Microorganism sampling device and method applied to deep sea gene sequencing instrument

Abstract

The invention relates to the field of deep-sea gene sequencing, in particular to a microorganism sampling device and method applied to a deep-sea gene sequencing instrument.The microorganism sampling device is characterized in that when a through valve is located at a first position, a seawater introduction pump, the through valve, a pressure-resistant filtering and enriching device and the through valve are sequentially communicated; the seawater introduction pump is started, the seawater sequentially flows through the through valve, the pressure-resistant filtering and enriching device and the through valve and then is discharged, and when the seawater flows through, the pressure-resistant filtering and enriching device intercepts and enriches microorganisms in the seawater to form a microorganism sample; when the through valve is located at the second position, the plunger pump, the through valve, the pressure-resistant filtering and enriching device and the through valve are sequentially communicated; when the pressure sensor monitors that the pressure of the microorganism sample is reduced to the normal pressure environment, the plunger pump is started, and the microorganism sample is transmitted to an external gene sequencing instrument through the through valve for gene sequencing. The problems of insufficient sample degradation and enrichment and high-voltage transmission in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the field of deep-sea gene sequencing, and in particular to a microbial sampling device and method applied to a deep-sea gene sequencing instrument. Background Art

[0002] The deep sea contains rich microbial resources. These microorganisms not only play a vital role in the deep-sea ecosystem, but also have important significance for the origin of life, the evolution of the earth's environment and the development of natural products. With the advancement of science and technology, gene sequencing technology has gradually become an important means to explore microbial diversity, function and their interrelationships. However, due to the particularity of the deep-sea environment, traditional microbial sampling and analysis methods have many challenges; the in situ application of existing gene sequencing technology in the deep sea also faces many technical difficulties, especially in terms of adaptability under high-pressure environments and the effective collection and enrichment of microbial samples.

[0003] Foreign scientific research institutions have conducted pioneering work in deep-sea microbial sampling and analysis technology. For example, Marc Garel et al. from Aix-Marseille University in France designed a pressure-retaining sampler PRS (Pressure-Retaining Sampler), which can maintain the pressure environment in the sea and achieve microbial sampling; the Monterey Bay Oceanographic Institution (MBVRI) in the United States designed an environmental sample processor ESP (Environmental Sample Processor) and used it to complete quantitative PCR and other molecular determinations in situ. These technologies and equipment provide reliable technical support for deep-sea ecology and genomics research. Wang Yong's team at the Institute of Deep-sea Science and Engineering of the Chinese Academy of Sciences independently developed a set of microbial filtration and fixation devices ISMIFF (In Situ Microbial Filtration and Fixation), which successfully enriched microorganisms in deep-sea environments and fixed microbial RNA and DNA, providing high-quality samples for subsequent genetic analysis.

[0004] However, most of the current deep-sea microbial sampling devices have not yet achieved in situ gene sequencing. Although the technical means of the device can maintain the deep-sea in situ state or effectively fix the microbial samples, in the process of recovering samples from the deep sea to the laboratory, due to factors such as long transportation time and changes in environmental conditions, nucleic acids and proteins still face the problem of degradation, thus affecting the results of microbial gene sequencing.

[0005] With the continuous development of gene sequencing instruments, the existing technology has been able to achieve high-throughput and high-precision sequencing under low microbial biomass conditions. However, in the deep-sea environment, the content of microorganisms is usually low, far from meeting the detection limit of amplification. Therefore, efficient microbial enrichment techniques are still needed to ensure that sufficient sample volume can be obtained to support subsequent operations. At the same time, with the continuous progress of technology, gene sequencing instruments have been miniaturized and can be placed in a pressure-resistant sealed cabin, enabling in-situ operation in the deep-sea environment. This provides new possibilities for in-situ deep-sea gene sequencing and enables direct data collection in the deep-sea environment. However, due to the complex operations involved in the work of sequencing instruments, such as optical path analysis and laser excitation, the device accuracy requirements for these operations are relatively high and the pressure-bearing design is relatively difficult, which makes the sequencing work unable to be directly carried out under the high-pressure environment of the deep sea. Therefore, although through technologies such as pressure-resistant design and sealed cabins, gene sequencing instruments can work in-situ in the deep sea, the sample is still in a high-pressure state after being collected and enriched from the deep sea. Therefore, how to efficiently and safely transfer the sample from the high-pressure environment to the atmospheric pressure environment for subsequent analysis is still a technical problem to be solved urgently. Summary of the Invention

[0006] An embodiment of the present invention provides a microbial sampling device and method applied to a deep-sea gene sequencing instrument to at least solve the problems of sample degradation, insufficient enrichment, and high-pressure transmission in the prior art.

