Deep-sea in-situ macro-transcriptome rna multi-stage processing and reverse transcription stabilization method and system

Abstract

This invention discloses a method and system for multi-stage processing and reverse transcription stabilization of deep-sea in-situ metatranscriptomic RNA, relating to the fields of deep-sea biotechnology and marine instrumentation. Within a continuous, sealed fluid channel, the following components are connected in series: a filtration and enrichment chamber, a controlled decompression unit, a programmable control unit, and at least one multifunctional reaction chamber. The multifunctional reaction chamber integrates a temperature control system. At least one set of bidirectional opposing electromagnets is disposed on the outer wall of the multifunctional reaction chamber. The programmable control unit controls the on / off state and current of the bidirectional opposing electromagnets, generating a dynamically switchable spatial magnetic field gradient within the multifunctional reaction chamber. This drives magnetic microspheres within the chamber to perform at least one of the following operations without centrifugation: adsorption and aggregation, suspension and mixing, in-situ washing, inter-chamber transfer, and elution / desorption. All biochemical operations are completed within a continuous, sealed fluid channel, eliminating cross-contamination between the sample and the external environment and achieving fully automated sample processing in deep-sea environments.

Description

Technical Field

[0001] This invention relates to the fields of deep-sea biotechnology and marine instrumentation technology, and in particular to a method and system for multi-stage processing and reverse transcription stabilization of deep-sea in-situ metatranscriptome RNA. Background Technology

[0002] The deep-sea environment is characterized by extreme pressure, low temperature, and high salinity, and the physiological activity of deep-sea microorganisms is highly dependent on in-situ physicochemical conditions. Metatranscriptomics, by capturing community mRNA, can directly reflect the in-situ metabolic state and functional expression of microorganisms, and is an important technical means to understand the laws governing deep-sea life activities. However, mRNA usually has a short half-life in cells (typically on the order of minutes, depending on the species and environment). During the sampling process, factors such as temperature changes, pressure release, and RNase activity all contribute to RNA degradation, causing the final transcriptome data to deviate significantly from the true in-situ state.

[0003] Current deep-sea microbial nucleic acid sampling protocols mainly focus on the physical enrichment and total nucleic acid preservation stages, exhibiting the following systemic technical deficiencies: (1) Lack of in situ RNA fixation capability: Existing Niskin water sampling bottles, CTD frame water sampling devices, pressure-holding water sampling devices, etc., usually do not have the function of immediate nucleic acid fixation; RNA degradation is easily caused during the depressurization and ascent of the sample, resulting in distortion of the transcriptome map.

[0004] (2) The decompression process lacks precise control: the pressure transition from deep-sea high pressure to atmospheric pressure is not actively controlled, and pressure release may lead to the precipitation of dissolved gases (degassing), local bubble formation and enhanced fluid disturbance, which may affect cell integrity and nucleic acid stability. Although existing pressure-holding sampling technology can maintain pressure, it does not have the engineering implementation of controlled gradient decompression.

[0005] (3) RNA purification depends on centrifugation equipment: Traditional nucleic acid extraction methods such as Trizol / chloroform extraction and rotary column centrifugation require high-speed centrifuges with a rotation speed of ≥10000rpm, which are difficult to integrate into deep-sea in-situ instruments.

[0006] (4) rRNA removal is inefficient in the laboratory: rRNA usually accounts for 80-95% of the total RNA in deep-sea samples and needs to be effectively removed to improve sequencing efficiency; existing magnetic bead / probe hybridization removal schemes are all optimized under laboratory room temperature conditions, and the adaptability of probe hybridization under deep-sea low temperature conditions has not been specifically studied.

[0007] (5) Difficulty in temperature control of multi-step enzymatic reactions: Enzymatic reactions such as reverse transcription have high requirements for temperature control, but it is difficult to achieve stable thermal management under the low temperature background of the deep sea environment.

[0008] (6) Each step is independent and difficult to automate: There is currently no integrated deep-sea instrument that couples controlled decompression, magnetic bead RNA separation, rRNA removal, mRNA enrichment and in situ reverse transcription into a fully automated closed-loop process, which is the key technical difficulty that this invention aims to solve. Summary of the Invention

[0009] To overcome the aforementioned problems in the existing technology, this invention proposes a method and system for multi-level processing and reverse transcription stabilization of deep-sea in-situ metatranscriptome RNA.

[0010] The technical solution adopted by the present invention to solve its technical problem is: a deep-sea in-situ metatranscriptionomic RNA multi-level processing and reverse transcription stabilization system, including a continuous closed fluid channel, wherein the continuous closed fluid channel is connected in series with: a filter enrichment chamber for retaining microorganisms in seawater samples and performing in-situ fixation; The controlled decompression unit is used to smoothly reduce the pressure of the trapped microbial samples from the deep-sea environment to the working pressure; The program control unit is used to coordinate the control system and achieve unattended, fully automated operation. At least one multifunctional reaction chamber is provided, wherein a temperature control system is integrated within the multifunctional reaction chamber, and at least one set of bidirectional opposing electromagnets is provided on the outer side of the chamber wall. The on / off state and current of the bidirectional opposing electromagnets are controlled by a program control unit to generate a dynamically switchable spatial magnetic field gradient inside the multifunctional reaction chamber, thereby driving the magnetic microspheres inside the chamber to perform at least one of the following operations without relying on centrifugation: adsorption and aggregation, suspension and mixing, in-situ cleaning, inter-chamber transfer, and elution and desorption.

