A long-term experimental platform and detection method for in situ nucleic acid detection in deep-sea microorganisms.

Abstract

This invention relates to the field of deep-sea microbial nucleic acid detection technology, specifically a long-term experimental platform and detection method for in-situ nucleic acid detection of deep-sea microorganisms. In this invention, a tangential flow filtration and concentration method is used to concentrate water samples via a filtration sampling module to obtain concentrated microbial samples. The samples are then transferred under reduced pressure to a lysis and purification module for cell lysis. The lysed samples are injected into a chromatography column, where impurities are filtered out while DNA is retained. The samples are then rinsed with elution buffer to obtain a high-purity DNA sample. A pipette tip is attached to the elution pipette, and the high-purity DNA sample is mixed with an enzyme solution and added to an amplification and detection module for reaction. Fluorescently labeled genes are amplified, and an amplification curve is obtained using a fluorescence detector. Changes in the amplification curve are used to detect in-situ nucleic acids of deep-sea microorganisms. The filtration sampling module incorporates an automatic cleaning component, and the lysis purification module uses a sampling, cleaning, and lysis chamber switching mechanism to prevent cross-contamination from multiple in-situ deep-sea detections.

Description

Technical Field

[0001] This invention relates to the field of deep-sea microbial nucleic acid detection technology, specifically to a long-term experimental platform and detection method for in-situ nucleic acid detection of deep-sea microorganisms. Background Technology

[0002] Deep-sea functional microorganisms exhibit exceptionally high species diversity and metabolic mechanism diversity, participating in highly complex networks of matter and energy conversion and transfer. Furthermore, environmental changes significantly alter functional microbial communities, and conversely, changes in functional microbial communities can also indicate environmental changes. Long-term, continuous monitoring of specific functional microbial groups can elucidate the patterns of environmental change in typical deep-sea ecosystems, reveal the ecological functions of microorganisms, and provide a theoretical basis for assessing the disturbance effects of human activities on typical deep-sea ecosystems and developing practical and effective deep-sea ecological security mechanisms.

[0003] For a long time, the monitoring of microorganisms in the deep-sea environment has typically relied on a sampling-to-laboratory analysis method. On the one hand, due to changes in environmental conditions and long-term transportation, the microorganisms in the samples can change, especially low-abundance microorganisms which may die off and become difficult to detect. On the other hand, this method struggles to obtain time-series biological data. Furthermore, it is difficult to acquire data on microorganisms that cannot be isolated and cultured in the laboratory, particularly those with low abundance, and the changes in microbial abundance during environmental changes. Therefore, there is an urgent need to develop instruments and methods for in-situ, real-time detection and time-series analysis, especially microbial detection instruments that can be deployed on the seabed for periodic, multiple tests, to provide data support for deep-sea biological research. Achieving long-term, continuous in-situ detection of deep-sea functional microorganisms requires solving technical challenges such as fully automated, integrated low-abundance microbial concentration sampling, cell lysis and DNA purification, real-time gene detection and analysis, and prevention of cross-contamination. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art, namely, the difficulty in obtaining high-purity DNA samples from microbial samples with low to medium abundance in the sea, and the complexity of the system due to the numerous retrieval steps. This invention provides a long-term experimental platform and detection method for in situ nucleic acid detection of deep-sea microorganisms.

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

[0006] A long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms includes a platform frame, a filtration and sampling module, and a pressure-resistant instrument chamber. The pressure-resistant instrument chamber is equipped with a lysis and purification module and an amplification and detection module.

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

[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 microorganism and elute it with the chromatography column and elution buffer to obtain a high-purity DNA sample.

[0009] The amplification detection module includes a microfluidic chip and a fluorescence detector. The amplification detection module is used to inject the high-purity DNA sample and the reaction agent into the microfluidic chip for fluorescent 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 in this invention, the deep-sea in-situ tangential flow filtration and circulation assembly includes a tangential flow filtration membrane pack, a quantitative chamber, a deep-sea circulation pump, a deep-sea sample injection pump, a three-way valve, and a circulation pipeline.

[0011] The inlet of the tangential flow filter membrane pack is connected to the deep-sea circulation pump through the circulation pipe, and the outlet of the tangential flow filter membrane pack is connected to the metering chamber and the pressure-resistant instrument chamber through the circulation pipe and one inlet / outlet of the three-way valve.