[0007] According to an embodiment of the present invention, there is provided a microbial sampling device applied to a deep-sea gene sequencing instrument, including: a seawater inlet pump, a switching valve, a pressure sensor, a pressure-resistant filtration and enrichment device, and a plunger pump;

[0008] The seawater inlet pump, the pressure-resistant filtration and enrichment device, and the plunger pump are all connected to the switching valve, and the switching valve can perform position switching;

[0009] When the switching valve is in the first position, the seawater inlet pump, the switching valve, the pressure-resistant filtration and enrichment device, and the switching valve are sequentially connected in series; when the seawater inlet pump is started, seawater flows through the switching valve, the pressure-resistant filtration and enrichment device, and the switching valve in sequence and then is discharged. When the seawater flows through, the pressure-resistant filtration and enrichment device intercepts and enriches the microorganisms in the seawater to form a microbial sample;

[0010] When the switching valve is in the second position, the plunger pump, the switching valve, the pressure-resistant filtration and enrichment device, and the switching valve are sequentially connected in series; the pressure sensor is connected to the pressure-resistant filtration and enrichment device to monitor the pressure environment of the microbial sample; when the pressure sensor monitors that the microbial sample is depressurized to the atmospheric pressure environment, the plunger pump is started to transfer the microbial sample to an external gene sequencing instrument through the switching valve for gene sequencing.

[0011] Furthermore, the pressure-resistant filtration and enrichment device uses a filter membrane with a small pore diameter, and the filter membrane is fixed by two pressing plates and clips, and then fixed in the microbial sampling device through a fixed iron ring.

[0012] Furthermore, the communication valve includes a two-position six-way valve.

[0013] Furthermore, the two-position six-way valve includes a rotor and a stator. The position switching of the two-position six-way valve is achieved by the movement of the rotor. The rotor rotates 60 degrees each time, and the positions include a first position and a second position. The rotor switches between the first position and the second position.

[0014] Furthermore, all parts of the microbial sampling device that come into contact with the deep-sea environment are made of high-pressure-resistant materials. At the same time, the communication valve, pressure sensor, pressure-resistant filtration and enrichment device, and plunger pump in the microbial sampling device are placed in a high-pressure-resistant sealed cavity.

[0015] Furthermore, the microbial sampling device further includes a pushing device. The pushing device pushes PBS solution or deionized water to flow through the plunger pump and the pressure-resistant filtration and enrichment device to wash the microorganisms in front of the membrane.

[0016] Furthermore, the communication valve includes a three-way valve and a four-way valve.

[0017] Furthermore, the seawater introduction pump includes a peristaltic pump, a diaphragm pump or a gear pump.

[0018] Furthermore, the pore diameter of the filter membrane is 0.22 μm.

[0019] According to another embodiment of the present invention, there is provided a microbial sampling method applied to a deep-sea gene sequencing instrument, including:

[0020] When the communication valve is in the first position, the seawater introduction pump, the communication valve, the pressure-resistant filtration and enrichment device, and the communication valve are connected in sequence; the seawater introduction pump is started, and the seawater flows through the communication valve, the pressure-resistant filtration and enrichment device, and the communication valve in sequence and then is discharged. When the seawater flows through, the pressure-resistant filtration and enrichment device intercepts and enriches the microorganisms in the seawater to form a microbial sample;

[0021] When the communication valve is in the second position, the plunger pump, the communication valve, the pressure-resistant filtration and enrichment device, and the communication valve are connected in sequence; when the pressure sensor monitors that the microbial sample is depressurized to the ambient pressure environment, the plunger pump is started, and the microbial sample is transmitted to an external gene sequencing instrument through the communication valve for gene sequencing.

