A fully-automatic nucleic acid extraction method for seawater bacteria
By combining a fully automated nucleic acid extraction workstation with polymer-modified magnetic beads, highly efficient and automated seawater bacterial nucleic acid extraction was achieved. This solved the problem of low efficiency in high-salt environments using traditional methods, improved nucleic acid purity and recovery rate, and reduced labor costs and experimental errors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEFEI INSTITUTE OF PHYSICAL SCIENCE CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2026-03-12
- Publication Date
- 2026-05-29
Smart Images
Figure CN122104679A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fully automated nucleic acid extraction technology for marine bacteria, and particularly relates to a fully automated nucleic acid extraction method for marine bacteria. Background Technology
[0002] Marine microorganisms possess rich biodiversity and are an important component of marine ecosystems. Since the 20th century, isolation and culture methods have been used to study the diversity of marine microorganisms. However, as research has deepened, it has been discovered that many microorganisms cannot be cultured and reproduced, posing new challenges to further molecular biology research. In recent years, the rapid development of molecular biology techniques has opened up new avenues for marine microbiology research, freeing it from dependence on traditional culture techniques. Due to the low concentration of microorganisms in seawater, nucleic acid extraction from marine pathogens generally requires pretreatment to enrich sufficient biomass in order to achieve adequate biomass.
[0003] Traditional magnetic bead / membrane column methods for nucleic acid extraction are limited by osmotic pressure imbalances in high-salt environments and adsorption competition in complex matrices, making them unsuitable for processing samples in extreme marine environments. Manual extraction not only incurs high labor costs but also suffers from low error rates and reproducibility, increasing the probability of sample contamination and hindering high-throughput sample processing. Fully automated nucleic acid extractors, due to their standardization and low labor costs, are gradually becoming essential equipment in major medical testing service institutions, and are operated using matching nucleic acid extraction kits.
[0004] Meanwhile, in the process of extracting bacterial nucleic acid, the existing technology still relies on manual operation. Manual operation is not only very inefficient, but also lacks precision in operations such as pipetting, leading to significant deviations in experimental results. Summary of the Invention
[0005] Based on the above background, the purpose of this invention is to provide a fully automated method for nucleic acid extraction from marine bacteria.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A fully automated method for extracting nucleic acids from marine bacteria includes a fully automated nucleic acid extraction workstation, which is equipped with a sample plate for placing seawater samples, an enrichment plate for enriching bacteria in the seawater samples, and a collection plate for collecting the filtration products of the enrichment plate. The collection plate is detachably covered on top of the enrichment plate, and a filter membrane for filtering bacteria is installed at the bottom of the enrichment plate. A pyrolysis solution tank for holding pyrolysis solution, a binding solution plate for holding binding solution, and a magnetic bead plate for holding magnetic beads. It also includes a magnetic sleeve that works in conjunction with the nucleic acid extractor; And detergent trays for holding detergents, and suspension trays for holding suspensions; The lysis buffer includes any one or a mixture of two or more of the following: guanidine isothiocyanate, hexadecyltrimethylammonium bromide, lysozyme, TritiumX-100, proteinase K, and sodium dodecyl sulfonate. The extraction method includes the following steps: (1) Dispose of the lysis buffer, binding solution, detergent, and suspension into the corresponding plate positions, and place the seawater sample into the corresponding sample plate position; (2) The fully automated nucleic acid extraction and amplification workstation shall be operated according to the following steps: 2.1 The enrichment of marine bacteria includes the following steps: 2.1.1 Use a pipette to draw seawater from the sample plate and add it to the enrichment plate and collection plate structure; 2.1.2 Place the enrichment plate and collection plate structure into the centrifuge. After centrifugation, remove the enrichment plate and place it on the collection plate. Reset the collection plate. Use a pipette to remove the waste liquid from the collection plate and discharge it into the waste container. After repeating the above steps several times, marine bacteria were enriched. 2.2 Bacterial lysis includes the following steps: 2.2.1 Use a pipette to take lysis solution from the lysis solution tank and add it to the enrichment plate and collection plate structure; 2.2.2 Place the enrichment plate and collection plate structure in a constant temperature incubator for heating; 2.2.3 After heating, place the enrichment plate and the collection plate into a centrifuge for centrifugation. 2.2.4 After centrifugation, remove the enrichment plate and place it in the waste storage tank, and remove the collection plate and put it back in its original position; 2.2.5 Use a pipette to remove the liquid from the collection plate and transfer it to the binding plate to obtain bacterial lysis products; 2.3 Bacterial nucleic acid purification includes the following steps: 2.3.1 During the heating process in step 2.2.2, the magnetic sleeve, magnetic bead plate, washing liquid plate, and suspension liquid plate are placed into the nucleic acid extractor using electric grippers; 2.3.2 Place the binding liquid plate from step 2.2.4 into a fully automated nucleic acid extractor for nucleic acid extraction; 2.3.3 After nucleic acid extraction, return the binding liquid plate, magnetic sleeve, magnetic bead plate, washing liquid plate and suspension plate to their corresponding positions.
[0007] Preferably, the lysis buffer comprises the following components: The following were used: 4M guanidine isothiocyanate, 5 mg / mL lysozyme, 2% TritiumX-100, and 40 μg / mL proteinase K. Sodium dodecyl sulfonate with a mass fraction of 1%; The total volume of the lysis solution is 500 μL.
[0008] Preferably, the binding solution is 200 μL of isopropanol with a mass fraction of 30%.
[0009] Preferably, the suspension includes a nucleic acid protectant, wherein the nucleic acid protectant is Tris-HCl with a concentration of 10 mM and EDTA with a concentration of 1 mM.
[0010] Preferably, the fully automated nucleic acid extraction workstation is equipped with a first washing liquid plate, a second washing liquid plate and a third washing liquid plate, wherein the first washing liquid plate contains a type I detergent; The second washing liquid tray contains Class II detergent, and the third washing liquid tray contains Class III detergent. The composition of the Class I detergent is as follows: Class I detergent includes any one or a mixture of two or more of guanidine hydrochloride, sodium acetate, acetic acid, and ethanol with a mass fraction of 55%. The composition formula of Class II detergents is as follows: Class II detergents include any one or a mixture of two of sodium acetate and 75% ethanol by mass. The composition of Class III detergents is as follows: Class III detergents include 70%-80% ethanol by mass.