[0012] The quantitative chamber, the deep-sea circulation pump, and the tangential flow filter membrane form a deep-sea tangential flow concentration circulation system.

[0013] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms in this invention, the filtration sampling module also includes a deep-sea in-situ automatic cleaning component, which uses filtered seawater to clean and protect the tangential flow filtration circulation component to prevent cross-contamination from 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 in this invention, the deep-sea in-situ automatic cleaning component includes a water filter chamber, a cleaning agent chamber, a deep-sea three-inlet-one-outlet reversing valve, and a deep-sea shut-off valve.

[0015] The filtered water collection chamber is connected to the outlet of the tangential flow filter membrane pack;

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

[0017] The deep-sea shut-off valve is connected to the end cap of the metering chamber via 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 in this invention, the deep-sea in-situ tangential flow filtration circulation component also includes a flow meter and a coarse filter.

[0019] Both the flow meter and the coarse filter are located at the front end of the quantitative chamber inlet. The flow meter is used to record the amount of water injected into the quantitative chamber and output the data in real time. The coarse filter is used to prevent larger particles from entering the deep-sea tangential flow concentration cycle.

[0020] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms in this 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, multiple plunger pumps and flow lines, wherein the multiple plunger pumps include a first plunger pump, a second plunger pump and a third plunger pump;

[0021] The sample chamber is connected to the pyrolysis chamber via the first plunger pump, and a semiconductor heating plate is provided on the wall of the pyrolysis chamber;

[0022] The alcohol reagent bag is connected to the pyrolysis chamber via the second plunger pump. The outlet of the pyrolysis chamber is equipped with the one-way valve and 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 connected to the DNA collection tube and the waste liquid chamber respectively through the reversing valve;

[0024] 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.

[0025] As an improvement to the technical solution of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms in this invention, the amplification and detection module includes a robotic arm, an electric pipette, a constant temperature amplification chamber, a fluorescence detector, multiple pipette tips, the microfluidic chip, and a chip compartment, wherein the chip compartment includes two chip storage compartments and one chip waste compartment; the robotic arm includes a support frame, a Z-axis motor, a radial motor, a rotary spindle, and an electric gripper.

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

[0027] The pressure-resistant instrument chamber is equipped with a sample high- and low-pressure transfer module on its end cap, which is used to transfer the lysed concentrated microbial sample into the pressure-resistant instrument chamber.

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

[0029] The filtration sampling module uses tangential flow filtration to enrich microorganisms in situ underwater and capture the microorganisms remaining in the water.

[0030] The microorganisms were lysed using a lysis purification module, and high-purity DNA samples were obtained through chromatography and elution.

[0031] High-purity DNA samples are injected into a microfluidic chip, and fluorescence signals are detected simultaneously to obtain amplification curves for the detection of specific microorganisms.

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

[0033] S1. Microorganisms are concentrated in situ underwater using tangential flow filtration, and samples containing concentrated microorganisms are obtained through a filtration sampling module.

[0034] S2. The concentrated microbial sample enters the lysis and purification module in the pressure chamber through the high-low pressure switching valve;

[0035] S3. Rinse and protect the tubing and membrane of the filtration sampling module with nucleic acid digestion.

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

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

[0038] S6. Rinse with elution buffer to obtain a high-purity DNA sample and transfer the DNA sample into the sample pool.

[0039] S7. Load the pipette tip with an electric pipette, draw DNA sample from the sample cell, inject it into the sample cell containing nucleic acid amplification reagent, mix, and then use a pipette to draw the mixed solution into the microfluidic chip.

[0040] S8. The microfluidic chip undergoes a second centrifugation process to uniformly distribute a quantitative mixed solution into the detection chamber of the microfluidic chip;

[0041] S9. The microfluidic chip is placed in a constant temperature chamber to amplify DNA and simultaneously detect fluorescence signals;

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

[0043] The beneficial effects of this invention are:

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

[0045] In this invention, the tubing in the tangential flow filtration sampling component is cleaned after each run by using a deep-sea in-situ automatic cleaning component, thus avoiding cross-contamination of the long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms due to repeated enrichment of microorganisms in the deep sea.