[0011] The aforementioned deep-sea in-situ metatranscriptionomic RNA multi-stage processing and reverse transcription stabilization system also includes a reagent storage unit and a cryopreservation unit. The reagent storage unit integrates a multi-channel micro-injection pump and a pre-cooling / room temperature storage chamber, and injects liquid quantitatively into each chamber according to the instructions of the program control unit. The cryopreservation unit is used to store the airtight micro-storage bag for encapsulating cDNA.

[0012] The aforementioned deep-sea in-situ metatranscriptome RNA multi-stage processing and reverse transcription stabilization system comprises a multifunctional reaction chamber including a tandem RNA capture chamber, an rRNA depletion chamber, and an mRNA enrichment and reverse transcription chamber. The RNA capture chamber is used to capture and purify total RNA using silanol-functionalized magnetic microspheres. The rRNA depletion chamber is used to remove rRNA using a cascade of biotinylated probes and streptavidin-functionalized magnetic microspheres. The mRNA enrichment and reverse transcription chamber is used to purify mRNA a second time using nucleic acid purification magnetic microspheres and to perform in-situ reverse transcription within the same chamber.

[0013] The aforementioned deep-sea in-situ metatranscriptionomic RNA multi-stage processing and reverse transcription stabilization system includes a temperature control system comprising a high thermal conductivity substrate attached to the outer wall of the chamber, a ceramic heating element embedded in the substrate, and a temperature sensor fixedly connected to the substrate; the temperature control system is controlled by PID closed-loop control.

[0014] The aforementioned deep-sea in-situ macrotranscriptional RNA multi-stage processing and reverse transcription stabilization system includes a controlled decompression unit comprising a high-pressure plunger pump and a fuzzy adaptive PID controller. The fuzzy adaptive PID controller dynamically adjusts the displacement rate of the high-pressure plunger pump to achieve decompression based on three-dimensional state variables: pressure deviation, pressure change rate, and plunger displacement.

[0015] A method for multi-stage processing and reverse transcription stabilization of deep-sea in-situ metatranscriptome RNA, based on the aforementioned system, is performed within a continuous closed fluid channel and includes the following steps: Step 1: Under the target deep-sea environmental pressure conditions, seawater samples are introduced into the filtration enrichment chamber, where microorganisms are trapped and pre-washing and RNA lysis are completed within the filtration enrichment chamber. Step 2: Activate the controlled decompression unit to perform three-stage decompression, which will steadily reduce the pressure of the fluid containing microorganisms from the deep-sea environment to the working pressure of 0.1 MPa. Step 3: The fluid passes sequentially through the RNA capture chamber, the rRNA depletion chamber, and the mRNA enrichment and reverse transcription chamber. The RNA capture chamber captures and purifies total RNA. The rRNA depletion chamber removes rRNA in a cascade. The mRNA enrichment and reverse transcription chamber purifies mRNA a second time and performs in situ reverse transcription to obtain a stable cDNA product. Step 4: Transfer the cDNA product obtained in Step 3 to a cryopreservation unit for storage.

[0016] The above-mentioned deep-sea in-situ macrotranscriptional RNA multi-stage processing and reverse transcription stabilization method includes a three-stage decompression process in step 2, comprising a high-pressure rapid release stage, an intermediate stable transition stage, and a low-pressure slow equilibrium stage. The pressure and plunger displacement are collected in real time by a fuzzy adaptive PID controller, and the decompression rate is dynamically corrected by combining a fluid volume change model that includes temperature and salinity compensation.

[0017] In the aforementioned deep-sea in-situ macrotranscriptional RNA multi-stage processing and reverse transcription stabilization method, the controlled decompression unit is equipped with dual redundant pressure sensors for cross-validation, and automatically pauses the plunger pump and triggers an alarm when an abnormal pressure is detected.