[0011] Preferably, the magnetic beads are Fe3O4, and their surfaces are modified with a polymer (the polymer includes any one of polydiallyldimethylammonium chloride PDDA, 3-aminopropyltriethoxysilane, or chitosan). The surface is positively charged, and after the magnetic beads are mixed with a binding solution containing nucleic acids, the positively charged magnetic beads adsorb the negatively charged nucleic acids.
[0012] Preferably, the fully automated nucleic acid extraction workstation further includes a nucleic acid extraction module, which includes a nucleic acid extraction module base, a rotating platform, and two lifting modules. The rotating platform is rotatably connected to the nucleic acid extraction module base, and the rotating platform has multiple workstations. Each workstation has a functional component corresponding to the process. A rotary drive motor is fixedly connected inside the nucleic acid extraction module base. The output end of the rotary drive motor is connected to the rotating platform and can drive the rotating platform to rotate around its axis. A magnetic rod sleeve buckle is fixedly connected to the moving end of one lifting module, and a magnetic rod frame is fixedly connected to the moving end of the other lifting module. The magnetic rod frame can face or move away from the magnetic rod sleeve buckle so that the magnetic rod sleeve buckle can be engaged or disengaged from the magnetic rod frame. The two lifting modules are designated as Lifting Module 1 and Lifting Module 2. An annular baffle is fixedly connected to the base of the nucleic acid extraction module. Lifting Module 1 includes an oscillating rear plate, a drive motor 1, a lead screw 1, and an oscillating front plate. The oscillating rear plate and the oscillating front plate are slidably fitted together. The oscillating rear plate is fixedly connected to the inner wall of the annular baffle. A drive motor 1 is fixedly connected to the oscillating rear plate. A lead screw 1 is fixedly connected to the output end of the drive motor 1. The oscillating front plate is screwed onto the lead screw 1. The bottom of the oscillating front plate is fixedly connected to the magnetic rod sleeve buckle. The lifting module also includes a slide rail. Two parallel slide rails are fixedly connected to one end of the oscillating rear plate facing the oscillating front plate, and are slidably connected to the oscillating front plate through the slide rails. The front plate of the oscillation is provided with a second lifting module, which includes a second drive motor and a second lead screw. The second drive motor is fixedly connected to the top of the front plate of the oscillation, and the output end of the second drive motor is connected to the second lead screw. The second lead screw is screwed to the magnetic rod frame.
[0013] Preferably, the fully automated nucleic acid extraction workstation also includes a main chassis and an operating table installed inside the main chassis; The operating table is equipped with a three-axis moving module, a centrifugation module, a sealing module, a low-temperature refrigeration module, a PCR amplification chamber, and a clamp-type incubation module; It also includes plate holders, pipette tip holders, and reagent holders installed on the operating table; The plate holder is used to place PCR deep well plates, PCR reaction plates and reagent deep well plates, the pipette tip holder is used to place tip holders, the reagent holder is used to place reagent solution plates, and the waste storage compartment is used to collect waste deep well plates and tip holders.
[0014] Preferably, the three-axis moving module includes a left moving module and a right moving module with the same structure. The left moving module includes an X-axis linear module, a Y-axis linear module, and a Z-axis linear module. The Z-axis linear module is fixedly connected to the moving end of the Y-axis linear module, and the Y-axis linear module is fixedly connected to the moving end of the X-axis linear module. The X-axis linear module is fixed inside the main body of the chassis. A variable pitch pipetting module is fixedly connected to the moving end of the Z-axis linear module of the left moving module. An ADP pipette is connected to the moving end of the variable pitch pipetting module. An electric gripper and an identification module are fixedly connected to the output end of the Z-axis linear module of the right moving module. The recognition module includes a CCD camera, a lens, and a light source. The CCD camera is fixed at the moving end of the Z-axis linear module of the right moving module, the lens is fixed at the front end of the CCD camera, and the light source is located between the CCD camera and the lens.
[0015] Preferably, the PCR amplification chamber includes a chamber body, an upper door, a front door, a PCR amplification module, and an amplification module entry / exit conveyor module. The chamber body has openings adapted to the upper door and the front door. The top of the chamber body has an opening and closing upper door, and the front part has an opening and closing front door. The upper door is slidably connected to the chamber body via an upper door slide rail. A synchronous belt drive module is fixedly connected to the inner wall of the chamber body. The synchronous belt drive module can drive the front door to open and close. The front door is driven to the chamber body via the amplification module entry / exit slide rail module and can drive the front door to open and close. The synchronous belt drive module includes an upper door drive motor, a synchronous belt, and two synchronous pulleys. The output end of the upper door drive motor is connected to one synchronous pulley and can drive the synchronous pulley to rotate. The synchronous pulley is connected to the other synchronous pulley through the synchronous belt. The other synchronous pulley is rotatably connected to the cabin body.
[0016] The present invention has the following beneficial effects: 1. The present invention provides a seawater bacterial nucleic acid extraction reagent. The enrichment plate in the nucleic acid extraction reagent can enrich bacteria on the filter membrane by centrifugation, so that no pretreatment is required before seawater extraction. The surface of the magnetic beads is modified with a high molecular polymer (polydiallyldimethylammonium chloride (PDDA), 3-aminopropyltriethoxysilane (APTES) or chitosan), and the surface is positively charged. Through the principle of charge adsorption, the adsorption efficiency of nucleic acid is higher than that of ordinary magnetic beads, so as to extract seawater samples with low concentration of pathogenic microorganisms.
[0017] 2. The automated nucleic acid extraction workstation provided by this invention, when used in conjunction with seawater bacterial nucleic acid extraction reagent, features high automation, fast speed, high nucleic acid purity, and high nucleic acid recovery rate, solving the problem of processing high-salt and low-biomass samples.
[0018] 3. In the automated nucleic acid extraction workstation, by setting the nucleic acid extraction module as a turntable structure, the relative movement of the magnetic rod sleeve buckle and the magnetic rod holder can be realized through two drive modules. This meets the needs of nucleic acid purification such as automatic switching of sample reagents, loading and unloading of magnetic rod sleeves, adsorption and transfer of magnetic beads, heating and lysing of samples, and shaking and mixing of reagents. It reduces the space occupied by the nucleic acid extraction module and realizes the miniaturization of the entire micro-volume nucleic acid extraction and amplification device.
