Aquatic microorganism nucleic acid automatic collection method

Abstract

The invention discloses a method for automatically collecting nucleic acid of aquatic microorganisms. According to the invention, a water sample is injected into a coagulation reaction bin with a temperature control function, and microbial cell lysis is realized through intermittent low-speed stirring; injecting a surface modification magnetic bead suspension into the reaction bin, starting a multi-shaft stirrer to realize efficient combination of a nucleic acid-magnetic bead compound, and synchronously starting temperature control in the bin to maintain RNA stability; by integrating high-precision positioning, pressure sensing and temperature control cracking core technologies, whole-process standardized operation is realized. The device adopts a dual-mode positioning and pressure sensor to ensure millimeter-level precision of geohydrological data of a sampling point; the three-dimensional turbulent flow field is combined with the magnetic separation array, so that the nucleic acid capture rate is greatly improved, and the risk of nucleic acid degradation is reduced as much as possible. According to the modularized flow path system, through three-stage gradient washing and nanometer ultraviolet sterilization, the problems that in traditional water sample collection, the sample size is large, information is prone to being lost, and treatment lags are effectively solved, and the overall collection efficiency and collection accuracy are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial nucleic acid collection, and specifically relates to an automated method for collecting nucleic acids of aquatic microorganisms. Background Art

[0002] Sampling nucleic acids of aquatic microorganisms is a method for studying and monitoring the microbial community structure and function in water bodies. It involves collecting water samples, extracting microbial DNA or RNA therefrom, and then analyzing the types, quantities, and activities of microorganisms using molecular biology techniques such as PCR and gene sequencing. During the sampling process, sterile containers and tools are required to avoid external contamination. At the same time, factors such as sampling location, depth, and time need to be considered to ensure the representativeness and accuracy of the samples. Sampling nucleic acids of aquatic microorganisms is of great significance in the fields of water quality monitoring, ecosystem health assessment, pathogen detection, and microbial resource development.

[0003] However, there are three major defects in traditional aquatic microorganism sampling: First, the low positioning accuracy, distorted depth data, and easy confusion of sample information caused by manual sampling; second, the nucleic acid degradation and changes in microbial activity caused by long-distance transportation of water samples; third, the insufficient manual lysis efficiency during offshore processing. Summary of the Invention

[0004] The purpose of the present invention is to provide an automated method for collecting nucleic acids of aquatic microorganisms in order to solve the above-mentioned problems.

[0005] The technical solution adopted by the present invention is as follows: An automated method for collecting nucleic acids of aquatic microorganisms, the method comprising the following steps:

[0006] S1: Automatically record the longitude and latitude of the sampling point through an integrated GPS / Beidou module, and at the same time measure and record the water depth using a pressure sensor or an ultrasonic ranging system;

[0007] S2: Pump the water body at the target depth into an anti-pollution flow path system through a telescopic sampling tube, and collect a quantitative water sample after pre-rinsing the pipeline;

[0008] S3: Inject the water sample into a coagulation reaction chamber with a temperature control function, automatically add a lysis solution in proportion, and achieve lysis of microbial cells through intermittent low-speed stirring;

[0009] S4: Inject a surface-modified magnetic bead suspension into the reaction chamber, start a multi-axis stirrer to achieve efficient binding of the nucleic acid-magnetic bead complex, and simultaneously turn on the temperature control in the chamber to maintain the stability of RNA;

[0010] S5: Insert a programmable magnetic rod array to complete the directional aggregation of magnetic beads, and transfer the magnetic bead-nucleic acid complex to a low-temperature storage cavity through a precision fluid control system;

[0011] S6: Use the in-situ water body at the sampling point for three-stage pulsed pipeline flushing to prevent sample cross-contamination and maintain the continuous operation stability of the system;

[0012] S7: Inject the captured magnetic beads into a cryopreservation tube with a pre-set two-dimensional code, and automatically write an EPC electronic tag containing metadata such as geographical coordinates, sampling depth, and timestamp;

[0013] S8: Cool the sample tube to 4°C for temporary storage through a Peltier temperature control module, and automatically arrange them into a standardized 96-well deep-well plate configuration after accumulating to a set quantity;

[0014] S9: Conduct laboratory joint processing. Transfer the whole plate of samples to a nucleic acid extraction workstation through a robotic arm, directly perform the washing-elution process, output PCR-ready nucleic acid templates, and then end the entire automated collection process of aquatic microbial nucleic acids.