[0018] In the aforementioned deep-sea in situ metatranscriptome RNA multi-stage processing and reverse transcription stabilization method, step 3 specifically comprises: Step 3.1: Inject SG magnetic bead suspension containing silanol-functionalized magnetic beads into the RNA capture chamber, start suspension mixing and incubation for 10 min, switch to adsorption state, inject washing buffer, and wash in situ to remove impurities; switch to suspension mixing state and wash with DNase buffer; switch to desorption elution state and inject elution buffer; the temperature control module raises the chamber temperature to the elution temperature, and the suspension is mixed; the magnetic field adsorbs the magnetic beads, and the elution buffer containing total RNA is collected; Step 3.2: The liquid obtained in step 3.1 and the biotin-labeled specific probe targeting the conserved regions of prokaryotic 16S and 23S rRNA are injected into the rRNA depletion chamber. After denaturation at 70°C under the control of a temperature control system, the temperature is lowered to 37°C for hybridization. The probe and rRNA binding is promoted by using a suspension and mixing state. Streptavidin-functionalized magnetic beads are injected to capture the rRNA-probe complex at 37°C, and the complex is separated by adsorption. Step 3.3: Inject the liquid obtained in Step 3.2 and the nucleic acid purification magnetic beads into the mRNA enrichment and reverse transcription chamber, start suspension mixing, capture mRNA onto the surface of the nucleic acid purification magnetic beads, switch the adsorption state to fix the magnetic beads, wash to remove impurities, inject nuclease-free water, adjust the temperature control module to 70℃±0.5℃, desorption mixing to complete mRNA elution; after magnetic field fixation of the magnetic beads, collect the elution containing high-purity mRNA; inject the reverse transcription premixed system into the eluted mRNA solution, heat to 50~55℃, maintain for 10~60min, so that the reverse transcriptase reacts rapidly at high temperature; heat to 80℃, maintain for 7min, heat-inactivate the reverse transcriptase, and obtain a stable cDNA solution.

[0019] The beneficial effects of the present invention are: (1) High fidelity throughout the entire process: The present invention realizes the in-situ processing of the entire process from deep-sea microorganism collection to stable cDNA preservation, eliminating the risk of mRNA degradation during sample transportation. Compared with the traditional scheme, the effective mRNA mapping ratio can be significantly improved and the rRNA removal rate can be significantly increased.

[0020] (2) Controlled decompression protects nucleic acid integrity: Based on the three-segment decompression curve of fuzzy adaptive PID algorithm, the decompression rate is precisely controlled (fastest 2MPa / min → slowest 0.05MPa / min), achieving stable decompression from 60MPa to normal pressure, and the integrity of nucleic acid is effectively improved.

[0021] (3) Closed-loop non-centrifugal magnetic bead control: In a completely sealed fluid chamber, the magnetic microspheres are controlled by the programmable switching of the upper and lower bidirectional electromagnet groups to achieve five control modes: adsorption, suspension and mixing, in-situ cleaning, precise transfer and elution and desorption. This can replace centrifugal separation to the greatest extent and make in-situ automated nucleic acid processing in the deep sea possible.

[0022] (4) Multi-level RNA purification: A four-level processing system integrating SG magnetic beads primary capture, DNaseI DNA removal treatment, probe-RM magnetic beads rRNA depletion and NA magnetic beads mRNA secondary purification is used to obtain high-purity mRNA.

[0023] (5) Multi-zone precision temperature-controlled enzymatic reaction: The temperature control architecture of aluminum nitride substrate + ceramic heating element + PT100 sensor + PID closed loop achieves a steady-state temperature control accuracy of ±0.2℃ and a temperature rise response of <30s in the micro reaction chamber, meeting the precision requirements of multi-step sequence such as lysis, hybridization, reverse transcription, and thermal inactivation.

[0024] (6) Modular integration and multi-platform compatibility: The system is encapsulated with an oil-filled pressure compensation chamber, with a working water depth of 500~6000m. It can be adapted to ROV, AUV and bottom-mounted deep-sea instruments, and supports unattended continuous sampling multiple times.

[0025] (7) Clear differentiation from existing technologies: Existing technologies lack effective solutions for deep-sea in-situ sealing systems that integrate controlled decompression, magnetic field-driven magnetic bead manipulation, multi-stage separation and purification, and precise temperature-controlled enzymatic reactions. This invention addresses an urgent need in the field. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the system of the present invention; Figure 2 This is a schematic diagram of a closed fluid channel and multi-chamber topology; Figure 3 This is a block diagram of the fuzzy adaptive PID control logic of the controlled decompression unit (DPU); Figure 4 This is a schematic diagram of a three-segment controlled decompression curve (Pt curve); Figure 5 This is a schematic diagram of the upper and lower bidirectional electromagnet assembly structure in chambers A / B / C; Figure 6 This is a multimodal manipulation state diagram of magnetic microspheres, where (a) is the adsorption state, (b) is the suspension and mixing state, (c) is the washing state, (d) is the transfer state, and (e) is the desorption and elution state. Figure 7 This is a flowchart of the RNA capture and purification process for chamber A: SG magnetic beads; Figure 8 This is a flowchart of the probe-RM bead cascade rRNA depletion process in chamber B. Figure 9 This is a flowchart of the secondary purification and reverse transcription process for mRNA in chamber C. Figure 10 This is a schematic diagram of the chamber precision temperature control system. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] This embodiment discloses a metatranscriptome RNA in situ processing system suitable for deep-sea high-pressure and low-temperature environments, such as... Figure 1 As shown, the system is composed of the following (1)-(8) functional units connected in series through pressure-resistant microfluidic tubing. All microfluidic tubing between chambers is connected by pressure-resistant capillary stainless steel tubes and dead-volume-free connectors. The inner surfaces of the tubing and chambers are silanized to reduce nucleic acid adsorption. The closed fluid channels and multi-chamber topology are as follows: Figure 2 As shown.