[0019] 4. In the automated nucleic acid extraction workstation, by integrating a triaxial moving module, centrifugation module, sealing module, low-temperature refrigeration module, PCR amplification chamber, clamp-type incubation module, and identification module into the micro-volume nucleic acid extraction and amplification device, the limitation of the single extraction function of the current nucleic acid extraction device is broken, and the applicability of the device is improved. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0021] Figure 1 This is a schematic diagram of the structure of components such as enrichment plate, collection plate, sample plate, binding liquid plate, washing liquid plate, and centrifugation module installed on the operating table in an embodiment of the present invention; Figure 2 This is a schematic diagram of the enrichment plate and collection plate structures in an embodiment of the present invention; Figure 3 This is a graph showing the results of CT value detection in Embodiment 4 of the present invention; Figure 4 This is a graph showing the results of CT value detection in Embodiment 5 of the present invention; Figure 5 This is a graph showing the results of PCR amplification detection in Example 6 of the present invention; Figure 6 This is a schematic diagram of the overall structure of the fully automated nucleic acid extraction workstation in an embodiment of the present invention; Figure 7 This is a schematic diagram of the nucleic acid extraction module structure provided in an embodiment of the present invention; Figure 8 This is a schematic diagram of the low-temperature refrigeration module structure provided in an embodiment of the present invention; Figure 9 This is a schematic diagram of the PCR amplification chamber structure provided in an embodiment of the present invention; Figure 10 This is a schematic diagram of the variable-pipette module structure provided in an embodiment of the present invention; Figure 11 This is a schematic diagram of the identification module structure provided in an embodiment of the present invention.
[0022] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0024] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.
[0025] Furthermore, in this invention, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.
[0026] Example 1 like Figures 1-11 As shown, a fully automated method for extracting nucleic acids from marine bacteria includes a fully automated nucleic acid extraction workstation, which performs nucleic acid extraction from marine bacteria in an automated manner.
[0027] Specifically, in order to facilitate experimental operations, the fully automated nucleic acid extraction workstation is equipped with a sample plate 400 for placing seawater samples, an enrichment plate 1600 for enriching bacteria in seawater samples, and a collection plate 600 for collecting the filtered products of the enrichment plate 1600. The collection plate 600 is detachably fitted onto the top of the enrichment plate 1600, and a filter membrane 1601 for filtering bacteria is installed at the bottom of the enrichment plate 1600; the pore size of the filter membrane 1601 is 1μm. According to the existing installation method of the filter membrane 1601, the filter membrane is assembled and fixed to the bottom of the enrichment plate 1600 by hot-press welding.
[0028] The enrichment plate 1600 and the collection plate form an enrichment plate 1600 and collection plate structure. During the experiment, the enrichment plate 1600 and collection plate 600 on the enrichment plate 1600 and collection plate 600 structure are merged or separated according to the experimental requirements.
[0029] It also includes a pyrolysis liquid tank 1200 for holding pyrolysis liquid, a binding liquid plate 700 for holding binding liquid, and a magnetic bead plate 1800 for holding magnetic beads; it also includes a magnetic sleeve 200 that works in conjunction with the magnetic bead plate 1800; a detergent plate for holding detergent; and a suspension plate 1700 for holding suspension.
[0030] Specifically, to meet experimental needs, the fully automated nucleic acid extraction workstation is equipped with a first washing solution plate 1100, a second washing solution plate 1300, and a third washing solution plate 1400. The first washing solution plate 1100 contains Class I detergent; the second washing solution plate 1300 contains Class II detergent; and the third washing solution plate 1400 contains Class III detergent.
[0031] Example 2 like Figures 1-11 As shown in the example, this embodiment, based on the previous embodiment, specifically discloses the components and formulations of the lysis buffer, combined solution, Class I detergent, Class II detergent, and Class III detergent, as shown in the table below:
[0032] Example 3 like Figures 1-11 As shown, this embodiment, based on Embodiment 2, specifically discloses the following method for automated extraction of bacterial nucleic acid: Place the different reagent plates into their corresponding slots.
[0033] Sample addition: Add the seawater sample to be extracted to the corresponding sample plate 400 and number the sample.
[0034] The specific steps of the fully automated nucleic acid extraction workstation are as follows: 3.1 Enrichment of marine bacteria 3.1.1 Use an 8-channel pipette to take 1 mL of seawater from sample plate 400 and add it to enrichment plate 1600 (including collection plate 600). 3.1.2 Then, using the electric gripper 4, the enrichment plate 1600 (including the collection plate 600) is placed into the centrifuge 5 and centrifuged at 3000 rpm / min for 3 min. 3.1.3 After centrifugation, the enrichment plate 1600 is removed by the electric gripper 4 and placed into the collection plate 600. The collection plate 600 is then removed and placed back in its original position. The liquid in the collection plate 600 is then aspirated by a pipette (i.e., the ADP pipette 19 described below) and discharged into the waste storage tank 10.
[0035] 3.1.4 Repeat steps 3.1.1-3.1.3 four times.
[0036] 3.2 Bacterial lysis in the filter membrane of enrichment plate 1600 3.2.1 Use a pipette to take 500 μL of lysis buffer from the lysis buffer tank 1200 and add it to the enrichment plate 1600 (including the collection plate 600). 3.2.2 Then, using the electric gripper 4, the enrichment plate 1600 (including the collection plate 600) is placed in the constant temperature incubator 5 and heated at 60°C for 5 minutes; 3.2.3 After heating, the enrichment plate 1600 (including the collection plate 600) is placed into the centrifuge 5 using the electric gripper 4, and centrifuged at 3000 rpm / min for 3 min; 3.2.4 After centrifugation, the enrichment plate 1600 is removed by the electric gripper 4 and placed into the waste storage tank 10, and the collection plate 600 is removed and placed in its original position; 3.2.4 Use a pipette to take 500 μL of liquid from the collection plate 600 and transfer it to the binding liquid plate 700.
[0037] 3.3 Bacterial Nucleic Acid Purification Steps 3.3.1 During the heating process in step 3.2.2, the magnetic sleeve 200, magnetic bead plate 1800, washing liquid plates 1, 2, and 3, and suspension plate 1700 are placed into the nucleic acid extractor by the electric gripper 4.
[0038] 3.3.2 The binding liquid plate 700 from 3.2.4 is placed into the fully automated nucleic acid extractor using the electric gripper 4.