[0015] In a preferred embodiment, in step S1, the sampling device integrates a high-precision dual-mode positioning module, which supports both GPS and Beidou navigation systems, and real-time collects and records the specific values of longitude and latitude. The depth measurement uses a fully sealed piezoresistive pressure sensor with a range covering 0 to 60 MPa, corresponding to a water depth range of 0 to 6000 meters. During sampling, pressure data is continuously collected at a high frequency of 500 times per second, and the actual depth value is converted through the physical formula of water density and gravitational acceleration. The system built-in temperature compensation algorithm automatically eliminates the water density fluctuation caused by water temperature changes. The positioning data is transmitted to the main control unit in real time through the RS485 communication protocol.

[0016] In a preferred embodiment, in step S2, the device is configured as a telescopic composite sampling tube. The outer layer uses a 316L stainless steel hard tube with an outer diameter of 25 mm and a wall thickness of 2 mm, and a fluoropolymer hose with an inner diameter of 8 mm is nested inside. Before sampling, start the self-cleaning program, pump in in-situ water three times the volume of the pipeline at a pressure of 0.3 MPa for three-stage pulsed flushing, and then extract the target water sample at a constant flow rate of 0.5 liters per minute. The single-sampling volume is accurately controlled at 500 ml, with an error not exceeding plus or minus 10 ml. The lowering speed of the sampling tube is adjusted to 0.1 m per second through an electric winch, and a turbidity sensor is configured at the tube end to monitor the water body stratification characteristics in real time to ensure the collection of representative samples at the target depth.

[0017] In a preferred embodiment, in step S3, the water sample is injected into a titanium alloy coagulation reaction chamber with a volume of 1.2 liters, which is pre-filled with a GTC lysis buffer solution with a pH value of 7.8, containing guanidine isothiocyanate with a molar concentration of 4, sodium lauroyl sarcosinate with a mass concentration of 0.5%, and sodium citrate with a concentration of 25 mmol / L. The lysis solution and the water sample are automatically mixed at a volume ratio of 1:4, and a double-layer stainless steel impeller is started to perform intermittent low-speed stirring, maintaining the constant temperature in the chamber at 60 °C, with a fluctuation range not exceeding ±1 °C, and continuous lysis for 30 minutes. During the process, the conductivity of the solution is monitored in real time through a zirconia electrode until it stabilizes at a threshold of 15 mS / cm.

[0018] In a preferred embodiment, in step S4, a suspension of surface-modified silicon hydroxyl magnetic beads is injected into the reaction chamber. The magnetic beads have a particle size of 1 μm and a concentration of 5 mg / mL, and the addition amount is 20 μL per mL of the mixed solution. A three-axis eccentric stirring mechanism is started to form a three-dimensional turbulent flow field at a rotation speed of 300 revolutions per minute to ensure sufficient binding of nucleic acid and magnetic beads, and the action time is set to 20 minutes. The system automatically adjusts the temperature in the chamber according to a preset program. The temperature is maintained at room temperature during the DNA capture stage, and the temperature is switched to a low temperature mode of 4 °C during the RNA capture stage. The temperature control accuracy is achieved to be ±0.5 °C through a high-precision PT100 temperature sensor.

[0019] In a preferred embodiment, in step S5, 12 groups of neodymium iron boron permanent magnet arrays are configured. The magnetic flux density on the surface of the magnet is 0.5 Tesla. The magnetic rod extends into the reaction chamber liquid surface at a speed of 0.1 mm per second to a position 10 mm below the liquid surface and stays for 5 minutes to complete the aggregation of magnetic beads. During the transfer process, a precision stepper motor is used to drive a pipette with an inner diameter of 2 mm to transfer the magnetic bead-nucleic acid complex to an aluminum sample storage chamber at 4 °C at a flow rate of 0.2 mL per second. Nitrogen is continuously purged throughout the process to prevent sample oxidation, ensuring that the residual rate is lower than 0.5%.