[0029] (1) Filtration enrichment chamber (FC chamber): preferably a pressure-resistant composite material filter membrane or a metal support membrane structure, used to retain microorganisms in seawater samples.

[0030] (2) Controlled pressure reduction unit (DPU): It consists of a high-pressure plunger pump, a high-pressure three-way valve group, an ambient pressure sensor and an intracavity pressure sensor to form a controlled pressure reduction closed loop.

[0031] (3) RNA capture chamber (chamber A): a cylindrical sealed microreaction chamber with two sets of opposing electromagnets integrated in the chamber wall for precise position control of the magnetic microspheres.

[0032] (4) rRNA depletion chamber (chamber B): a sealed microreaction chamber of the same specifications as chamber A, with an integrated temperature control module, used for probe hybridization and rRNA magnetic separation.

[0033] (5) mRNA enrichment and reverse transcription chamber (chamber C): a sealed micro-reaction chamber with an integrated temperature control module, used for secondary purification of mRNA and in situ reverse transcription.

[0034] (6) Reagent storage module (RM): integrates a multi-channel micro-injection pump and a pre-cooled and room temperature storage chamber, used to accurately and quantitatively inject lysis buffer, magnetic bead suspension, washing solution, probe solution, reverse transcription mixture, etc. into each chamber according to the program.

[0035] (7) Low-temperature storage unit (CS): integrates Peltier cooling plate and combined with heat transfer oil circulation, and stores airtight micro liquid storage bag for encapsulating cDNA.

[0036] (8) Program control unit (MCU): ARM Cortex embedded microcontroller, running a real-time operating system, coordinating the timing execution of all functional units through the CAN bus, supporting unattended autonomous operation underwater.

[0037] In this embodiment, the controlled decompression unit is a core prerequisite for ensuring RNA integrity. This invention employs a fuzzy adaptive PID control algorithm based on three-dimensional state variables: "pressure deviation (e) – pressure change rate (Δe / Δt) – plunger displacement (d)" (e.g., ...). Figure 3 As shown in the figure, the displacement rate of the high-pressure plunger pump is precisely controlled to achieve controlled and stable pressure reduction throughout the entire process, from ambient pressure (up to 60 MPa) to the nucleic acid processing working pressure (0.1 MPa). The pressure reduction control accuracy is ≤0.05 MPa.

[0038] The decompression curve employs a three-stage strategy: a high-pressure rapid release stage, where RNA molecules are in a high-salt protective solution, and the large pressure difference drives high efficiency, resulting in rapid decompression; a stable transition stage, approaching the cavitation critical pressure range, where the decompression rate decreases to avoid dissolved gas precipitation; and a low-pressure slow equilibrium stage, where final pressure equilibrium is achieved at the slowest rate in the range closest to atmospheric pressure, reducing the risk of bubble precipitation and fluid disturbance. The control algorithm incorporates a fluid nonlinearity compensation model. Pressure control is achieved through real-time pressure feedback acquisition combined with a fluid volume change model for closed-loop regulation. This model considers the effects of temperature and salinity on fluid compressibility. The three-stage controlled decompression curve is shown below. Figure 4 As shown.

[0039] The pressure reduction module also features the following safety mechanisms: (a) cross-validation of dual redundant pressure sensors; (b) automatic pause and alarm triggering in case of pressure abnormality; (c) plunger displacement limit protection; and (d) automatic isolation and switching to the backup flow path in case of valve group failure. After pressure reduction, the system can perform pressure boosting and reset, and supports multiple consecutive samplings.

[0040] In this embodiment, the outer walls of the RNA capture chamber, rRNA depletion chamber, and mRNA enrichment and reverse transcription chamber are all equipped with bidirectional opposing electromagnets (such as...). Figure 5 As shown in the figure, the bidirectional opposing electromagnets generate a programmable spatial magnetic field gradient, which drives the magnetic microspheres to complete all biochemical operations in the chamber, replacing centrifugal separation.

[0041] The electromagnetic field driving module includes at least two sets of electromagnets disposed on opposite sides (e.g., upper and lower sides) of the reaction chamber; the program control module independently controls the on / off state and current magnitude of each electromagnet to generate a magnetic field in the vertical direction within the chamber that can achieve the following states: Adsorption state: When the lower electromagnet is energized, the magnetic microspheres are attracted to the bottom of the chamber to form a dense aggregate layer, and the liquid phase can be safely extracted; Suspension and mixing state: The upper and lower electromagnets are alternately switched on and off, driving the magnetic beads to reciprocate to achieve thorough mixing; Cleaning status: The lower group is energized to fix the magnetic beads, and the cleaning fluid is injected from the top and discharged to achieve in-situ cleaning; Transfer state: The upper group is energized to move the magnetic bead to the top, and then hydraulic pressure is injected to send it to the downstream chamber; Desorption and elution process: Power is turned off in both groups. Elution buffer is injected and the mixture is resuspended and mixed. After nucleic acid dissociation, the magnetic beads are re-adsorbed, and the eluent is collected. Through the above-mentioned magnetic field gradient switching, combined with fluid delivery, this system can complete the entire nucleic acid processing flow within a completely closed chamber. The multimodal manipulation state of the magnetic microspheres is as follows: Figure 6 As shown.