[0039] 3.3.3 The fully automated nucleic acid extractor begins automated nucleic acid extraction according to the set nucleic acid extraction steps.
[0040] 3.3.4 After nucleic acid extraction, the magnetic sleeve 200, magnetic bead plate 1800, binding liquid plate 700, washing liquid plates 1, 2, and 3, and suspension plate 1700 are returned to their corresponding positions using the electric gripper 4.
[0041] 4. After the nucleic acid extraction is complete, remove the suspension plate 1700, store the nucleic acid for later use, discard the remaining reagent plates and reagent tanks, and turn off the machine.
[0042] Example 4 like Figures 1-11 As shown, in order to increase the extraction efficiency, the magnetic beads of this invention are Fe3O4, and their surfaces are modified by a polymer and are positively charged. The magnetic beads are mixed with a binding solution containing nucleic acids, causing the magnetic beads to adsorb negatively charged nucleic acids.
[0043] Therefore, after modification with polymer, the extraction efficiency and effect are significantly improved. Thus, an extraction experiment was conducted to compare the results with those of conventional magnetic beads (unmodified) (as a control example). In the comparison experiment, except for the magnetic beads, all other experimental parameters and steps remained unchanged. Both the experimental example and the control example were tested simultaneously on 10 samples. 2 Nucleic acid was extracted from seawater samples containing *Vibrio vulnificus* at a concentration of cfμ / mL. The extracted nucleic acid was then detected by quantitative real-time PCR using a *Vibrio vulnificus* detection kit. The detection CT values of the nucleic acid extracted using the two reagents were compared, and the results are as follows: Figure 2 As shown.
[0044] Depend on Figure 3It is evident that the extraction efficiency and effect of the magnetic beads modified with polymers (including any one of polydiallyldimethylammonium chloride PDDA, 3-aminopropyltriethoxysilane, or chitosan) are significantly better than those of conventional unmodified magnetic beads.
[0045] Example 5 like Figures 1-11 As shown, in order to further verify the extraction effect of the extraction reagents and extraction methods disclosed in this invention on seawater bacteria, this embodiment conducts a seawater bacterial nucleic acid extraction experiment.
[0046] The experimental procedure is as follows: Using 10 1 cfμ / mL and 10 3 Seawater simulated samples of Vibrio vulnificus (cfμ / mL) were used to extract nucleic acid from Vibrio vulnificus using the seawater bacterial nucleic acid extraction reagent according to the steps in Example 2 and the commercial magnetic bead bacterial nucleic acid extraction kit, following the experimental method in Example 4.
[0047] The extracted nucleic acid was detected by real-time PCR using a Vibrio vulnificus detection kit. The detection CT values of nucleic acid extracted by the two reagents were compared. Figure 4 .
[0048] Depend on Figure 4 It can be seen that the modified magnetic beads used in this invention can significantly improve the efficiency of nucleic acid extraction.
[0049] Example 6 like Figures 1-11 As shown, in order to further verify the extraction effect of the extraction reagents and extraction methods disclosed in this invention on seawater bacteria, this embodiment conducts a seawater bacterial nucleic acid extraction experiment.
[0050] 10 0 cfμ / mL, 10 1 cfμ / mL, 10 2 cfμ / mL, 10 3 cfμ / mL, 10 4 cfμ / mL and 10 5 A seawater-derived simulated sample of Vibrio vulnificus (cfμ / mL) was used. Nucleic acid was extracted from Vibrio vulnificus using a seawater bacterial nucleic acid extraction reagent following the steps in Example 4. The extracted nucleic acid was then detected by real-time quantitative PCR using a Vibrio vulnificus detection kit. The detection results are as follows: Figure 5 .
[0051] Depend on Figure 5 It can be seen that the modified magnetic beads used in this invention can significantly improve the efficiency of nucleic acid extraction.
[0052] Example 7 likeFigures 1-11 As shown, in order to achieve automated operation and solve the technical defects of traditional manual experimental operation, such as low efficiency and poor accuracy, this invention discloses the specific structure and working method of a fully automated nucleic acid extraction workstation.
[0053] The specific structure of the fully automated nucleic acid extraction workstation is as follows: The fully automated nucleic acid extraction workstation includes a main body 6, a front cover 1, an operating table 11, a three-axis moving module 2, a centrifugation module 10, a nucleic acid extraction module 9 (i.e., a nucleic acid extractor), a sealing module 14, a low-temperature refrigeration module 16, a PCR amplification chamber 15, and a clamp-type incubation module 5. The front cover 1 is connected to the main body 6 via a rotating connection device. The front cover 1 can rotate relative to the main body 6, thereby opening and closing the front cover 1, and causing the main body 6 to communicate with the outside or to be closed.
[0054] The main body 6 houses an operating table 11. The centrifugation module 10 (centrifuge), nucleic acid extraction module 9, sealing module 14, low-temperature refrigeration module 16, PCR amplification chamber 15, and clamp-type incubation module 5 are all fixed on the operating table 11. The centrifugation module 10 separates the nucleic acid supernatant from the sample carrier after sample incubation. The sealing module 14 seals the PCR reaction plate before amplification. A three-axis moving module 2 is fixed to the top of the operating table 11. The moving end of the three-axis moving module 2 is connected to an electric gripper 4 and a pipette 7. The electric gripper 4 is used to hold and transfer the reaction plate. The three-axis moving module 2 drives the electric gripper 4, pipette 7, and recognition module 12 to move in the X, Y, and Z directions, moving between the centrifugation module 10 (specifically the centrifuge), nucleic acid extraction module 9, sealing module 14, low-temperature refrigeration module 16, PCR amplification chamber 15, and clamp-type incubation module 5. A display 13 is fixed on the main body 6. It should be noted that the operating table 11 is equipped with plate holders, pipette tip holders, reagent holders, and a waste storage compartment 10. The operating table 11 has multiple plate holders, such as five, designated as Plate Holder One to Plate Holder Five. These plate holders are used to hold PCR deep-well plates, PCR reaction plates, and reagent deep-well plates. The pipette tip holders are used to hold tip tubes, and the reagent holders are used to hold reagent containers. That is, the sample plate 400, enrichment plate 1600, collection plate 600, lysis buffer container 1200, and binding buffer plate 700 on the workstation are all mounted on these holders with different functions.