[0046] In this invention, a quantitative amount of concentrated solution is used for cell lysis in the lysis and purification module. The lysed sample is then injected into a chromatography column, where impurities are filtered out while DNA is retained. The sample is then rinsed with elution buffer to obtain a high-purity DNA sample, ensuring the reliability and stability of the detection. Multiple high-purity DNA samples are collected independently using a multi-channel valve switching system, effectively preventing cross-contamination. A robotic arm drives a high-precision pipette for precise positioning, and the high-purity DNA sample is precisely and quantitatively added to the microfluidic chip in the amplification and detection module via automatic tip loading for amplification. Fluorescence values ​​are detected, and an amplification curve is obtained. Changes in the amplification curve are used to detect in-situ nucleic acids of deep-sea microorganisms, ensuring a high success rate.

[0047] In this invention, a fully automated process from sample collection to result output is achieved in situ in the deep sea without human intervention. Automatic cleaning and maintenance are implemented during microbial sampling, and independent tubing is used for different samples during DNA extraction. This enables the instrument to perform multiple in-situ tests in the deep sea. After a set interval, it can be deployed to the deep sea for long-term time-series microbial detection, providing scientists with data on the dynamic changes of deep-sea microorganisms. Attached Figure Description

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

[0049] Figure 2 This is a schematic diagram of the filtering sampling module structure in this invention;

[0050] Figure 3 This is a schematic diagram of the pyrolysis and purification module in this invention;

[0051] Figure 4 for Figure 3 A schematic diagram of the split structure;

[0052] Figure 5 This is a schematic diagram of the amplification detection module in this invention;

[0053] Figure 6 This is a flowchart of the deep-sea microbial in situ nucleic acid detection method of the present invention.

[0054] Figure Label Explanation: 1 - Platform Frame; 2 - Power Supply; 3 - Filtration Sampling Module; 4 - Lysis and Purification Module; 5 - Amplification and Detection Module; 6 - Pressure-Resistant Instrument Chamber; 31 - Deep-Sea In-Situ Tangential Flow Filtration and Circulation Assembly; 311 - Tangential Flow Filtration Membrane Pack; 312 - Quantitative Chamber; 313 - Deep-Sea Circulation Pump; 314 - Deep-Sea Sample Injection Pump; 315 - Flow Meter; 316 - Coarse Filter; 317 - Three-Way Valve; 321 - Filter Water Collection Chamber; 322 - Cleaning Agent Chamber; 323 - Deep-Sea Three-In-One-Out Reversing Valve; 324 - Deep-Sea Shut-Off Valve; 411 - Sample Chamber; 412 - Lysis Chamber; 413 - Chromatography Column; 414 - Gas Pump; 415 - Alcohol Reagent Bag; 416 - Eluent Reagent Bag; 417 - Multi-Channel Rotary Valve; 418 - 419 - Reversing valve (one-way valve); 420 - Waste liquid chamber; 421 - Alcohol adsorption chamber; 422 - DNA collection tube; 423 - First plunger pump; 424 - Second plunger pump; 425 - Third plunger pump; 51 - Robotic arm; 511 - Support frame; 512 - Z-axis motor; 513 - Radial motor; 514 - Rotary spindle; 515 - Electric gripper; 52 - Electric pipette; 53 - Isothermal amplification chamber; 54 - Fluorescence detector; 55 - Pipe tip; 56 - Microfluidic chip; 571 - Chip storage compartment; 572 - Chip waste compartment. Detailed Implementation

[0055] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0056] like Figures 1 to 5 As shown, 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] The filtration sampling module 3 is used to filter seawater. The filtration sampling module 3 uses tangential flow filtration to concentrate microorganisms in the water in situ to obtain a sample with concentrated microorganisms.

[0058] The lysis and purification module 4 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 from the chromatography column.

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

[0060] In this invention, the filtration sampling module 3 is used to perform in-situ underwater microbial filtration and enrichment by tangential flow filtration, and to intercept the microorganisms remaining in the water.

[0061] Microorganisms were lysed using the lysis and purification module 4, and high-purity DNA samples were obtained through chromatography column and elution buffer.

[0062] High-purity DNA samples are injected into microfluidic chip 56 and fluorescence signals are detected simultaneously to obtain amplification curves for the detection of specific microorganisms.