[0042] In this embodiment, the RNA capture chamber, rRNA depletion chamber, and mRNA enrichment and reverse transcription chamber are all integrated with a temperature control system (such as...). Figure 10 As shown), different temperatures can be switched sequentially within the chamber to execute a complete biochemical reaction program: The outer wall of the chamber is made of aluminum nitride with high thermal conductivity, which is closely attached to the outer surface of the chamber to ensure that heat is evenly conducted to the entire chamber. A miniature alumina ceramic heating element is embedded on the outside of the AlN substrate. The PT100 platinum resistance temperature sensor is directly welded to the AlN substrate, and the detection point is less than 0.5 mm away from the liquid area of ​​the chamber. The PID controller runs in the MCU, with a sampling frequency of 2 Hz and a control accuracy of ≤ ±0.2℃. The entire chamber is wrapped with a 3 mm thick polyurethane foam insulation layer. Under the background temperature of 1~5℃ in the deep sea, the steady-state heating power required to cover the temperature control range of 37~80℃ is only 2.0~3.0W.

[0043] In this embodiment, the MCU runs a pre-programmed timing protocol to coordinate all operations of the DPU, chambers A / B / C, the syringe pump, the temperature control module, and the cryopreservation unit, achieving unattended, fully automated operation. A complete sampling and processing cycle (1L of seawater, processed to cDNA preservation) takes approximately 130 minutes, distributed as follows: enrichment and fixation 10 minutes, controlled decompression approximately 80 minutes, chamber A processing 60 minutes, chamber B processing 50 minutes, and chamber C processing 45 minutes (some steps are executed in parallel with other chambers), with a total serial time of approximately 120 minutes (after parallel execution of key steps).

[0044] After a sampling cycle is completed, the system performs automatic cleaning (RNase-free water flushing of all tubing and chambers × 3 times) and pressure boosting reset, preparing for the next sampling. Each sampling uses a disposable magnetic bead reagent pack (pre-filled in an independent sealed chamber) to avoid cross-contamination; the system is designed to support 4-6 consecutive autonomous sampling cycles (limited by on-chip storage capacity), enabling time-series metatranscriptomics analysis.

[0045] Based on the above system, this embodiment also discloses a method for in situ processing of metatranscriptomic RNA, including the following steps: Step 1: Under the target deep-sea environmental pressure conditions, seawater samples are introduced into the filtration enrichment chamber. Microorganisms are retained in the filtration enrichment chamber, and the pre-washing (injecting pre-cooled PBS to remove high salt and enzyme inhibitors from the sample; discharging waste liquid; repeating the washing once) and RNA lysis release (injecting lysis buffer to complete cell lysis; transferring the lysis buffer containing nucleic acids to chamber A) processes are completed in the filtration enrichment chamber. Step 2: Activate the controlled decompression unit to perform three-stage decompression, which will steadily reduce the pressure of the fluid containing microorganisms from the deep-sea environment to the working pressure of 0.1 MPa. Step 3: The fluid passes sequentially through the RNA capture chamber, the rRNA depletion chamber, and the mRNA enrichment and reverse transcription chamber. The RNA capture chamber captures and purifies total RNA. The rRNA depletion chamber removes rRNA in a cascade. The mRNA enrichment and reverse transcription chamber purifies mRNA a second time and performs in situ reverse transcription to obtain a stable cDNA product. Step 4: Transfer the cDNA product obtained in Step 3 to a cryopreservation unit for storage.

[0046] In this embodiment, the processing flow of chamber A (such as...) Figure 7 As shown in the figure: (a) RNA adsorption of SG magnetic beads: Inject SG magnetic bead suspension (silanol-functionalized magnetic beads, which mainly utilize high-salt, low-pH binding buffer to achieve selective binding of RNA to silanol magnetic beads) into chamber A; start suspension mixing and incubation for 10 min; (b) Cleaning with cleaning solution: Switch to adsorption state, and the magnetic beads will gather at the bottom of the chamber within 30 seconds; wash solution 1 and wash solution 2 are injected in sequence, each time from the top and discharged from the bottom, to remove impurities such as proteins, lipids and salt ions in situ. (c) DNase washing: Switch to suspension and mix, inject DNase buffer and keep for 5 min; add rebinding buffer and isopropanol; wash again with washing buffer 2; (d) Thermal elution of total RNA: Switch to desorption elution mode and inject elution buffer; the temperature control module raises the chamber temperature to the elution temperature and suspends and mixes for a certain time; magnetic beads are adsorbed by magnetic field and the elution buffer containing total RNA is collected; RNA with high integrity can be obtained (RIN value depends on sample type); it is transferred to chamber B via microfluidic tubing.