[0020] In a preferred embodiment, in step S6, a three-stage gradient flushing mode is adopted. For the first flushing, 1 liter of in-situ water body is used to perform pulsed flushing for 30 seconds at a pressure of 0.4 MPa; for the secondary flushing, a sodium hydroxide solution with a concentration of 0.1 mol / L is injected for cyclic cleaning for 10 minutes; for the final flushing, ultrapure water is used for negative pressure suction for 5 minutes. An ultraviolet sterilization module is configured at the pipeline interface, with an emission wavelength of 254 nm and an intensity reaching 8000 μW / cm², ensuring that the system cleanliness meets the biosafety level II standard.

[0021] In a preferred embodiment, in step S7, a two-dimensional code is laser engraved on the surface of the pre-set cryotube, which conforms to the QRCode Version 10 standard with an error correction rate of 15%. At the same time, an electronic tag conforming to the ISO18000-6C standard is embedded, and the storage capacity is 4 kilobytes. The tag is automatically written with a 32-bit unique sample number, geographical coordinates in the WGS84 coordinate system, depth value with a resolution of 0.01 meters, UTC timestamp, and device serial number. Data encryption uses the AES-256 algorithm. After the writing is completed, the green LED indicator is triggered to indicate that the operation is completed.

[0022] In a preferred embodiment, in step S8, the semiconductor refrigeration module is combined with a liquid cooling system, which can cool the sample tube from room temperature to 4 degrees Celsius within 10 minutes and maintain the temperature fluctuation range within plus or minus 0.3 degrees Celsius. The sample storage turntable is configured with 96 magnetic positioning slots. After every 12 samples are collected, it automatically rotates 15 degrees. When the full plate is reached, an audible and visual alarm is triggered. The weight of the full plate is strictly controlled at 2.2 kilograms, with an error not exceeding plus or minus 0.1 kilograms, so as to adapt to the standard automation equipment interface.

[0023] In a preferred embodiment, in step S9, the standardized 96-well plate is docked with a magnetic rod nucleic acid extractor through a six-degree-of-freedom robotic arm, and the repeated positioning accuracy of the robotic arm reaches plus or minus 0.1 millimeters. The elution procedure is set as follows: for the first wash, 500 microliters of 70% ethanol is used for 30 seconds of oscillation; for the second wash, 300 microliters of 80% ethanol is used for 1 minute of standing; finally, 50 microliters of TE buffer with a pH of 8.0 is used for elution at 65 degrees Celsius for 15 minutes, and the output nucleic acid concentration is not less than 20 nanograms per microliter, and the OD260 / 280 ratio is stable between 1.8 and 2.0.

[0024] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0025] 1. In the present invention, through the integration of high-precision positioning, pressure sensing, and temperature-controlled lysis core technologies, full-process standardized operation is achieved. The device adopts dual-mode positioning and pressure sensors to ensure millimeter-level accuracy of geographical and hydrological data at the sampling point; through a three-dimensional turbulent flow field combined with a magnetic separation array, the nucleic acid capture rate is greatly improved, and the risk of nucleic acid degradation is minimized as much as possible. The modular flow path system uses three-stage gradient flushing and nano-ultraviolet sterilization to make the cross-contamination rate lower than 0.01%, effectively solving the problems of large sample volume, easy loss of information, and lag in processing existing in traditional water sample collection, and improving the overall collection efficiency and collection accuracy.

[0026] 2. In the present invention, a seamless connection system from field sampling to laboratory analysis is constructed. The millimeter-level docking accuracy between the 96-well standardized storage module and the robotic arm improves the laboratory processing efficiency. Combined with the encrypted EPC electronic tag system, the sample traceability data can be traced. The 1-micron magnetic bead particle size can simultaneously capture the nucleic acids of bacteria and plankton. Combined with the multi-platform deployment ability, the sampling period for large-scale water area microbiome research is shortened. Therefore, the present invention not only compresses the traditional manual operation that takes several hours into a fully automated process, but also provides a standardized data source with high spatio-temporal resolution for the research on the geographical distribution and ecological functions of aquatic microorganisms through multi-dimensional data fusion technology, significantly enhancing the scientific value of environmental monitoring and biological resource development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the process principle of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Embodiment:

[0030] Referring to Figure 1 , an automated method for collecting nucleic acids of aquatic microorganisms, the method comprising the following steps:

[0031] S1: Automatically record the longitude and latitude of the sampling point by integrating a GPS / Beidou module, and at the same time measure and record the water depth using a pressure sensor or an ultrasonic ranging system (hard pipe structure / hose + iron ball counterweight scheme);