[0063] In detail, this invention collects a certain amount of environmental water sample through the filtration sampling module 3, which is then filtered and concentrated to obtain a concentrated solution. A quantitative amount of the concentrated solution is taken and introduced into the sample chamber 411 of the pressure-resistant instrument chamber 6 through the high-low pressure conversion module, and then introduced into the lysis and purification module 4 for cell lysis. The lysed sample flows through a chromatography column, where impurities are filtered out while DNA is retained. Then, it is washed with elution buffer to obtain a high-purity DNA sample. The high-purity DNA sample is then mixed with an enzyme solution and quantitatively added to the amplification and detection module 5 for isothermal amplification to increase the amount of fluorescently labeled DNA. The amplification curve is obtained by passing the sample through a fluorescence detector, thus realizing the detection of in-situ nucleic acids of deep-sea microorganisms.

[0064] In existing technologies, negative pressure vertical filtration is generally used to concentrate microorganisms in situ underwater. The amount of microorganisms in the filter membrane and the concentrate is different. Whether rinsing the filter membrane or collecting the concentrate, it is impossible to accurately quantify the concentration of microorganisms in the sample.

[0065] In this invention, tangential flow filtration is used to concentrate microorganisms in water in situ. During the filtration process, the flow direction of the sample is parallel to the surface of the filter membrane, which can wash the surface of the filter membrane and effectively alleviate the accumulation of cells or particles on the filter membrane. On the one hand, it avoids insufficient water filtration caused by filter membrane blockage, and on the other hand, it allows the collected microorganisms to be evenly retained in the concentrate, which is convenient for post-processing.

[0066] The invention also includes a power supply 2, which is electrically connected to the filter sampling module 3, the pressure-resistant instrument chamber 6, the amplification detection module 5, and the lysis purification module 4 to supply power to them.

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

[0068] The deep-sea in-situ tangential flow filtration and circulation assembly 31 includes a tangential flow filtration membrane pack 311, a quantitative chamber 312, a deep-sea circulation pump 313, a deep-sea sample injection pump 314, a deep-sea pressure reducing valve, a flow meter 315, and a coarse filter 316. The inlet of the tangential flow filtration membrane pack is connected to the deep-sea circulation pump 313 via pipes, and the outlet of the tangential flow filtration membrane pack is connected to the quantitative chamber 312 and the pressure-resistant instrument chamber 6 via pipes and a three-way valve 317. The quantitative chamber 312, the deep-sea circulation pump 313, and the tangential flow filtration membrane pack 311 form a tangential flow filtration circulation.

[0069] The quantitative chamber 312 is equipped with a flow meter 315 and a coarse filter 316 at the front end of the sample inlet. The flow meter 315 is used to record the sample volume and output data in real time; the coarse filter 316 is used to prevent larger particles from entering the tangential flow filtration circulation and clogging the tangential flow membrane pack. Preferably, the tangential flow filter membrane pack uses Millipore P2GVPPV05 to achieve a seawater filtration capacity of more than 30L and meet a concentration ratio of ≥100:1.

[0070] In some embodiments of the present invention, multiple cross-contamination-free sampling is achieved by using a deep-sea in-situ automatic cleaning assembly; the deep-sea in-situ automatic cleaning assembly includes a filtered water collection chamber 321, a cleaning agent chamber 322, a deep-sea three-inlet-one-outlet reversing valve 323, and a deep-sea shut-off valve 324.

[0071] Furthermore, the filtered water collection chamber 321 is provided with an interface connected to the outlet of the tangential flow filter membrane package 311; the deep-sea three-inlet-one-outlet reversing valve 323 is installed on the connecting pipeline between the deep-sea sampling pump 314 and the quantitative chamber 312, with one interface connected to the filtered water collection chamber 321 and the other interface connected to the cleaning agent chamber 322; the deep-sea shut-off valve 324 is installed on the end cap of the quantitative chamber 312; the cleaning agent chamber 322 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, in which the lysis and purification module 4 and the amplification and detection module 5 are encapsulated together; the pressure environment inside the pressure-resistant instrument chamber 6 is an atmospheric pressure environment; the end cap of the pressure-resistant instrument chamber 6 is provided with a sample high and low pressure transfer module for transferring concentrated microbial samples to the atmospheric pressure environment inside the pressure-resistant instrument chamber 6.