[0022] The microbial sampling device and method applied to deep-sea gene sequencing instruments in the embodiments of the present invention are deployed on a deep-sea in-situ experimental platform to achieve in-situ collection and enrichment of microorganisms in the deep-sea high-pressure environment. Subsequently, the enriched samples are transmitted to the gene sequencing instruments in the deep-sea in-situ. Combined with the gene sequencing instruments, the present invention can complete efficient sampling and sequencing tasks without unattended operation, realizing automated analysis and integrated collaborative work of deep-sea microbial sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present invention and form a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0024] Figure 1 is a schematic structural diagram of the device in the present invention;

[0025] Figure 2 is a schematic position diagram of the six-way valve in the present invention;

[0026] Figure 3 is a schematic diagram of the sampling enrichment flow path and the sample backflush transmission flow path in the present invention;

[0027] Figure 4 is a schematic internal structure diagram of the pressure-resistant filter in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above drawings are used to distinguish similar objects and do not necessarily need to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices.

[0030] The present invention aims to provide a device and method capable of efficiently collecting and enriching microbial samples in a deep-sea high-pressure environment and safely transporting them to an atmospheric pressure environment for gene sequencing, so as to solve the problems of sample degradation, insufficient enrichment, and high-pressure transmission in the prior art.

[0031] As Figure 1 shown is the structural diagram of the overall device of the present invention, which mainly includes a peristaltic pump 2, a two-position six-way valve 3, two three-way connectors 4, two pressure sensors 5, a pressure-resistant filtration and enrichment device 6, a passage 1 of a gene sequencing instrument, and a plunger pump 8 for transporting samples and a pushing device 7. The design of the entire device focuses on stable operation in a high-pressure environment. All parts in contact with the deep-sea environment are made of high-pressure-resistant materials, and at the same time, the devices of key parts are placed in a high-pressure-resistant sealed cavity to ensure the long-term stable operation of the entire device.

[0032] As Figure 2 shown is the two-position six-way valve 3. The two-position six-way valve 3 includes a rotor 31 and a stator 32. The position switching of the two-position six-way valve 3 is achieved by the movement of the rotor 31. Each time the rotor 31 rotates 60 degrees, the positions include position 33 and position 34, and the rotor 31 switches between position 33 and position 34.

[0033] When realizing microbial sampling and enrichment, as Figure 3 shown in the sampling and enrichment flow path, control the two-position six-way valve 3 to be in position 33, and at the same time control the peristaltic pump 2 to work to introduce seawater samples into the entire device. After the seawater passes through the peristaltic pump 2, it flows through the two-position six-way valve 3, the three-way connector 4, and the pressure-resistant filtration and enrichment device 6 in sequence for the interception and enrichment of microorganisms.

[0034] As Figure 4 shown, the pressure-resistant filtration and enrichment device 6 uses a filter membrane 62 with a pore size of 0.22um, which can effectively intercept microorganisms in seawater. At the same time, the filter membrane 62 is fixed by two pressing plates 63, and then fixed in the device through a fixing iron ring 61. Under the continuous operation of the peristaltic pump 2, when seawater flows through the filter membrane 62, microorganisms are intercepted on the filter membrane 62, and the enrichment process is gradually completed. As the microbial samples are enriched, enough microbial samples are stored on the filter membrane 62 to support subsequent gene sequencing analysis. When the enrichment process is completed, as Figure 3 shown in the transmission flow path, the two-position six-way valve 3 is switched to position 44, and the front pressure sensor 5 monitors whether the microbial sample is depressurized to the atmospheric pressure environment. After depressurization, the microbial sample is transported to the gene sequencing instrument through the passage 1 by the plunger pump 8 for further analysis. In this process, deionized water or PBS solution is injected into the back end of the filter membrane 62 to push the enriched microbial samples into the gene sequencing instrument to complete the subsequent analysis process.