[0032] S2: Pump the water body at the target depth into the anti-pollution flow path system through a retractable sampling tube (hard / soft pipe composite structure), and collect a quantitative water sample after pre-flushing the pipeline;

[0033] S3: Inject the water sample into the coagulation reaction chamber with temperature control function, automatically add lysis solution (such as GITC buffer system) in proportion, and achieve microbial cell lysis through intermittent low-speed stirring;

[0034] S4: Inject the surface-modified magnetic bead suspension into the reaction chamber, start the multi-axis stirrer to achieve efficient binding of the nucleic acid-magnetic bead complex, and simultaneously turn on the temperature control in the chamber to maintain the stability of RNA;

[0035] S5: Insert the programmable magnetic rod array to complete the directional aggregation of magnetic beads, and transfer the magnetic bead-nucleic acid complex to the low-temperature storage cavity through a precision fluid control system;

[0036] S6: Use the in-situ water body at the sampling point for three-stage pulsed pipeline flushing to prevent sample cross-contamination and maintain the continuous operation stability of the system;

[0037] S7: Inject the captured magnetic beads into a cryopreservation tube with a pre-set QR code, and automatically write an EPC electronic tag containing metadata such as geographical coordinates, sampling depth, and timestamp;

[0038] S8: Cool the sample tube to 4°C for temporary storage through the Peltier temperature control module, and automatically arrange them into a standardized 96-well deep-well plate configuration after accumulating to the set quantity;

[0039] S9: Conduct laboratory joint processing. Transfer the whole plate of samples to the nucleic acid extraction workstation through the robotic arm, directly perform the washing-elution process, output the PCR-ready nucleic acid template, and then the entire automated collection process of aquatic microbial nucleic acids can be completed.

[0040] In step S1, the sampling device integrates a high-precision dual-mode positioning module, which supports both GPS and Beidou navigation systems, and real-time collects and records the specific values of longitude and latitude. The depth measurement uses a fully sealed piezoresistive pressure sensor with a measurement range covering 0 to 60 MPa, corresponding to a water depth range of 0 to 6000 meters. During sampling, pressure data is continuously collected at a high frequency of 500 times per second, and the actual depth value is converted through the physical formula of water density and gravitational acceleration. The system built-in temperature compensation algorithm automatically eliminates the water density fluctuation caused by water temperature changes. The positioning data is transmitted to the main control unit in real time through the RS485 communication protocol.

[0041] In step S2, the device is configured with a telescopic composite sampling tube. The outer layer is made of 316L stainless steel hard tube with an outer diameter of 25 mm and a wall thickness of 2 mm, and a fluoropolymer hose with an inner diameter of 8 mm is nested inside. Before sampling, start the self-cleaning program, pump in three times the pipeline volume of in-situ water at a pressure of 0.3 MPa for three-stage pulsed flushing, and then extract the target water sample at a constant flow rate of 0.5 liters per minute. The single sampling volume is accurately controlled at 500 ml, with an error not exceeding plus or minus 10 ml. The lowering speed of the sampling tube is adjusted to 0.1 m per second through an electric winch, and a turbidity sensor is configured at the tube end to monitor the water body stratification characteristics in real time to ensure that a representative sample at the target depth is collected.

[0042] In step S3, the water sample is injected into a titanium alloy coagulation reaction chamber with a volume of 1.2 liters. The chamber is pre-filled with a GTC lysis buffer with a pH value of 7.8, which contains guanidine isothiocyanate with a molar concentration of 4 mol / L, sodium dodecyl sarcosinate with a mass concentration of 0.5%, and sodium citrate with a concentration of 25 mmol / L. The lysis solution and the water sample are automatically mixed at a volume ratio of 1:4, and a double-layer stainless steel impeller is started to perform intermittent low-speed stirring, maintaining a constant temperature of 60 °C in the chamber with a fluctuation range not exceeding ±1 °C, and continuously lysing for 30 minutes. During the process, the conductivity of the solution is monitored in real time through a zirconia electrode until it stabilizes at a threshold of 15 mS / cm.