[0073] As a specific embodiment of this method, the deep-sea in-situ automatic cleaning process is as follows: During tangential flow filtration circulation, the discharged filtered seawater is collected in the filtered water collection chamber 321; after the tangential flow filtration circulation is completed, the deep-sea three-inlet-one-outlet reversing valve 323 is switched to the interface of the filtered water collection chamber 321, and the deep-sea circulation pump 313 is started to inject the seawater in the filtered water collection chamber 321 into the pipeline and tangential flow filter membrane package 311 of the deep-sea in-situ tangential flow filtration circulation assembly 31, while the deep-sea shut-off valve 324 is opened. Drain the seawater to rinse away the sample remaining in the deep-sea in-situ tangential flow filtration and circulation assembly 31; close the deep-sea shut-off valve 324 and switch the deep-sea three-in-one-out reversing valve 323 to the cleaning agent tank 322 interface, injecting a certain amount of NaOH solution to dissolve residual microorganisms or DNA; before the next sampling and concentration, switch the deep-sea three-in-one-out reversing valve 323 to the deep-sea sample pump 314 interface, delay opening the deep-sea circulation pump 313 and the deep-sea shut-off valve 324, and rinse away the NaOH solution with in-situ seawater.

[0074] In some embodiments 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, multiple plunger pumps, and flow channels; the multiple plunger pumps include a first plunger pump 422, a second plunger pump 423, and a third plunger pump 424.

[0075] Furthermore, the sample chamber 411 is connected to the lysis chamber 412 via a first plunger pump 422. The wall of the lysis chamber 412 is equipped with a semiconductor heating plate to achieve high-temperature cell lysis. The alcohol reagent bag is connected to the lysis chamber 412 via a second plunger pump 423 to flush the lysed sample into the chromatography column 413. The outlet of the lysis chamber 412 is equipped with a one-way valve and is connected to a 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. The reversing valve 418 is connected to the DNA collection tube 421 and the waste liquid chamber 419, respectively.

[0076] The lysed sample flows through the chromatography column 413 into the waste chamber 419, where DNA is retained by the chromatography column 413 to achieve DNA purification. The eluent reagent bag 416 is connected to the multi-channel rotary valve 417 via the third plunger pump 424. After the reversing valve 418 reverses the flow, the eluent flows through the chromatography column 413 to elute the adsorbed DNA and is sent to the DNA collection tube 421. The chromatography column 413, DNA collection tube 421, reversing valve 418, and their connected tubing are multiple independent tubing systems. The multi-channel rotary valve 417 is used to switch between systems for each test to prevent cross-contamination.

[0077] Furthermore, in this invention, microbial lysis is illustrated using a high-temperature lysis method; DNA purification is illustrated using a silica gel membrane chromatography column 413 method.

[0078] As a specific embodiment of this method, the automated DNA extraction and purification process is as follows: The concentrated microbial sample in sample chamber 411 enters lysis chamber 412 through first plunger pump 422. The lysis chamber 412 is heated to 95°C and maintained for 10 minutes by a semiconductor heating element to destroy the microbial cells, and the DNA in the cells enters the liquid. Anhydrous ethanol is injected into lysis chamber 412 through second injection pump, forming a 70% ethanol environment mixture in lysis chamber 412. The mixture is then passed through silica gel membrane chromatography column 413 by air pump 414, and the overflow liquid enters waste liquid chamber 419. The DNA is retained by chromatography column 413. Then, 70% ethanol is injected using second plunger pump 423 to wash chromatography column 413 1-2 times to better remove impurities. Air pump 414 runs continuously for 5 minutes to blow away the ethanol in chromatography column 413 and tubing. The gas flows through alcohol adsorption chamber 420 containing activated carbon, and the alcohol is adsorbed to prevent excessive alcohol in the instrument chamber and avoid danger. Finally, the DNA is eluted from the chromatography column 413 by adding elution buffer through the third plunger pump 424 to achieve DNA purification; after the reversing valve 418 is switched, the air pump 414 is started, and the airflow 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, DNA collection tube 421, reversing valve 418 and its connected tubing are 24 independent components. Each component is connected to one channel of the multi-channel rotary valve 417, so that the sample after each sample lysis enters a new tubing and chromatography column 413, and DNA purification of 24 samples can be completed to avoid contamination from multiple tests.