[0035] Deploy the device of the present invention on the deep - sea in - situ experimental platform to achieve the collection and enrichment of in - situ microorganisms in the deep - sea high - pressure environment. Subsequently, transfer the enriched samples to the gene sequencing instrument in the deep - sea in - situ. The combination of the present invention and the gene sequencing instrument can complete the high - efficiency sampling and sequencing tasks without unattended operation, realizing the automated analysis and integrated collaborative work of deep - sea microbial sequencing.

[0036] The advantages or beneficial effects of the present invention are as follows:

[0037] (1) By using a 0.22 - μm pore - size filter membrane 62 for microbial enrichment, the present invention can effectively intercept microorganisms in seawater, ensuring that the concentration of microorganisms in the sample reaches the level required for gene sequencing. At the same time, the linkage control of the peristaltic pump 2 and the two - position six - way valve 3 ensures the stable flow of seawater samples during the enrichment process, effectively avoiding the loss of microorganisms during transmission and the problem of sample degradation.

[0038] (2) The present invention realizes pressure reduction through the flow - path switching of the two - position six - way valve 3 after enrichment, ensuring that the sample can be reduced from the high - pressure environment to normal pressure, providing a reliable guarantee for subsequent sample transfer and gene sequencing.

[0039] (3) The present invention pushes PBS solution or deionized water from the back end of the filter membrane 62 through the pushing device 7 and the plunger pump 8 to wash the microorganisms in front of the membrane, ensuring the transmission of microbial samples and meeting the sample requirements of the gene sequencing instrument.

[0040] (4) Through the automated valve control and pump system, the present invention simplifies the operations in the sample collection and transmission process, avoids the errors that may be brought by manual operations, and can collect and enrich samples without unattended operation, further improving the reliability and efficiency of the system.

[0041] The alternative technical solutions of the present invention are as follows:

[0042] 1. Use other types of valves (such as combinations of three - way valves, four - way valves, etc.) to replace the two - position six - way valve to achieve the functions of pipeline switching and pressure reduction.

[0043] 2. Use other types of pumps (such as diaphragm pumps or gear pumps) to replace the peristaltic pump to achieve seawater sample injection and sample transmission.

[0044] The technical key points and points to be protected of the present invention are as follows:

[0045] 1. By controlling the switching of different positions through the two - position six - way valve 3, the present invention realizes the pressure reduction of microbial samples in the system and simultaneously realizes the switching of the flow paths of collection and transmission.

[0046] 2. The pressure-resistant filtration and enrichment device 6 uses a filter membrane 62 with a pore size of 0.22 μm, combined with the design of two pressing plate clips 63 and a fixed iron ring 61, which can effectively intercept microorganisms and ensure the fixation of the filter membrane 62 and the stability of the enrichment process.

[0047] 3. A plunger pump 8 is used for precise transmission of the sample, ensuring that the microbial sample can be smoothly and stably transmitted from the sampling system to the gene sequencing instrument after pressure reduction.

[0048] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0049] In the above embodiments of the present invention, the descriptions of each embodiment have their own focuses. For the parts not detailed in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0050] In several embodiments provided by the present application, it should be understood that the disclosed technical content can be implemented in other ways. Among them, the system embodiments described above are only illustrative. For example, the division of units can be a logical function division. In actual implementation, there can be other division methods. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the displayed or discussed coupling or direct coupling or communication connection between each other can be through some interfaces. The indirect coupling or communication connection of units or modules can be in an electrical or other form.

[0051] The units described as separate components may or may not be physically separated. The components displayed as units may or may not be physical units, that is, they can be located in one place or distributed to multiple units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0052] In addition, the functional units in each embodiment of the present invention can be integrated into a processing unit, or each unit can exist physically alone, or two or more units can be integrated into one unit. The above integrated units can be implemented in the form of hardware or in the form of software functional units.

[0053] When the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or all or part of this technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions for causing a computer device (which can be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the methods in various embodiments of the present invention. The aforementioned storage medium includes: various media that can store program codes, such as USB flash drives, read-only memories (ROMs), random access memories (RAMs), mobile hard disks, magnetic disks, or optical discs.