[0043] In step S4, a suspension of surface-modified silica hydroxyl magnetic beads is injected into the reaction chamber. The magnetic beads have a particle size of 1 μm and a concentration of 5 mg / mL, and the addition amount is 20 μL per mL of the mixed solution. The three-axis eccentric stirring mechanism is started to form a three-dimensional turbulent flow field at a rotation speed of 300 revolutions per minute to ensure full binding of nucleic acids and magnetic beads, and the action time is set to 20 minutes. The system automatically adjusts the temperature in the chamber according to the preset program, maintaining room temperature during the DNA capture stage and switching to a low-temperature mode of 4 °C during the RNA capture stage, and achieving a temperature control accuracy of ±0.5 °C through a high-precision PT100 temperature sensor.

[0044] In step S5, 12 groups of neodymium iron boron permanent magnet arrays are configured. The magnetic flux density on the surface of the magnet is 0.5 Tesla. The magnetic rod extends into the reaction chamber liquid surface at a speed of 0.1 mm per second to a position 10 mm below the liquid surface and stays for 5 minutes to complete the aggregation of magnetic beads. During the transfer process, a precision stepper motor is used to drive a pipette with an inner diameter of 2 mm to transfer the magnetic bead-nucleic acid complex to an aluminum sample storage chamber at 4 °C at a flow rate of 0.2 mL per second. Nitrogen is continuously purged throughout the process to prevent sample oxidation, ensuring that the residual rate is less than 0.5%.

[0045] In step S6, a three-stage gradient flushing mode is adopted. For the first flushing, 1 liter of in-situ water body is used to perform pulsed flushing for 30 seconds at a pressure of 0.4 MPa; for the second flushing, a 0.1 mol / L sodium hydroxide solution is injected for cyclic cleaning for 10 minutes; for the final flushing, ultrapure water is used for negative pressure suction for 5 minutes. An ultraviolet sterilization module is configured at the pipeline interface, with an emission wavelength of 254 nm and an intensity reaching 8000 μW / cm², ensuring that the system cleanliness meets the biosafety level II standard.

[0046] In step S7, a QR code conforming to the QRCode Version 10 standard with a 15% error correction rate is laser-engraved on the surface of the pre-set cryotube. Meanwhile, an electronic tag conforming to the ISO18000-6C standard with a storage capacity of 4 kilobytes is embedded. The tag is automatically written with a 32-bit unique sample number, geographical coordinates in the WGS84 coordinate system, depth value with a resolution of 0.01 meters, UTC timestamp, and device serial number. Data encryption uses the AES-256 algorithm. After writing is completed, a green LED indicator is triggered to indicate that the operation is completed.

[0047] In step S8, a semiconductor refrigeration module combined with a liquid cooling system can cool the sample tube from room temperature to 4 degrees Celsius within 10 minutes and maintain the temperature fluctuation range within plus or minus 0.3 degrees Celsius. The sample storage turntable is configured with 96 magnetic positioning slots. After every 12 samples are collected, it automatically rotates 15 degrees. When the full plate is reached, an audible and visual alarm is triggered. The weight of the full plate is strictly controlled at 2.2 kilograms, with an error not exceeding plus or minus 0.1 kilograms to adapt to the standard automation equipment interface.

[0048] In step S9, a standardized 96-well plate is docked with a magnetic rod nucleic acid extractor through a six-degree-of-freedom robotic arm, and the repeat positioning accuracy of the robotic arm reaches plus or minus 0.1 millimeters. The elution program is set as follows: for the first wash, 500 microliters of 70% ethanol is used for 30 seconds of oscillation; for the second wash, 300 microliters of 80% ethanol is used for 1 minute of standing; finally, 50 microliters of TE buffer with a pH of 8.0 is used for elution at 65 degrees Celsius for 15 minutes. The output nucleic acid concentration is not less than 20 nanograms per microliter, and the OD260 / 280 ratio is stable between 1.8 and 2.0.

[0049] In the present invention, through the integration of high-precision positioning, pressure sensing, and temperature-controlled lysis core technologies, full-process standardized operation is achieved. The device uses dual-mode positioning and pressure sensors to ensure millimeter-level accuracy of geographical and hydrological data at the sampling point; through a three-dimensional turbulent flow field combined with a magnetic separation array, the nucleic acid capture rate is greatly improved, and the risk of nucleic acid degradation is minimized as much as possible. The modular flow path system uses three-stage gradient flushing and nano-ultraviolet sterilization to make the cross-contamination rate less than 0.01%, effectively solving the problems of large sample volume, easy loss of information, and lag in processing existing in traditional water sample collection, and improving the overall collection efficiency and collection accuracy.

