, the electric claw gripper 515 grabs the microfluidic chip 56 and moves it to the chip waste compartment 572 to discard the microfluidic chip 56.

[0083] More specifically, as a specific embodiment of the present invention, in order to achieve 24 - time timing detection, 2 chip storage compartments 571 and 1 chip waste compartment 572 are designed. Each chip storage compartment 571 holds 12 microfluidic chips 56. After 12 detections are completed, the first chip storage compartment 571 becomes the chip waste compartment 572. The transfer of the chips is performed by a 3 - degree - of - freedom translation manipulator 51, which can reach any position in the planar area through rotational axes and linear motion. Two motors are installed on the Z - axis. One motor controls the gripper of the manipulator to pick up the chip and place it into the constant - temperature detection chamber, and after the detection is completed, take out the chip and discard it into the waste compartment. The manipulator 51 also performs operations such as grasping the magnetic cover and the thermal insulation cover. The other motor controls a high - precision electronic pipette, which performs operations such as loading the pipette tip 55, adding liquid to the chip, and discarding the pipette tip 55. Using disposable pipette tips 55 for liquid addition can avoid cross - contamination during multiple detections.

[0084] In addition, as a specific embodiment of the present invention, the amplification detection of the target DNA is carried out on a microfluidic chip 56 driven by a two - stage centrifuge. After the DNA sample and the buffer solution containing Mg ions are injected into the sample chamber, the first - stage centrifuge is started at 1500 rpm. The sample mixture is evenly distributed into 8 quantitative pools of 20 μl. Then the second - stage centrifuge is started at 3000 rpm to transfer the sample to the amplification chamber.

[0085] The isothermal amplification reagent containing the enzyme solution, primers, and probes are made into freeze - dried microspheres and pre - placed in the amplification chamber. After the DNA and the buffer enter the amplification chamber, they dissolve the freeze - dried microspheres and carry out an amplification reaction with the intervention of Mg ions. A heater with high - precision temperature feedback control is designed at the bottom of the amplification chamber to achieve a constant temperature of 39°C. After the amplified DNA fragment binds to the probe, it emits fluorescence and is detected by the detector. The fluorescence values of the entire reaction process can be plotted into an amplification curve. There are 3 reaction zones on each chip, enabling parallel detection and blank detection. Each zone has 8 detection channels. By pre - setting different primers and probes, simultaneous detection of up to 8 genes can be achieved.

[0086] An example of the overall working process of the present invention: Collect a certain amount of environmental water sample, filter and concentrate it. The concentrated solution enters the instrument compartment through high - low pressure conversion. Take a quantitative amount of the concentrated solution for cell lysis (high temperature or adding a lysis solution). The lysed sample is injected into high - concentration alcohol and passed through a chromatography column. The impurities are filtered out while the DNA is retained by the chromatography column. Flush with a quantitative eluent to obtain a high - purity DNA sample. DNA amplification uses the RPA isothermal amplification method. The high - purity DNA sample is first mixed with the enzyme solution, quantitatively added to the microfluidic chip 56, and primers, probes, and magnesium ions are pre - set in the chip. React at a constant temperature of 39°C with the chip rotating at a low speed. The light intensity is recorded by a developed fluorescence detector during the reaction process to obtain an amplification curve.

[0087] As Figure 6 shown, on the other hand, the present invention provides a method for in-situ nucleic acid detection of deep-sea microorganisms, using the long-term experimental platform for in-situ detection of deep-sea microorganisms as described above, including the following steps:

[0088] Utilize the filtration sampling module 3 to perform negative pressure vertical filtration to enrich microorganisms in-situ underwater, and intercept the microorganisms remaining in the water body;

[0089] Lyse the microorganisms through the lysis and purification module 4, and obtain a high-purity DNA sample through a chromatography column and eluent;

[0090] Inject the high-purity DNA sample into the microfluidic chip 56, and synchronously detect the fluorescence signal to obtain an amplification curve for detecting specific microorganisms.