[0054] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A microbial sampling device applied to deep-sea gene sequencing instruments, characterized in that, Comprising: Seawater inlet pump, on-off valve, pressure sensor, pressure-resistant filtration and enrichment device, plunger pump; The seawater inlet pump, pressure-resistant filtration and enrichment device, and plunger pump are all connected to the on-off valve, and the on-off valve can perform position switching; When the on-off valve is in the first position, the seawater inlet pump, on-off valve, pressure-resistant filtration and enrichment device, and on-off valve are connected in sequence; when the seawater inlet pump is started, seawater flows through the on-off valve, pressure-resistant filtration and enrichment device, and on-off valve in sequence and then is discharged. When the seawater flows through, the pressure-resistant filtration and enrichment device intercepts and enriches the microorganisms in the seawater to form a microorganism sample; When the on-off valve is in the second position, the plunger pump, on-off valve, pressure-resistant filtration and enrichment device, and on-off valve are connected in sequence; the pressure sensor is connected to the pressure-resistant filtration and enrichment device to monitor the pressure environment of the microorganism sample; when the pressure sensor monitors that the pressure of the microorganism sample drops to the atmospheric pressure environment, the plunger pump is started, and the microorganism sample is transmitted through the on-off valve to an external gene sequencing instrument for gene sequencing.

2. The microbial sampling device applied to the deep-sea gene sequencing instrument according to claim 1, characterized in that The pressure-resistant filtration and enrichment device uses a filter membrane with a small pore diameter, and the filter membrane is fixed by two pressing plates and clips, and then fixed in the microorganism sampling device by a fixing iron ring.

3. The microbial sampling device applied to the deep-sea gene sequencing instrument according to claim 1, characterized in that, The on-off valve includes a two-position six-way valve.

4. The microbial sampling device applied to the deep-sea gene sequencing instrument according to claim 3, characterized in that, The two-position six-way valve includes a rotor and a stator. The position switching of the two-position six-way valve is realized by the movement of the rotor. The rotor rotates 60 degrees each time, and the positions include the first position and the second position. The rotor switches between the first position and the second position.

5. The microbial sampling device applied to the deep-sea gene sequencing instrument according to claim 1 is characterized in that, All parts of the microorganism sampling device in contact with the deep-sea environment are made of high-pressure-resistant materials, and at the same time, the on-off valve, pressure sensor, pressure-resistant filtration and enrichment device, and plunger pump in the microorganism sampling device are placed in a high-pressure-resistant sealed chamber.

6. The microbial sampling device applied to the deep-sea gene sequencing instrument according to claim 1, characterized in that, The microorganism sampling device further includes a pushing device, and the pushing device pushes PBS solution or deionized water to flow through the plunger pump and pressure-resistant filtration and enrichment device to wash the microorganisms in front of the membrane.

7. The microbial sampling device applied to a deep-sea gene sequencing instrument according to claim 1, wherein The on-off valve includes a three-way valve and a four-way valve.

8. The microbial sampling device applied to a deep-sea gene sequencing instrument according to claim 1, wherein The seawater inlet pump includes a peristaltic pump, a diaphragm pump or a gear pump.

9. The microbial sampling device applied to the deep-sea gene sequencing instrument according to claim 2, wherein The pore diameter of the filter membrane is 0.22 μm.

10. A method for microbial sampling applied to the microbial sampling device according to claim 1, characterized in that, Comprising: When the on-off valve is in the first position, the seawater inlet pump, on-off valve, pressure-resistant filtration and enrichment device, and on-off valve are connected in sequence; when the seawater inlet pump is started, seawater flows through the on-off valve, pressure-resistant filtration and enrichment device, and on-off valve in sequence and then is discharged. When the seawater flows through, the pressure-resistant filtration and enrichment device intercepts and enriches the microorganisms in the seawater to form a microorganism sample; When the on-off valve is in the second position, the plunger pump, on-off valve, pressure-resistant filtration and enrichment device, and on-off valve are connected in sequence; when the pressure sensor monitors that the pressure of the microorganism sample drops to the atmospheric pressure environment, the plunger pump is started, and the microorganism sample is transmitted through the on-off valve to an external gene sequencing instrument for gene sequencing.

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