[0055] Waste storage compartment 10 is used to collect waste deep hole plates and tip heads. That is, during the experiment, the above consumables are discarded in waste storage compartment 10 after use.
[0056] The three-axis moving module 2 includes a left moving module 3 and a right moving module 23. The left moving module 3 and the right moving module 23 have the same structure but different arrangement positions. The left moving module 3 includes an X-axis linear module (not shown in the figure), a Y-axis linear module, and a Z-axis linear module. The Z-axis linear module is fixedly connected to the moving end of the Y-axis linear module, and the Y-axis linear module is fixedly connected to the moving end of the X-axis linear module. The X-axis linear module is fixed inside the main body 6 of the chassis. The moving end of the Z-axis linear module of the left moving module 3 is fixedly connected to a variable pitch pipetting module 8. The moving end of the variable pitch pipetting module 8 is connected to an ADP pipette 19. The variable pitch pipetting module 8 is a commercially available part, and its specific structure will not be described in detail. In this embodiment, the moving end of the variable pitch pipetting module 8 is connected to eight ADP pipettes 19 arranged side by side. The variable pitch pipetting module 8 can drive the ADP pipettes 19 arranged side by side to adjust the pitch. An electric gripper 4 is fixedly connected to the output end of the Z-direction linear module of the right moving module 23. An identification module 12 is also provided on the outside of the electric gripper 4. The identification module 12 is also fixed to the output end of the Z-direction linear module of the right moving module 23.
[0057] It should be noted that the X-axis linear module, Y-axis linear module, and Z-axis linear module are all commercially available lead screw linear modules (i.e., conventional lead screw modules disclosed in the prior art, such as the most commonly used cross lead screw module). Those skilled in the art can consult technical manuals and dictionaries to learn about the specific structure of the above-mentioned X-axis linear module, Y-axis linear module, and Z-axis linear module and how to cooperate with experimental operation actions to perform the corresponding action process.
[0058] Similar to the structure of the intelligent recognition device in existing automated experimental equipment, the aforementioned recognition module 12 includes a CCD camera 20, a lens 22, and a light source 21. The CCD camera 20 is fixed on the moving end of the Z-direction linear module of the right moving module 23, and the lens 22 is fixed on the front end of the CCD camera 20 for taking pictures and collecting reagents or consumables on various plate holders, reagent tank holders, tip head holders, etc. The light source 21 is located between the CCD camera 20 and the lens 22 to provide a light source for taking pictures and collecting data, and to reduce interference from reflections.
[0059] The clamp-type incubation module 5 is used for heating and lysing the sample, as well as assisting in the separation of the sample processing kit's sample storage plate and filtrate receiving plate. The clamp-type incubation module 5 is a commercially available component, that is, it is a conventional constant temperature incubator disclosed in the prior art. Those skilled in the art can consult technical manuals and dictionaries to understand its specific structure and working principle.
[0060] Similar to existing thermostatic incubators, thermostatic incubators can shake, heat, and fix samples; The nucleic acid extraction module 9 is used to purify nucleic acids. The nucleic acid extraction module 9 includes a nucleic acid extraction module base, a rotating platform 29, a heating module 35, a magnetic rod holder 28, a lifting module one 30, a lifting module two 26, a magnetic rod sleeve buckle 34, and a rotary drive motor 36. The top of the nucleic acid extraction module base has a groove for accommodating the rotating platform 29 and the rotary drive motor 36. The rotating platform 29 is rotatably connected to the nucleic acid extraction module base, and the rotary drive motor 36 is fixedly connected to the inner wall of the bottom of the nucleic acid extraction module base. The output end of the rotary drive motor 36 is connected to the rotating platform 29 for transmission. The rotary drive motor 36 can drive the rotating platform 29 to rotate around its axis. The rotating platform 29 has multiple fixing slots, one of which is fixedly connected to a heating module 35, and the other fixing slots are respectively equipped with corresponding functional components.
[0061] Each fixed slot forms a workstation. The rotation of the rotating platform 29 enables the positioning and switching of the sample and reagent deep well plate. Each rotation completes the process of one workstation, thereby improving purification efficiency and saving space. In this embodiment, a total of eight fixed slots are set to form eight workstations. The nucleic acid extraction module 9 achieves rapid nucleic acid purification by automatically switching sample reagents, loading and unloading magnetic rod sleeves, adsorbing and transferring magnetic beads, heating and lysing the sample, and shaking and mixing the reagents.
[0062] It is mainly used to place multiple reagent plates, which are used to place magnetic bead plate 1800, binding liquid plate 700, first cleaning liquid plate, second cleaning liquid plate, third cleaning liquid plate, suspension plate 1700, magnetic sleeve plate and spare plate position respectively; the reagent plates on different work stations form functional components on different work stations.
[0063] During the operation, the rotating platform 29 rotates to the magnetic sleeve plate station for magnetic sleeve loading; then, it rotates to the magnetic bead plate station for magnetic bead enrichment; next, it rotates to the binding liquid plate 700 station for nucleic acid enrichment; then, it rotates sequentially to the first washing liquid plate, the second washing liquid plate, and the third washing liquid plate for magnetic bead cleaning; then, it rotates sequentially to the magnetic bead suspension station for magnetic bead suspension; finally, it rotates to the magnetic sleeve plate station for magnetic sleeve disposal.
[0064] The heating module 35 is a commercially available component (i.e., the heating module 35 is a heating device for conventional nucleic acid extraction experiments disclosed in the prior art). It can be a nucleic acid heating module, which has a rectangular heating membrane inside for heating in the lysis or elution steps.