[0080] In some embodiments of the present invention, the amplification detection module 5 includes a robotic arm 51, an electric pipette 52, a constant temperature amplification chamber 53, a fluorescence detector 54, multiple pipette tips 55, a microfluidic chip 56, and a chip compartment; the robotic arm 51 includes a support frame 511, a Z-axis motor 512, a radial motor 513, a rotary spindle 514, and an electric gripper 515; the chip compartment includes two chip storage compartments 571 and one chip waste compartment 572;

[0081] As a specific embodiment of this method, the automated DNA amplification detection process is as follows: a radial motor 513 drives a rotating spindle 514 to move and position the electronic pipette and electric gripper 515 in a planar manner; a Z-axis motor 512 drives the electronic pipette and electric gripper 515 to move vertically to the position where actions such as picking up the chip, loading the pipette tip 55, and aspirating and adding liquid are performed. The chip compartment includes two chip storage compartments 571 and one chip waste compartment 572; each chip storage compartment 571 holds multiple microfluidic chips 56; the multiple microfluidic chips 56 are transferred by a robotic arm 51, and the electric gripper 515 is moved to a designated position by a moving device; the electric gripper 515 picks up the microfluidic chip 56 from the chip storage compartment 571 and places it into the isothermal amplification chamber 53; after detection, the electric gripper 515 picks up the microfluidic chip 56 and moves it to the chip waste compartment 572 to discard the microfluidic chip 56.

[0082] In detail, the radial motor 513 and the rotary spindle 514 are used to drive the electronic pipette and the electric gripper 515 to achieve planar movement and positioning; the Z-axis motor 512 is used to drive the electronic pipette and the electric gripper 515 to achieve vertical movement, reaching the position to perform 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 holds multiple microfluidic chips 56. The multiple microfluidic chips 56 are transferred by a robotic arm 51, and a moving device moves an electric gripper 515 to above the chip storage compartment 571. The electric gripper 515 picks up the microfluidic chip 56 and places it into a 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 chamber of the microfluidic chip 56 by centrifugal force and mixed with pre-placed reagents. The mixture is heated to maintain a specific temperature for amplification reaction. A fluorescence detector 54 detects the fluorescence value of the reaction area and plots the detected fluorescence value as a reaction curve after a certain time. If the reaction curve exceeds a certain threshold, it is considered that microorganisms have been detected, and the time to reach the threshold is converted into DNA concentration. After the detection is completed, the electric gripper 515 picks up 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, to achieve 24 time-series detections, two chip storage compartments 571 and one chip waste compartment 572 are designed. Each chip storage compartment 571 holds 12 microfluidic chips 56. After completing 12 detections, the first chip storage compartment 571 becomes the chip waste compartment 572. Chip transfer is performed by a 3-DOF translational manipulator 51, which can reach any position in the planar area through rotational axis and linear motion. Two motors are mounted on the Z-axis. One motor controls the manipulator to grasp the chip and place it into the temperature-controlled detection compartment, and remove the chip after detection and discard it into the waste compartment. The manipulator 51 also performs operations such as grasping magnetic caps and insulated caps. The other motor controls a high-precision electronic pipette to perform operations such as loading pipette tips 55, adding liquid to the chip, and discarding pipette tips 55. Using disposable pipette tips 55 for liquid addition avoids cross-contamination during multiple detection processes.

[0084] In another specific embodiment of the present invention, the amplification and detection of the target DNA is performed on a two-stage centrifugation-driven microfluidic chip 56. After the DNA sample and the Mg-containing buffer are injected into the sample chamber, the first-stage centrifugation is started at 1500 rpm. The sample mixture is evenly distributed into eight 20 μl quantitative cells. Then, the second-stage centrifugation is started at 3000 rpm, and the sample is transferred to the amplification chamber.

[0085] Isothermal amplification reagents containing enzyme solution, primers, and probes are pre-prepared as lyophilized microspheres and placed in the amplification chamber. DNA and buffer are introduced into the amplification chamber to dissolve the lyophilized microspheres, and the amplification reaction occurs with the presence of Mg ions. The bottom of the amplification chamber is designed with a high-precision temperature feedback-controlled heater to achieve a constant temperature of 39°C. The amplified DNA fragments bind to the probes, emitting fluorescence that is detected by the detector. The fluorescence values ​​throughout the reaction process can be plotted as an amplification curve. Each chip has three reaction zones, enabling parallel detection and blank detection. Each zone has eight detection channels, allowing for the simultaneous detection of up to eight genes by pre-preparing different primers and probes.