[0047] Within chamber B, specific probe hybridization, capture, and removal of rRNA are completed. The core technologies include probe design and hybridization / magnetic separation steps (such as...). Figure 8 (as shown) Probe design: Targeting the rRNA diversity of deep-sea microbial communities (including bacteria, archaea, and a small number of eukaryotic microorganisms), probe sequences can be designed and optimized according to the target community, and denaturation-renaturation control can be performed using known nucleic acid hybridization principles.

[0048] Hybridization and magnetic separation steps: Inject total RNA solution and probe mixture into chamber B. The probe is designed for the conserved regions of prokaryotic 16S, 23S and 5S rRNA (biotinylated rRNA hybridization probe, which mainly uses biotinylated locked nucleic acid probes to specifically hybridize with rRNA, and then removes it by streptavidin magnetic beads). The temperature control module heats chamber B to 70℃ (±0.5℃) for denaturation for 10 min, then cools it to 37℃ (±0.3℃). At 37℃, hybridization is performed for 20 min using magnetic bead suspension mixing mode (0.5Hz). Subsequently, streptavidin-functionalized RM magnetic beads are injected, and incubation continues at 37℃ for 15 min (suspension mixing at 1Hz). The biotin-probe-rRNA complex binds to the surface of the RM magnetic beads via biotin-streptavidin. Switching to adsorption mode, the rRNA-RM magnetic bead complex settles and is fixed at the bottom of the chamber (aggregation time <60s). The supernatant containing mRNA is pressurized to chamber C through a microfluidic channel, achieving an rRNA removal rate of not less than 90%.

[0049] Chamber C completes the secondary purification and reverse transcription process of mRNA (e.g.) Figure 9 (as shown) (a) mRNA adsorption to NA magnetic beads: Nucleic acid purification magnetic beads (NA magnetic beads, surface-modified with polyethylene glycol-silyl) and binding buffer are injected into the solution containing mRNA; suspension mixing mode is initiated; mRNA is captured onto the surface of NA magnetic beads through PEG-assisted precipitation and electrostatic interaction; (b) Double washing: Switch to adsorption mode to fix the magnetic beads; inject 75% ethanol and washing buffer three times in sequence to remove formamide, salt ions and proteins; (c) Heat elution of mRNA: Inject nuclease-free water; adjust the temperature control module to 70℃±0.5℃, desorb and mix to complete mRNA elution; after fixing the magnetic beads with a magnetic field, collect the eluted mRNA containing high purity.

[0050] (d) Reverse transcription: Inject the reverse transcription premixed system (thermostable reverse transcriptase) into the eluted mRNA solution. The MCU controls chamber C to perform reverse transcription according to the following temperature program: heat up to 50~55℃ and hold for 10~60 min to allow the reverse transcriptase to react rapidly at high temperature; heat up to 80℃ and hold for 7 min to inactivate the reverse transcriptase and obtain a stable cDNA solution. The micro-peristaltic pump transfers the cDNA-containing solution to the cryopreservation unit through the microfluidic tubing.

[0051] Based on the above methods and systems, this embodiment performs in-situ deep-sea macrotranscriptome data collection in the western Pacific abyss (6000m depth, 60MPa pressure). The deep-sea instrument is mounted on an ROV robotic arm (backup plan: a bottom-mounted fixed platform). After reaching the target water depth (6000m, 60MPa, 2℃), the MCU automatically initiates the following operation sequence according to a preset program: ① Enrichment and fixation stage (0~10min): Turn on the peristaltic pump in the FC chamber to draw 5L of seawater at 3L / h. Microorganisms are retained in the 0.22μm polycarbonate filter membrane (retention efficiency ≥99%). After stopping the pump, inject 4mL of RNAlater into the FC chamber (adapted to the high-pressure sealed reservoir chamber, effectively maintaining RNA fixation activity under 60MPa conditions). Switch the valve group to the closed state and incubate for 5min. Then perform the complete PBS washing-lysis step.

[0052] ② Controlled decompression stage (10~100min): The valve connecting the FC chamber to the seawater is closed, forming a closed flow path; the DPU plunger pump executes three decompression curves according to the MCU instructions (60→10MPa: 2MPa / min; 10→2MPa: 0.5MPa / min; 2→0.1MPa: 0.05MPa / min); dual pressure sensors monitor in real time, and the fuzzy PID algorithm corrects the plunger displacement rate in real time; the entire decompression process takes about 90 minutes. ③ Chamber A treatment (95~155min, partly in parallel with decompression): Refer to Section (III).3 of the technical plan for the SG magnetic bead capture-DNaseI treatment-elution process to obtain total RNA; ④ Chamber B treatment (155~205min): Refer to Section (III).4 of the technical plan to perform probe-RM magnetic bead cascade rRNA depletion, denature at 70℃±0.3℃ for 10min, hybridize at 37℃ for 20min, incubate with RM magnetic beads for 15min to obtain mRNA enrichment solution; ⑤ Chamber C processing (205~260min, including parallel processing): NA magnetic bead secondary purification (10min) + in situ reverse transcription; reverse transcription is performed with precise temperature control at 50~55℃±0.2℃; reverse transcriptase is inactivated at 80℃; cDNA is transferred to the 4℃ refrigeration unit; the total time for the whole machine is about 260min, and the instrument can continue to perform the next sampling cycle or wait for ROV recovery.