[0065] A ring-shaped baffle is fixedly connected to the circumferentially of the base of the nucleic acid extraction module. A lifting module 30 is fixedly connected to the ring-shaped baffle. The lifting module 30 is a linear screw module and includes an oscillating rear plate, a drive motor 24, a lead screw 31, an oscillating front plate, and a slide rail 32. The slide rail 32 is fixedly connected to the end of the oscillating rear plate facing the oscillating front plate, and the slide rail 32 slides in cooperation with the oscillating front plate. In this embodiment, two parallel slide rails 32 are fixedly connected to the oscillating rear plate. The oscillating rear plate is fixedly connected to the inner wall of the ring-shaped baffle. The drive motor 24 is fixedly connected to the oscillating rear plate. The output end is fixedly connected to a lead screw 31, and an oscillating front plate is screwed onto the lead screw 31. A magnetic rod sleeve buckle 34 is fixedly connected to the bottom of the oscillating front plate. The oscillating front plate is provided with a lifting module 26. The lifting module 26 is used to drive the magnetic rod holder 28 to oscillate up and down and to load and unload auxiliary consumables. The lifting module 26 includes a drive motor 25, a lead screw 27, and a magnetic rod holder 28. The top of the oscillating front plate is fixedly connected to the drive motor 25. The output end of the drive motor 25 is driven by the lead screw 27. The magnetic rod holder 28 is screwed onto the lead screw 27. The magnetic rod holder 28 can move up and down along the height direction of the oscillating front plate. It should be noted that the rotating platform 29 is made of aluminum alloy, and the magnetic rod holder 28 is made of magnetic material. The magnetic rod holder 28 includes a number of magnetic rods arranged in an array. In this embodiment, the magnetic rods are arranged in a matrix and are equally spaced. The number can be 32, 48, 96 or other required quantities.
[0066] The low-temperature refrigeration module 16 is used to provide the low-temperature storage temperature required for long-term preservation of the extract, preventing the extract from losing its activity at room temperature for a long time. The low-temperature refrigeration module 16 includes a PCR plate adapter block 37, an anti-condensation cover 38, a refrigeration component 39, and a heat preservation module support 41. The refrigeration component 39 is equipped with a refrigeration semiconductor, a heat sink 40, and a cooling fan 42 to provide low-temperature storage conditions for the entire module. The heat preservation module support 41 is fixed on the operating table 11 and is used to provide installation support for the refrigeration component 39. The anti-condensation cover 38 is fixedly connected to the top of the heat preservation module support 41, and the PCR plate adapter block 37 is fixedly connected inside the anti-condensation cover 38.
[0067] The PCR amplification chamber 15 is used for thermal cycling amplification of nucleic acids. The PCR amplification chamber 15 is equipped with a PCR amplification module 44. The PCR amplification chamber 15 is an independent and sealed space, thereby avoiding cross-contamination of samples during the amplification process. The PCR amplification chamber 15 includes a chamber body 43, an upper door 46, a front door 50, a PCR amplification module 44, and an amplification module in / out transfer module 17. The top of the chamber body 43 is equipped with an upper door 46 that can be opened and closed, and the front is equipped with a front door 50 that can be opened and closed. The chamber body 43 has a cubic structure. When the upper door 46 and the front door 50 are closed, the PCR amplification chamber 15 forms an independent and sealed chamber. At this time, the PCR amplification module 44 is located in this sealed chamber and is isolated from other parts such as the previous extraction. It is used to complete the PCR reaction plate injection, PCR amplification reaction, and PCR reaction plate effluent, which can effectively avoid cross-contamination caused by the amplification process.
[0068] The upper hatch 46 is slidably connected to the cabin body 43 via the upper hatch slide rail 48. A synchronous belt drive module 47 is fixedly connected to the inner wall of the cabin body 43. The synchronous belt drive module 47 is a commercially available synchronous belt drive module. The synchronous belt drive module 47 includes an upper hatch drive motor 49, a synchronous belt, and two synchronous pulleys. The output end of the upper hatch drive motor 49 is connected to one synchronous pulley and can drive the synchronous pulley to rotate. The synchronous pulley is connected to the other synchronous pulley via the synchronous belt. The other synchronous pulley is rotatably connected inside the cabin body 43. The synchronous belt is fixedly connected to the upper hatch 46. The upper hatch drive motor 49 drives the synchronous pulley to rotate, which can drive the synchronous belt to rotate between the two synchronous pulleys, thereby realizing the movement of the upper hatch 46. An expansion compartment inlet is opened at the top of the cabin body 43. The front door 50 is connected to the cabin body 43 via the amplification module entry / exit slide rail module 53. The amplification module entry / exit slide rail module 53 is located below the synchronous belt drive module 47. The amplification module entry / exit slide rail module 53 is equipped with an amplification module entry / exit drive motor 18, an amplification module entry / exit conveyor module 17, and a PCR amplification module lower base plate 54. The amplification module entry / exit conveyor module 17 and the synchronous belt drive module 47 have the same structure and are both commercially available synchronous belt drive modules. The synchronous belt of the amplification module entry / exit conveyor module 17 is fixedly connected to the PCR amplification module lower base plate 54. The PCR amplification module lower base plate 54 is fixedly connected to the front door 50. The PCR amplification module 44 is fixed on the top of the PCR amplification module lower base plate 54. The PCR amplification module 44 can be a commercially available PCR amplification instrument. The PCR amplification module 44 is equipped with a heat cover 45, a PCR thermal circulation component 51, and a heat dissipation module 52.