[0086] An example of the overall workflow of this invention: A certain amount of environmental water sample is collected, filtered, and concentrated. The concentrate is then transferred to the instrument chamber via a high-low pressure conversion process. A quantitative amount of the concentrate is used for cell lysis (high temperature or with added lysis buffer). The lysed sample is injected into a high-concentration ethanol chromatography column, where impurities are filtered out while DNA is retained. A quantitative amount of elution buffer is used to rinse the sample to obtain a high-purity DNA sample. DNA amplification is performed using the RPA isothermal amplification method. The high-purity DNA sample is first mixed with an enzyme solution, and a quantitative amount is added to a microfluidic chip 56, which contains pre-placed primers, probes, and magnesium ions. The reaction is carried out at a constant temperature of 39°C with the chip rotated at a low speed. The reaction process is monitored using a specially developed fluorescence detector to record the light intensity, resulting in an amplification curve.

[0087] like Figure 6 As shown, another aspect of 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] The filtration sampling module 3 is used to perform in-situ underwater microbial filtration and enrichment by tangential flow filtration, and to intercept the microorganisms remaining in the water.

[0089] Microorganisms were lysed using the lysis and purification module 4, and high-purity DNA samples were obtained through chromatography column and elution buffer.

[0090] High-purity DNA samples are injected into microfluidic chip 56 and fluorescence signals are detected simultaneously to obtain amplification curves for the detection of specific microorganisms.

[0091] In detail, it includes the following steps:

[0092] S1. Microorganisms are concentrated in situ underwater using tangential flow filtration, and a sample containing concentrated microorganisms is obtained through the filtration sampling module 3.

[0093] S2. The concentrated microbial sample enters the lysis and purification module 4 in the pressure chamber through the high-low pressure switching valve;

[0094] S3. Rinse and protect the tubing and membrane of the filtration sampling module 3 with nucleic acid digestion.

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

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

[0097] S6. Rinse with elution buffer to obtain a high-purity DNA sample and transfer the DNA sample into the sample pool.

[0098] S7. Load the pipette tip 55 with an electric pipette, draw DNA sample from the sample cell, inject it into the sample cell containing nucleic acid amplification reagent, mix, and then use a pipette to draw the mixed solution into the microfluidic chip 56.

[0099] S8. After two centrifugations, the microfluidic chip 56 uniformly distributes a quantitative mixed solution into the detection chamber of the microfluidic chip 56.

[0100] S9 and microfluidic chip 56 are placed in a constant temperature chamber to amplify DNA and simultaneously detect fluorescence signals.

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

[0102] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are 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 frame, a filtering and sampling module, and a pressure-resistant instrument chamber, wherein the pressure-resistant instrument chamber is equipped with a lysis purification module and an amplification detection module; The filtration sampling module includes a deep-sea in-situ tangential flow filtration and circulation component. The filtration sampling module is used to filter and enrich microorganisms in seawater, discharge seawater and retain microorganisms in the water. 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 microorganism and elute it with the chromatography column and elution buffer to obtain a high-purity DNA sample. The amplification detection module includes a microfluidic chip and a fluorescence detector. The amplification detection module is used to inject the high-purity DNA sample and the reaction agent into the microfluidic chip for fluorescent labeling amplification of microbial genes. The deep-sea in-situ tangential flow filtration and circulation assembly includes a tangential flow filtration membrane pack, a quantitative chamber, a deep-sea circulation pump, a deep-sea sample injection pump, a three-way valve, and circulation pipelines. The inlet of the tangential flow filter membrane pack is connected to the deep-sea circulation pump through the circulation pipe, and the outlet of the tangential flow filter membrane pack is connected to the metering chamber and the pressure-resistant instrument chamber through the circulation pipe and one inlet / outlet of the three-way valve. The quantitative chamber, the deep-sea circulation pump, and the tangential flow filtration membrane form a deep-sea tangential flow concentration circulation system. The filtration sampling module also includes a deep-sea in-situ automatic cleaning component, which uses filtered seawater to clean and protect the tangential flow filtration circulation component to prevent cross-contamination from multiple samplings in the deep-sea environment. 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, multiple plunger pumps, and flow lines, wherein the multiple plunger pumps include a first plunger pump, a second plunger pump, and a third plunger pump. The sample chamber is connected to the pyrolysis chamber via the first plunger pump, and a semiconductor heating plate is provided on the wall of the pyrolysis chamber; The alcohol reagent bag is connected to the pyrolysis chamber via the second plunger pump. The outlet of the pyrolysis chamber is equipped with the one-way valve and 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 connected to the DNA collection tube and the waste liquid chamber respectively 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; The amplification and detection module includes a robotic arm, an electric pipette, a constant temperature amplification chamber, a fluorescence detector, multiple pipette tips, the microfluidic chip, and a chip compartment, wherein the chip compartment includes two chip storage compartments and one chip waste compartment; the robotic arm includes a support frame, a Z-axis motor, a radial motor, a rotary spindle, and an electric gripper.