[0053] To verify the control accuracy of the magnetic bead in the bidirectional electromagnet assembly, the following verification experiments can be conducted in a transparent chamber model simulating deep-sea temperature and pressure (2℃, simulating a 1MPa laboratory environment) (a substitute of chamber A specification, with optical glass walls): (a) Adsorption time verification: 200 μg SG magnetic beads (particle size 2.5 μm) were uniformly dispersed in 600 μL solution, the magnetic field was turned on, and the time required for the magnetic beads to completely aggregate at the bottom was recorded by high-speed camera; (b) Mixing efficiency verification: A fluorescent dye solution (Rhodamine B, 5 μM) was injected into a chamber containing 100 μg of fluorescently labeled magnetic beads. Suspension mixing was initiated by alternating on and off cycles (1 Hz), and the solution fluorescence intensity homogenization time was measured using a fluorescence imaging system. (c) Transfer accuracy verification: Magnetic bead transfer from chamber A to chamber B (transfer mode, magnetic bead is attracted to the top and then liquid is pushed for transfer); repeated 10 times, with a target transfer volume of 5 μL each time; verify the transfer volume and recovery rate from the magnetic bead; (d) Verification of cleaning removal rate: After the magnetic beads at the bottom of chamber A are fixed by adsorption, a cleaning solution containing fluorescent protein (BSA-FITC, 1 mg / mL) is injected. After three in-situ ethanol cleanings, the residual fluorescence intensity of BSA-FITC in the chamber is measured.

[0054] The thermal performance of the precision temperature control system in chamber C was tested in a 2℃ constant temperature water bath (simulating the temperature of the deep sea environment). (a) Temperature rise response: From 2℃ to 50℃, with a ceramic heating element input power of 2.5W under PID control, the time required to reach the target temperature range of ±0.5℃ is 26±2s (better than the design specification of 30s). (b) Steady-state accuracy: After holding at 50℃ for 60 min, the PT100 collects the temperature every 5 s and the steady-state temperature fluctuation range is 49.82~50.18℃ (i.e. ±0.18℃, which meets the design requirement of ≤±0.2℃). (c) Temperature zone switching: Switching time from 50°C to 80°C (enzyme inactivation): 18±3s; (d) Thermal uniformity: The uniformity of liquid temperature in the chamber was verified by distributed fiber optic temperature sensors (3 measurement points). Under steady state, the temperature difference at each point was <0.4℃, which meets the requirements of biochemical reaction.

[0055] This invention employs a "closed fluid system + multi-physics field collaborative control" architecture. All biochemical operations are completed within a continuous sealed fluid channel, eliminating cross-contamination between the sample and the external environment. The magnetic microspheres are driven to complete all biochemical operations—adsorption, cleaning, transfer, and desorption—within the chamber through the collaborative control of three physical fields: pressure (plunger pump + high-pressure valve group), magnetic field (upper and lower bidirectional electromagnet groups), and temperature (multi-region independent micro-thermal control units). This achieves separation operations without centrifugation within the closed system, enabling fully automated sample processing in deep-sea environments.

[0056] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its scope and spirit, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization system, characterized in that, It includes a continuous closed fluid channel, in which a filtration enrichment chamber is connected in series for retaining microorganisms in seawater samples and performing in-situ fixation. The controlled decompression unit is used to smoothly reduce the pressure of the trapped microbial samples from the deep-sea environment to the working pressure; The program control unit is used to coordinate the control system and achieve unattended, fully automated operation. At least one multifunctional reaction chamber is provided, wherein a temperature control system is integrated within the multifunctional reaction chamber, and at least one set of bidirectional opposing electromagnets is provided on the outer side of the chamber wall. The on / off state and current of the bidirectional opposing electromagnets are controlled by a program control unit to generate a dynamically switchable spatial magnetic field gradient inside the multifunctional reaction chamber, thereby driving the magnetic microspheres inside the chamber to perform at least one of the following operations without relying on centrifugation: adsorption and aggregation, suspension and mixing, in-situ cleaning, inter-chamber transfer, and elution and desorption.

2. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization system according to claim 1, characterized in that, It also includes a reagent storage unit and a cryogenic storage unit. The reagent storage unit integrates a multi-channel micro-injection pump and a pre-cooling / room temperature storage chamber, and injects liquid into each chamber quantitatively according to the instructions of the program control unit. The cryogenic storage unit is used to store the airtight micro-storage bag for encapsulating cDNA.

3. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization system according to claim 1, characterized in that, The multifunctional reaction chamber includes a series of RNA capture chambers, rRNA depletion chambers, and mRNA enrichment and reverse transcription chambers. The RNA capture chamber is used to capture and purify total RNA using silanol-functionalized magnetic microspheres. The rRNA depletion chamber is used to remove rRNA using a cascade of biotinylated probes and streptavidin-functionalized magnetic microspheres. The mRNA enrichment and reverse transcription chamber is used to purify mRNA a second time using nucleic acid purification magnetic microspheres and to perform in situ reverse transcription within the same chamber.

4. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization system according to claim 1, characterized in that, The temperature control system includes a high thermal conductivity substrate attached to the outer wall of the cavity, a ceramic heating element embedded in the substrate, and a temperature sensor fixedly connected to the substrate; the temperature control system is controlled by PID closed-loop control.

5. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization system according to claim 1, characterized in that, The controlled pressure reduction unit includes a high-pressure plunger pump and a fuzzy adaptive PID controller; the fuzzy adaptive PID controller dynamically adjusts the displacement rate of the high-pressure plunger pump to achieve pressure reduction based on the three-dimensional state variables of pressure deviation, pressure change rate and plunger displacement.

6. A method for multi-stage processing and reverse transcription stabilization of deep-sea in situ metatranscriptome RNA, characterized by, Based on the system according to any one of claims 1-5, the method is carried out within a continuous closed fluid channel and includes the following steps: Step 1: Under the target deep-sea environmental pressure conditions, seawater samples are introduced into the filtration enrichment chamber, where microorganisms are trapped and pre-washing and RNA lysis are completed within the filtration enrichment chamber. Step 2: Activate the controlled decompression unit to perform three-stage decompression, which will steadily reduce the pressure of the fluid containing microorganisms from the deep-sea environment to the working pressure of 0.1 MPa. Step 3: The fluid passes sequentially through the RNA capture chamber, the rRNA depletion chamber, and the mRNA enrichment and reverse transcription chamber. The RNA capture chamber captures and purifies total RNA. The rRNA depletion chamber removes rRNA in a cascade. The mRNA enrichment and reverse transcription chamber purifies mRNA a second time and performs in situ reverse transcription to obtain a stable cDNA product. Step 4: Transfer the cDNA product obtained in Step 3 to a cryopreservation unit for storage.

7. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization method according to claim 6, characterized in that, The three-stage decompression in step 2 includes a high-pressure rapid release stage, an intermediate stable transition stage, and a low-pressure slow balancing stage. The pressure and plunger displacement are collected in real time by a fuzzy adaptive PID controller, and the decompression rate is dynamically corrected by combining a fluid volume change model that includes temperature and salinity compensation.

8. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization method according to claim 6, characterized in that, The controlled pressure reduction unit is equipped with dual redundant pressure sensors for cross-verification, and automatically stops the plunger pump and triggers an alarm when an abnormal pressure is detected.

9. The deep-sea in-situ macro-transcriptome RNA multi-stage processing and reverse transcription stabilization method according to claim 6, characterized in that, Step 3 specifically involves: Step 3.1: Inject SG magnetic bead suspension containing silanol-functionalized magnetic beads into the RNA capture cavity, start suspension mixing and incubation for 10 min, switch to adsorption state, inject washing buffer, and wash in situ to remove impurities; switch to suspension mixing state and wash with DNase buffer; switch to desorption elution state and inject elution buffer. The temperature control module raises the chamber temperature to the elution temperature and suspends the mixture; the magnetic field adsorbs magnetic beads and collects the elution buffer containing total RNA. Step 3.2: The liquid obtained in step 3.1 and the biotin-labeled specific probe targeting the conserved regions of prokaryotic 16S and 23S rRNA are injected into the rRNA depletion chamber. After denaturation at 70°C under the control of a temperature control system, the temperature is lowered to 37°C for hybridization. The probe and rRNA binding is promoted by using a suspension and mixing state. Streptavidin-functionalized magnetic beads are injected to capture the rRNA-probe complex at 37°C, and the complex is separated by adsorption. Step 3.3: Inject the liquid obtained in Step 3.2 and the nucleic acid purification magnetic beads into the mRNA enrichment and reverse transcription chamber, start suspension mixing, capture mRNA onto the surface of the nucleic acid purification magnetic beads, switch the adsorption state to fix the magnetic beads, wash to remove impurities, inject nuclease-free water, adjust the temperature control module to 70℃±0.5℃, desorption mixing to complete mRNA elution; after magnetic field fixation of the magnetic beads, collect the elution buffer containing high-purity mRNA; inject the eluted mRNA solution into the reverse transcription premixed system, heat to 50~55℃, maintain for 10~60min, so that the reverse transcriptase reacts rapidly at high temperature; heat to 80℃, maintain for 7min, heat-inactivate the reverse transcriptase, and obtain a stable cDNA solution.

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