[0069] The procedure for nucleic acid extraction and amplification in this implementation is as follows: S1. Preparation of reagents and consumables: such as lysis buffer, binding buffer, magnetic bead plate 1800, washing buffer, magnetic bead suspension and PCR system plate, etc., place them on the corresponding labeled plate holders (reagent plates); place proteinase K, lysis factor, washing buffer, processing buffer, tip, etc. on the corresponding labeled plate holders (reagent plates). S2. Intelligent self-test: The three-axis moving module 2 moves above the operating table 11 to detect each plate holder, reagent tank holder, tip head holder, etc., to determine whether the corresponding reagents or consumables are placed accurately, avoiding human error such as omission or misplacement, so as to improve the accuracy of the test. S3. Sample pretreatment: Using the three-axis moving module 2, the variable-distance pipetting module 8 is controlled by the pipette module 7. The tip is loaded and a certain amount of proteinase K is aspirated into the corresponding sample plate and mixed by suction. S4. Cell lysis: The sample processing sleeve on the plate holder is transferred to the corresponding clamping incubation module 5 using the three-axis moving module 2 and the moving electric gripper 4. Then, the clamping incubation module 5 is heated and shaken for several minutes to fully lyse the cells in the sample. S5. Cell lysis buffer collection: Using the electric gripper 4 of the triaxial moving module 2, the sample processing plates on the corresponding clamping incubation module 5 are transferred in pairs to the basket of the centrifugation module 10 for centrifugation at a speed of 3500 r / min. After centrifugation, the electric gripper 4 of the triaxial moving module 2 is used to transfer the sample processing plates in pairs to the corresponding clamping incubation module 5. Then, the electric grippers of each clamping incubation module 5 clamp the sample processing plates. Using the electric gripper 4 of the triaxial moving module 2, the sample storage plates on each sample processing plate are transferred to the waste storage chamber 10. After the sample storage plates are discarded, the electric grippers of each clamping incubation module 5 will automatically release. At this time, the electric gripper 4 of the triaxial moving module 2 is used to transfer the filtrate receiving plate to the plate holder one to the plate holder four in sequence. At this time, the collection of lysis buffer has been completed. S6. Nucleic acid purification preparation: During the centrifugation process in S5, the three-axis moving module 2 moves the electric gripper 4 to transfer the magnetic rod sleeve, magnetic bead plate 1800, washing liquid plate (in this embodiment, there are three washing liquid plates), magnetic bead suspension, etc. to the second to seventh plates in the purification module for use in nucleic acid purification. S7. Transfer of lysate to binding plate 700: The three-axis moving module 2 drives the variable pitch pipetting module 8 to load the tip, and then the variable pitch pipetting module 8 moves the tip to transfer the lysate in the sample wells (24 wells / set × 4 sets) of the filtrate receiving plate on the four plate positions to the ninety-six sample wells on the lysate binding plate 700 on the plate position five, and add a certain amount of treatment liquid to each sample well. It should be noted that in S7, each plate holder is equipped with twenty-four filtrate receiving plate sample holes. S8. Cell nucleic acid purification: Using the three-axis moving module 2 to move the electric gripper 4, the binding liquid plate 700 is transferred to the first plate position of the nucleic acid extraction module 9. The nucleic acid extraction module 9 will sequentially install the magnetic sleeve, add magnetic beads, wash three times, air dry, resuspend, and discard the magnetic sleeve. Finally, the purified nucleic acid is stored in the magnetic bead suspension plate 1700. S9. Nucleic acid cryopreservation: Using the three-axis moving module 2 to move the electric gripper 4, the magnetic bead suspension plate 1700 storing purified nucleic acid is transferred to the low-temperature refrigeration module 16 for storage and later use. The temperature of the low-temperature refrigeration module 16 is 4°C. S10. PCR amplification preparation: Use the triaxial moving module 2 to move the variable-distance pipetting module 8, load the tip, and transfer the nucleic acid-magnetic bead binding solution in the magnetic bead suspension plate 1700 to the PCR reaction plate; then, use the triaxial moving module 2 to move the electric gripper 4 to transfer the PCR reaction plate to the sealing module 14 for sealing the PCR reaction plate to avoid cross-contamination of samples. S11. PCR Amplification Process: After sealing the PCR reaction plate, the upper door 46 of the PCR amplification chamber 15 is opened. The electric gripper 4 is moved using the three-axis moving module 2 to transfer the PCR reaction plate from the sealing module 14 to the PCR amplification module 44 in the PCR amplification chamber 15. The upper door 46 is then closed to allow PCR amplification to proceed. After PCR amplification is completed, the front door 50 will be opened to allow the operator to remove the PCR reaction plate. S12. Remove the PCR reaction plate: After completing the above operations, remove the PCR reaction plate from the PCR sample outlet of the device. At this point, nucleic acid extraction and amplification have been completed.
[0070] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.
Claims
1. A fully automated method for nucleic acid extraction from marine bacteria, characterized in that, It includes a fully automated nucleic acid extraction workstation, which is equipped with a sample plate for placing seawater samples, an enrichment plate for enriching bacteria in seawater samples, and a collection plate for collecting the filtration products of the enrichment plate. A pyrolysis solution tank for holding pyrolysis solution, a binding solution plate for holding binding solution, and a magnetic bead plate for holding magnetic beads. Among them, magnetic beads are modified by polymer; It also includes a magnetic sleeve that works in conjunction with the nucleic acid extractor; And detergent trays for holding detergents, and suspension trays for holding suspensions; The lysis buffer includes any one or a mixture of two or more of the following: guanidine isothiocyanate, hexadecyltrimethylammonium bromide, lysozyme, TritiumX-100, proteinase K, and sodium dodecyl sulfonate. The extraction method includes the following steps: (1) Dispose of the lysis buffer, binding solution, detergent, and suspension into the corresponding plate positions, and place the seawater sample into the corresponding sample plate position; (2) The fully automated nucleic acid extraction and amplification workstation shall be operated according to the following steps: 2.1 The enrichment of marine bacteria includes the following steps: 2.1.1 Use a pipette to draw seawater from the sample plate and add it to the enrichment plate and collection plate structure; 2.1.2 Place the enrichment plate and collection plate structure into the centrifuge. After centrifugation, remove the enrichment plate and place it on the collection plate. Reset the collection plate. Use a pipette to remove the waste liquid from the collection plate and discharge it into the waste container. After repeating the above steps several times, marine bacteria were enriched. 2.2 Bacterial lysis includes the following steps: 2.2.1 Use a pipette to take lysis solution from the lysis solution tank and add it to the enrichment plate and collection plate structure; 2.2.2 Place the enrichment plate and collection plate structure in a constant temperature incubator for heating; 2.2.3 After heating, place the enrichment plate and the collection plate into a centrifuge for centrifugation. 2.2.4 After centrifugation, remove the enrichment plate and place it in the waste storage tank, and remove the collection plate and put it back in its original position; 2.2.5 Use a pipette to remove the liquid from the collection plate and transfer it to the binding plate to obtain bacterial lysis products; 2.3 Bacterial nucleic acid purification includes the following steps: 2.3.1 During the heating process in step 2.2.2, the magnetic sleeve, magnetic bead plate, washing liquid plate, and suspension liquid plate are placed into the nucleic acid extractor using electric grippers; 2.3.2 Place the binding liquid plate from step 2.2.4 into a fully automated nucleic acid extractor for nucleic acid extraction; 2.3.3 After nucleic acid extraction, return the binding liquid plate, magnetic sleeve, magnetic bead plate, washing liquid plate and suspension plate to their corresponding positions.
2. The fully automated nucleic acid extraction method for marine bacteria according to claim 1, characterized in that, The lysis buffer comprises the following components: The following were used: 4M guanidine isothiocyanate, 5 mg / mL lysozyme, 2% TritiumX-100, and 40 μg / mL proteinase K. Sodium dodecyl sulfonate with a mass fraction of 1%; The total volume of the lysis solution is 500 μL.