[0091] Specifically, it includes the following steps:

[0092] S1. Concentrate microorganisms in-situ underwater by using the negative pressure vertical filtration method, and obtain a sample with concentrated microorganisms through the filtration sampling module 3;

[0093] S2. The sample of concentrated microorganisms enters the lysis and purification module 4 in the pressure-resistant cabin through the high-low pressure conversion valve;

[0094] S3. Flush the pipeline and membrane package of the filtration sampling module 3 and perform nucleic acid digestion protection;

[0095] S4. Lyse the cells in the sample through the lysis and purification module 4;

[0096] S5. Use the air pump 414 to blow air to make the sample of lysed cells flow through the chromatography column 413, wash the chromatography column 413 with alcohol, and use the air pump 414 to dry the alcohol;

[0097] S6. Flush with eluent to obtain a high-purity DNA sample, and send the DNA sample into the sample pool;

[0098] S7. Use an electric pipette to load the tip 55, suck the DNA sample from the sample pool, inject it into the sample pool containing nucleic acid amplification reagents for mixing, and then use the pipette to suck the mixed solution and inject it into the microfluidic chip 56;

[0099] S8. The microfluidic chip 56 is centrifuged twice to evenly distribute and quantify the mixed solution into the detection chamber of the microfluidic chip 56;

[0100] S9. Place the microfluidic chip 56 in a constant temperature chamber to amplify the DNA and synchronously detect the fluorescence signal.

[0101] S10. Perform flow path cleaning on the lysis and purification module 4.

[0102] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms, characterized in that, It includes a platform framework, a filtration sampling module, and a pressure-resistant instrument cabin, and a lysis purification module and an amplification detection module are arranged in the pressure-resistant instrument cabin; The filtration detection module includes a deep-sea in-situ tangential flow filtration and circulation assembly, and the filtration detection module is used for filtering and enriching microorganisms in seawater, discharging seawater and intercepting microorganisms in the water body; The lysis purification module includes a lysis chamber and a chromatography column, and the lysis purification module is used for extracting DNA from the microorganisms and obtaining a high-purity DNA sample by eluting through the chromatography column and an eluent; The amplification detection module includes a microfluidic chip and a fluorescence detector, and the amplification detection module is used for injecting the high-purity DNA sample and a reagent into the microfluidic chip for fluorescence labeling amplification of microbial genes.

2. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 1, characterized in that, The deep-sea in-situ tangential flow filtration and circulation assembly includes a tangential flow filtration membrane package, a metering chamber, a deep-sea circulation pump, a deep-sea sampling pump, a three-way valve, and a circulation pipeline; The water inlet of the tangential flow filtration membrane package is connected to the deep-sea circulation pump through the circulation pipeline, and the water return port of the tangential flow filtration membrane package is connected to the metering chamber and the pressure-resistant instrument cabin through the circulation pipeline and one inlet and outlet of the three-way valve; The metering chamber, the deep-sea circulation pump, and the tangential flow filtration membrane package form a deep-sea tangential flow concentration circulation.

3. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 1, characterized in that, The filtration sampling module further includes a deep-sea in-situ automatic cleaning assembly, and the filtered seawater is used to clean and protect the tangential flow filtration and circulation assembly to prevent cross-contamination during multiple samplings in the deep-sea environment.

4. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 3, characterized in that, The deep-sea in-situ automatic cleaning assembly includes a filtered water collection cabin, a cleaning agent cabin, a deep-sea three-in-one-out reversing valve, and a deep-sea stop valve; The filtered water collection cabin is connected to the water outlet of the tangential flow filtration membrane package; The deep-sea three-in-one-out reversing valve is arranged on the pipeline between the deep-sea experience pump and the metering chamber, and the first interface of the deep-sea three-in-one-out reversing valve is connected to the filtered water collection cabin, and the second interface is connected to the cleaning agent cabin; The deep-sea stop valve is connected to the end cover of the metering chamber through a pipeline.

5. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 3, characterized in that The deep-sea in-situ tangential flow filtration and circulation assembly further includes a flowmeter and a coarse filter; The flowmeter and the coarse filter are both arranged at the front end of the inlet of the metering chamber. The flowmeter is used to record the water intake of the metering chamber and output data in real time; the coarse filter is used to prevent larger particles from entering the deep-sea tangential flow concentration circulation.

6. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 1, characterized in that, The lysis purification module includes a sample chamber, the lysis chamber, the chromatography column, an air pump, an alcohol reagent bag, an eluent reagent bag, a multi-channel rotary valve, a one-way valve, a reversing valve, a waste liquid chamber, an alcohol adsorption chamber, a DNA collection tube, a plurality of plunger pumps, and a flow-through pipeline, and the plurality of plunger pumps include a first plunger pump, a second plunger pump, and a third plunger pump; The sample chamber is connected to the lysis chamber through the first plunger pump, and a semiconductor heating sheet is arranged on the wall of the lysis chamber; The alcohol reagent bag is connected to the lysis chamber through the second plunger pump, and a one-way valve is arranged at the discharge port of the lysis chamber and is connected to the multi-channel rotary valve; The front end of the chromatography column is connected to the multi-channel rotary valve, and the rear end is respectively connected to the DNA collection tube and the waste liquid chamber through the reversing valve; The sample outlet of the lysis chamber is connected to the chromatography column and the waste liquid chamber, and the eluent reagent bag is connected to the multi-channel rotary valve through the third plunger pump.

7. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 1, characterized in that, The amplification and detection module includes a manipulator, an electric pipette, a constant temperature amplification chamber, a fluorescence detector, a plurality of tips, the microfluidic chip and a chip chamber, wherein the chip chamber includes two chip storage chambers and a chip waste chamber; The manipulator includes a support frame, a Z-axis motor, a radial motor, a rotating main shaft and an electric claw clamp.

8. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 1, wherein, The pressure-resistant instrument chamber is a cylindrical pressure-resistant instrument chamber, and the lysis and purification module and the amplification and detection module are jointly encapsulated in the pressure-resistant instrument chamber; A sample high and low pressure transfer module is arranged on the end cover of the pressure-resistant instrument chamber for transferring the concentrated microbial sample after lysis into the pressure-resistant instrument chamber.

9. A method for in-situ nucleic acid detection of deep-sea microorganisms, characterized in that, Using the long-term experimental platform for in-situ detection of deep-sea microorganisms according to any one of claims 1-8, comprising the following steps: Using the filtration sampling module to perform in-situ microbial filtration and enrichment underwater by negative pressure vertical filtration, and intercepting the microorganisms remaining in the water body; Lysing the microorganisms through the lysis and purification module, and obtaining a high-purity DNA sample through the chromatography column and the eluent; Injecting the high-purity DNA sample into the microfluidic chip, and synchronously detecting the fluorescence signal to obtain an amplification curve for detecting specific microorganisms.

10. The in-situ nucleic acid detection method for deep-sea microorganisms according to claim 9, characterized in that, Comprising the following steps: S1. Concentrate microorganisms in-situ underwater by negative pressure vertical filtration, and obtain a sample with concentrated microorganisms through the filtration sampling module; S2. The sample of concentrated microorganisms enters the lysis and purification module in the pressure-resistant chamber through the high and low pressure conversion valve; S3. Flush the pipeline and membrane package of the filtration sampling module and perform nucleic acid digestion protection; S4. Lyse the cells in the sample through the lysis and purification module; S5. Use an air pump to blow air to make the sample of lysed cells flow through the chromatography column, wash the chromatography column with alcohol, and dry the alcohol with an air pump; S6. Flush with the eluent to obtain a high-purity DNA sample, and send the DNA sample to the sample pool; S7. Use an electric pipette to load the tips, suck the DNA sample from the sample pool, inject it into the sample pool containing the nucleic acid amplification reagent for mixing, and then use the pipette to suck the mixed solution and inject it into the microfluidic chip; S8. The microfluidic chip is centrifuged twice to evenly distribute and quantify the mixed solution into the detection chamber of the microfluidic chip; S9. Place the microfluidic chip in a constant temperature chamber to amplify the DNA and synchronously detect the fluorescence signal. S10. Perform flow path cleaning on the lysis and purification module.

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