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 automatic cleaning assembly includes a filtered water collection chamber, a cleaning agent chamber, a deep-sea three-inlet-one-outlet reversing valve, and a deep-sea shut-off valve. The filtered water collection chamber is connected to the outlet of the tangential flow filter membrane pack; The deep-sea three-inlet-one-outlet reversing valve is installed on the pipeline between the deep-sea experience pump and the metering chamber, and the first port of the deep-sea three-inlet-one-outlet reversing valve is connected to the filtered water collection chamber, and the second port is connected to the cleaning agent chamber. The deep-sea shut-off valve is connected to the end cap of the metering chamber via a pipeline.

3. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 2, characterized in that, The deep-sea in-situ tangential flow filtration and circulation assembly also includes a flow meter and a coarse filter; Both the flow meter and the coarse filter are located at the front end of the quantitative chamber inlet. The flow meter is used to record the amount of water injected into the quantitative chamber and output the data in real time. The coarse filter is used to prevent larger particles from entering the deep-sea tangential flow concentration cycle.

4. The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms according to claim 1, characterized in that, The pressure-resistant instrument chamber is a cylindrical pressure-resistant instrument chamber, and the lysis purification module and the amplification detection module are jointly encapsulated in the pressure-resistant instrument chamber; The pressure-resistant instrument chamber is equipped with a sample high- and low-pressure transfer module on its end cap, which is used to transfer the lysed concentrated microbial sample into the pressure-resistant instrument chamber.

5. A method for in-situ nucleic acid detection of deep-sea microorganisms, characterized in that, The long-term experimental platform for in-situ nucleic acid detection of deep-sea microorganisms as described in any one of claims 1-4 includes the following steps: The filtration sampling module uses tangential flow filtration to enrich microorganisms in situ underwater and capture the microorganisms remaining in the water. The microorganisms were lysed using a lysis purification module, and high-purity DNA samples were obtained through chromatography and elution. High-purity DNA samples are injected into a microfluidic chip, and fluorescence signals are detected simultaneously to obtain amplification curves for the detection of specific microorganisms.

6. The method for in-situ nucleic acid detection of deep-sea microorganisms according to claim 5, characterized in that, It includes the following steps: S1. Microorganisms are concentrated in situ underwater using tangential flow filtration, and samples containing concentrated microorganisms are obtained through a filtration sampling module. S2. The concentrated microbial sample enters the lysis and purification module in the pressure chamber through the high-low pressure switching valve; S3. Rinse and protect the tubing and membrane of the filtration sampling module with nucleic acid digestion. S4. Lyse the cells in the sample using the lysis and purification module; S5. Use an air pump to blow air through the lysed sample to make it flow through the chromatography column, wash the chromatography column with alcohol, and then blow the alcohol dry with an air pump. S6. Rinse with elution buffer to obtain a high-purity DNA sample and transfer the DNA sample into the sample pool. S7. Load the pipette tip with an electric pipette, draw DNA sample from the sample cell, inject it into the sample cell containing nucleic acid amplification reagent, mix, and then use a pipette to draw the mixed solution into the microfluidic chip. S8. The microfluidic chip undergoes a second centrifugation process to uniformly distribute a quantitative mixed solution into the detection chamber of the microfluidic chip; S9. The microfluidic chip is placed in a constant temperature chamber to amplify DNA and simultaneously detect fluorescence signals; S10. Clean the flow path of the pyrolysis and purification module.

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