3. The fully automated nucleic acid extraction method for marine bacteria according to claim 1, characterized in that, The binding solution is 200 μL of isopropanol with a mass fraction of 30%.
4. The fully automated nucleic acid extraction method for marine bacteria according to claim 1, characterized in that, The suspension includes a nucleic acid protectant, which is Tris-HCl at a concentration of 10 mM and EDTA at a concentration of 1 mM.
5. The fully automated nucleic acid extraction method for marine bacteria according to claim 1, characterized in that, The fully automated nucleic acid extraction workstation is equipped with a first washing liquid plate, a second washing liquid plate and a third washing liquid plate, and the first washing liquid plate contains Class I detergent. The second washing liquid tray contains Class II detergent, and the third washing liquid tray contains Class III detergent. The composition of the Class I detergent is as follows: Class I detergent includes any one or a mixture of two or more of guanidine hydrochloride, sodium acetate, acetic acid, and ethanol with a mass fraction of 55%. The composition formula of Class II detergents is as follows: Class II detergents include any one or a mixture of two of sodium acetate and 75% ethanol by mass. The composition of Class III detergents is as follows: Class III detergents include 70%-80% ethanol by mass.
6. The fully automated nucleic acid extraction method for marine bacteria according to claim 1, characterized in that, The magnetic beads are Fe3O4 with a surface modified by a polymer. The surface is positively charged. When the magnetic beads are mixed with a binding solution containing nucleic acid, the positively charged magnetic beads adsorb the negatively charged nucleic acid. The polymer includes any one of the following: polydiallyldimethylammonium chloride (PDDA), 3-aminopropyltriethoxysilane, or chitosan.
7. The fully automated nucleic acid extraction method for marine bacteria according to claim 1, characterized in that, The fully automated nucleic acid extraction workstation also includes a nucleic acid extraction module, which comprises a nucleic acid extraction module base, a rotating platform, and two lifting modules. The rotating platform is rotatably connected to the nucleic acid extraction module base, and the rotating platform has multiple workstations. Each workstation has a functional component corresponding to the process. A rotary drive motor is fixedly connected inside the nucleic acid extraction module base, and the output end of the rotary drive motor is connected to the rotating platform for transmission, and can drive the rotating platform to rotate around its axis. A magnetic rod sleeve buckle is fixedly connected to the moving end of one lifting module, and a magnetic rod frame is fixedly connected to the moving end of the other lifting module. The magnetic rod frame can face or move away from the magnetic rod sleeve buckle so that the magnetic rod sleeve buckle can be engaged or disengaged from the magnetic rod frame. The two lifting modules are designated as Lifting Module 1 and Lifting Module 2. An annular baffle is fixedly connected to the base of the nucleic acid extraction module. Lifting Module 1 includes an oscillating rear plate, a drive motor 1, a lead screw 1, and an oscillating front plate. The oscillating rear plate and the oscillating front plate are slidably fitted together. The oscillating rear plate is fixedly connected to the inner wall of the annular baffle. A drive motor 1 is fixedly connected to the oscillating rear plate. A lead screw 1 is fixedly connected to the output end of the drive motor 1. The oscillating front plate is screwed onto the lead screw 1. The bottom of the oscillating front plate is fixedly connected to the magnetic rod sleeve buckle. The lifting module also includes a slide rail. Two parallel slide rails are fixedly connected to one end of the oscillating rear plate facing the oscillating front plate, and are slidably connected to the oscillating front plate through the slide rails. The front plate of the oscillation is provided with a second lifting module, which includes a second drive motor and a second lead screw. The second drive motor is fixedly connected to the top of the front plate of the oscillation, and the output end of the second drive motor is connected to the second lead screw. The second lead screw is screwed to the magnetic rod frame.
8. The fully automated nucleic acid extraction method for marine bacteria according to claim 7, characterized in that, The fully automated nucleic acid extraction workstation also includes the main chassis and the operating table installed inside the main chassis; The operating table is equipped with a three-axis moving module, a centrifugation module, a sealing module, a low-temperature refrigeration module, a PCR amplification chamber, and a clamp-type incubation module; It also includes plate holders, pipette tip holders, and reagent holders installed on the operating table; The plate holder is used to place PCR deep well plates, PCR reaction plates and reagent deep well plates, the pipette tip holder is used to place tip holders, the reagent holder is used to place reagent solution plates, and the waste storage compartment is used to collect waste deep well plates and tip holders.
9. The fully automated nucleic acid extraction method for marine bacteria according to claim 8, characterized in that, The three-axis moving module includes a left moving module and a right moving module with identical structures. The left moving module includes an X-axis linear module, a Y-axis linear module, and a Z-axis linear module. The Z-axis linear module is fixedly connected to the moving end of the Y-axis linear module, and the Y-axis linear module is fixedly connected to the moving end of the X-axis linear module. The X-axis linear module is fixed inside the main body of the chassis. A variable pitch pipetting module is fixedly connected to the moving end of the Z-axis linear module of the left moving module. An ADP pipette is connected to the moving end of the variable pitch pipetting module. An electric gripper and an identification module are fixedly connected to the output end of the Z-axis linear module of the right moving module. The recognition module includes a CCD camera, a lens, and a light source. The CCD camera is fixed at the moving end of the Z-axis linear module of the right moving module, the lens is fixed at the front end of the CCD camera, and the light source is located between the CCD camera and the lens.
10. The fully automated nucleic acid extraction method for marine bacteria according to claim 9, characterized in that, The PCR amplification chamber includes a chamber body, an upper door, a front door, a PCR amplification module, and an amplification module entry / exit conveyor module. The chamber body has openings adapted to the upper door and the front door. The top of the chamber body has an opening and closing upper door, and the front of the chamber body has an opening and closing front door. The upper door is slidably connected to the chamber body via an upper door slide rail. A synchronous belt drive module is fixedly connected to the inner wall of the chamber body. The synchronous belt drive module can drive the front door to open and close. The front door is driven to the chamber body via the amplification module entry / exit slide rail module and can drive the front door to open and close. The synchronous belt drive module includes an upper door drive motor, a synchronous belt, and two synchronous pulleys. The output end of the upper door drive motor is connected to one synchronous pulley and can drive the synchronous pulley to rotate. The synchronous pulley is connected to the other synchronous pulley through the synchronous belt. The other synchronous pulley is rotatably connected to the cabin body.