[0050] In the present invention, a seamless connection system from field sampling to laboratory analysis is constructed. The millimeter-level docking accuracy between the 96-well standardized storage module and the robotic arm improves the laboratory processing efficiency. Combined with the encrypted EPC electronic tag system, the sample traceability data can be traced. The 1-micron magnetic bead particle size can simultaneously capture the nucleic acids of bacteria and plankton. Combined with the multi-platform deployment ability, the sampling period for large-scale water microbial community research is shortened. Therefore, the present invention not only compresses the traditional manual operation that takes several hours into a fully automated process, but also provides a standardized data source with high spatio-temporal resolution for the study of the geographical distribution and ecological functions of aquatic microorganisms through multi-dimensional data fusion technology, significantly enhancing the scientific value of environmental monitoring and biological resource development.

[0051] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of additional identical elements in the process, method, article or device including the element.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for automatically collecting nucleic acid from aquatic microorganisms, characterized in that: The method comprises the following steps: S1: Automatically record the latitude and longitude of the sampling point through the integrated GPS / Beidou module, and use the pressure sensor or ultrasonic ranging system to measure and record the water depth; S2: The water body at the target depth is pumped into the anti-pollution flow path system through a retractable sampling tube, and quantitative water samples are collected after the pipeline is pre-flushed; S3: Inject the water sample into the coagulation reaction chamber with temperature control function, automatically add lysis solution according to proportion, and achieve microbial cell lysis through intermittent low-speed stirring; S4: inject the surface modified magnetic bead suspension into the reaction chamber, start the multi-axis stirrer to achieve efficient binding of nucleic acid-magnetic bead complex, and simultaneously start the temperature control in the chamber to maintain RNA stability; S5: Reach into the programmable magnetic rod array to complete the directional aggregation of magnetic beads, and transfer the magnetic bead-nucleic acid complex to the low-temperature temporary storage chamber through a precise fluid control system; S6: Use the in-situ water at the sampling point to perform three-stage pulse pipeline flushing to prevent sample cross-contamination and maintain the continuous operation stability of the system; S7: Inject the capture magnetic beads into the cryopreservation tube with a preset QR code, and automatically write the EPC electronic tag containing the geographical coordinates, sampling depth, and timestamp metadata; S8: The sample tubes are cooled to 4°C and temporarily stored by the Peltier temperature control module. After the set number of sample tubes is accumulated, they are automatically arranged into a standardized 96-well deep-well plate configuration; S9: Conduct laboratory joint processing, dock the entire plate of samples to the nucleic acid extraction workstation through a robotic arm, directly carry out the washing-elution process, output the PCR-ready nucleic acid template, and then the entire aquatic microorganism nucleic acid automated collection process can be completed.

2. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In the step S1, the sampling device integrates a high-precision dual-mode positioning module, supports GPS and Beidou navigation systems, collects and records specific values ​​of longitude and latitude in real time, and uses a fully sealed piezoresistive pressure sensor for depth measurement, with a range of 0 to 60 MPa, corresponding to a water depth of 0 to 6000 meters. During sampling, pressure data is continuously collected at a high frequency of 500 times per second, and is converted into an actual depth value through a physical formula of water density and gravitational acceleration. The system's built-in temperature compensation algorithm automatically eliminates water density fluctuations caused by changes in water temperature; the positioning data is transmitted to the main control unit in real time through the RS485 communication protocol.

3. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In the step S2, the device is configured as a retractable composite sampling tube, the outer layer of which is a 316L stainless steel hard tube with an outer diameter of 25 mm and a wall thickness of 2 mm, and the inner layer is a fluoropolymer hose with an inner diameter of 8 mm. Before sampling, three times the volume of the pipeline is pumped into the in-situ water at a pressure of 0.3 MPa for three pulse flushings, and then the target water sample is extracted at a constant flow rate of 0.5 liters per minute. The single collection volume is accurately controlled at 500 ml, and the error does not exceed plus or minus 10 ml. The lowering speed of the sampling tube is adjusted to 0.1 meters per second by an electric winch, and a turbidity sensor is configured at the end of the tube to monitor the water stratification characteristics in real time to ensure that representative samples of the target depth are collected.

4. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In the step S3, the water sample is injected into a titanium alloy coagulation reaction chamber with a volume of 1.2 liters, and the chamber is pre-loaded with a GTC lysis buffer with a pH value of 7.8, which contains 4 molar concentrations of guanidine isothiocyanate, 0.5% mass concentration of sodium lauryl sarcosinate and 25 millimolar concentration of sodium citrate; the lysis solution and the water sample are automatically mixed in a volume ratio of 1 to 4, and the double-layer stainless steel impeller is started for intermittent low-speed stirring, maintaining a constant temperature of 60 degrees Celsius in the chamber with a fluctuation range of no more than plus or minus 1 degree Celsius, and continuing the lysis for 30 minutes; during the process, the solution conductivity is monitored in real time by a zirconium oxide electrode until it stabilizes at a threshold value of 15 millisiemens per centimeter.

5. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In step S4, a surface-modified silanol magnetic bead suspension is injected into the reaction chamber, the magnetic bead particle size is 1 micron, the concentration is 5 mg / ml, and the addition amount is 20 μl per ml of the mixed solution; The three-axis eccentric stirring mechanism is started to form a three-dimensional turbulent field at a speed of 300 revolutions per minute to ensure full combination of nucleic acid and magnetic beads. The action time is set to 20 minutes. The system automatically adjusts the temperature in the chamber according to the preset program, maintaining room temperature during the DNA capture stage and switching to 4 degrees Celsius low-temperature mode during the RNA capture stage. The temperature control accuracy of up to plus or minus 0.5 degrees Celsius is achieved through the high-precision PT100 temperature sensor.

6. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In step S5, 12 groups of NdFeB permanent magnet arrays are configured, the magnetic flux density on the magnet surface is 0.5 Tesla, and the magnetic rod is extended into the reaction chamber at a speed of 0.1 mm per second to a position 10 mm below the liquid surface, and maintained for 5 minutes to complete the aggregation of magnetic beads; during the transfer process, a precision stepper motor is used to drive a pipette with an inner diameter of 2 mm to transfer the magnetic bead-nucleic acid complex to an aluminum sample storage chamber at 4 degrees Celsius at a flow rate of 0.2 ml per second, and nitrogen is continuously purged throughout the process to prevent sample oxidation to ensure that the residual rate is less than 0.5%.

7. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In step S6, a three-level gradient flushing mode is adopted. The first flush uses 1 liter of in-situ water to perform a 30-second pulse flush at a pressure of 0.4 MPa; the second flush injects a 0.1 molar concentration of sodium hydroxide solution for 10 minutes of circulation cleaning; the final flush uses ultrapure water for 5 minutes of negative pressure suction; a UV sterilization module is configured at the pipeline interface, with an emission wavelength of 254 nanometers and an intensity of 8000 microwatts per square centimeter, to ensure that the system cleanliness meets the biosafety level 2 standard.

8. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In step S7, a QR code is laser engraved on the surface of the preset cryotube, and an electronic tag that complies with the ISO18000-6C standard is embedded with a storage capacity of 4 kilobytes; the tag automatically writes a 32-bit unique sample number, geographic coordinates in the WGS84 coordinate system, a depth value with a resolution of 0.01 meters, a UTC timestamp and a device serial number.

9. The method for automatically collecting nucleic acid from aquatic microorganisms according to claim 1, characterized in that: In step S8, the semiconductor refrigeration module is combined with the liquid cooling system to reduce the temperature of the sample tube from room temperature to 4 degrees Celsius within 10 minutes, and maintain the temperature fluctuation range within plus or minus 0.3 degrees Celsius; the sample storage turntable is equipped with 96 magnetic positioning slots, and automatically rotates 15 degrees after completing the collection of 12 samples.

10. The method for automatically collecting nucleic acid of aquatic microorganisms according to claim 1, characterized in that: In step S9, the standardized 96-well plate is docked with a magnetic rod nucleic acid extractor through a six-degree-of-freedom robotic arm, and the robotic arm has a repeatability accuracy of plus or minus 0.1 mm; the elution program is set as follows: the first wash uses 500 μL of 70% ethanol for 30 seconds, the second wash uses 300 μL of 80% ethanol for 1 minute, and finally 50 μL of pH 8.0 TE buffer is used for elution at 65 degrees Celsius for 15 minutes, the output nucleic acid concentration is not less than 20 ng / μL, and the OD260 / 280 ratio is stable between 1.8 and 2.0.

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