Real-time analysis system for interaction of multiple microorganisms

Abstract

The invention discloses a real-time analysis system for interaction of various microorganisms, which comprises a shell, and a connecting plate, an upper cover, a control mainboard, a sample moving device, a sample loading device, a temperature control device, a fluorescence detection device and a fixed frame which are arranged in the shell, and belongs to the field of microorganism detection. And the connecting plate, the fluorescence detection device and the temperature control device are all connected with the control mainboard. An external computer can be connected to the connecting plate access system in a wired manner to obtain detection data; the fluorescence detection device is used for carrying out fluorescence detection on a microorganism sample under the control of the control main board; the sample moving device is used for realizing accurate positioning of microorganism sample fluorescence detection under the control of the control mainboard; the temperature control device is used for providing an environment suitable for microbial incubation under the control of the control main board. The interaction research of microorganisms marked by two or three different fluorescent protein genes is realized. The system is high in automation degree, can be used for detecting various microorganisms in real time on line, and analyzing interspecific relationships of interspecific coexistence, intergrowth, symbiosis, antagonism, competition, parasitism, predation and the like among the microorganisms, and the detection efficiency is improved.

Description

Technical Field

[0001] The invention relates to the field of microbial detection, in particular to a real-time analysis system for the interaction of multiple microorganisms. Background Art

[0002] There are various complex relationships between microorganisms, including "interspecific coexistence", "mutualism", "symbiosis", "antagonism", "competition", "parasitism" and "predation". "Interspecific coexistence" is manifested as the peaceful coexistence of microorganisms, such as lactobacillus and streptococci; "mutualism" is a loose association between microorganisms, which may benefit both parties, such as ammonifying bacteria and nitrifying bacteria; "symbiosis" is manifested as the formation of special symbionts between microorganisms, resulting in new structures and physiological divisions of labor, including mutualistic symbiosis and partial symbiosis, such as lichens formed by algae and fungi; "antagonism" is a phenomenon in which a microorganism inhibits or kills another microorganism through metabolic products or environmental changes; "competition" occurs for limited resources, resulting in the inhibition of the growth of both, and is the driving force for the development and evolution of microorganisms; "parasitism" is manifested as one organism obtaining nutrients from another organism, which is usually harmful to the host, such as bacteriophages and bacteria; "predation" is a microorganism directly swallowing another microorganism, such as the predation of bacteria by protozoa, which plays an important role in controlling population density and ecological food chains.

[0003] At present, the research techniques used for the interaction between microorganisms include fluorescence microscopy and microbial co-cultivation. Fluorescence microscopy is a method that uses fluorescent dyes to label microorganisms, so that experimenters can observe the composition and activities of microorganisms. Microbial co-cultivation is a method that studies the interaction between microorganisms by co-culturing different types of microorganisms in the same culture medium. The results of these two methods are intuitive, but the experimental steps are complicated, the requirements for experimenters are high, and real-time online monitoring cannot be achieved, and the detection efficiency is low. Summary of the invention

[0004] The purpose of the present invention is to provide a real-time analysis system for multiple microbial interactions to solve the problem that the existing technology cannot realize real-time online monitoring of multiple microbial interactions and the detection efficiency is low. The core of this technology is to use computers (including microprocessors), sensors, automatic control and fluorescence detection technology to realize real-time online detection of multiple microbial interactions and improve detection efficiency.

[0005] In order to solve the above problems, the specific implementation scheme of this article is as follows:

[0006] The present invention provides a real-time analysis system for the interaction of multiple microorganisms, comprising a shell, and a connecting plate, an upper cover, a control main board, a sample moving device, a sample loading device, a temperature control device, a fluorescence detection device and a U-shaped fixing frame arranged in the shell; a hatch is arranged on the shell.

[0007] Specifically, the connection board includes a serial cable interface and a power supply interface and realizes a wired electrical connection with the control mainboard with a microprocessor (MCU). An external computer can be connected to the serial cable interface via a serial cable, and then connected to the control mainboard to obtain detection data.

[0008] Specifically, the sample loading device also includes a heat-conducting copper tube, a temperature sensor, a detection baffle, an exit baffle, a cover plate, and a sample loading base.

[0009] A 96-well plate can be placed in the sample loading device, and the temperature sensor is connected to the control main board to read the real-time temperature of the sample loading base.

[0010] Specifically, the sample moving device also includes a first U-shaped photoelectric switch, a second U-shaped photoelectric switch, a third U-shaped photoelectric switch, a fixed shaft, a movable base, a transmission wheel, a first motor, a driven wheel and a slide rail.

[0011] The first motor is electrically connected to the control main board via a wire. A transmission wheel is mounted on the first motor. The transmission wheel, the driven wheel and the sample loading device can be connected via a track.

[0012] The slide rail includes a loading rail and a fixed rail. The loading device is fixed on the loading rail. The loading rail can slide on the fixed rail, thereby realizing the movement of the sample driven by the first motor.

[0013] The first U-shaped photoelectric switch, the second U-shaped photoelectric switch, and the third U-shaped photoelectric switch are all electrically connected to the control main board by wire; the first U-shaped photoelectric switch and the exit door provide logic for the sample loading device to enter and exit the cabin door; the second U-shaped photoelectric switch and the detection baffle provide initial position information for the sample loading device; the third U-shaped photoelectric switch and the detection baffle provide precise positioning for sample detection.

[0014] Specifically, the fluorescence detection device also includes a second motor, a transmission belt, a first optical fiber holder, a detection rail, a slider, a detection device, an optical fiber and a second optical fiber holder.

[0015] The first optical fiber holder and the second optical fiber holder are capable of installing 8 optical fibers, the second optical fiber holder is installed under the movable base, the detection slide rail and the first optical fiber holder are installed on the U-shaped fixing frame, and the second motor is installed on the first optical fiber holder.

[0016] The detection device is installed on the slider, and the slider is connected to the second motor through the transmission belt. The second motor is electrically connected to the control main board via a wire, and the detection device is driven under the control of the control main board to detect the fluorescent signals in the eight optical fibers.

[0017] The detection device contains multiple optical path systems, which can monitor the interactions between multiple microorganisms at the same time.

[0018] Specifically, a single optical path principle in the detection device includes a light source, a first filter, a dichroic mirror, a first convex lens, an optical fiber, a second filter, a second convex lens and a photodetector.

[0019] The light source is aligned with the center of the first filter, thereby generating an excitation light signal of the required wavelength and reaching the dichroic mirror; the dichroic mirror is installed at a 45-degree angle to the light source end, thereby reflecting the excitation light signal to the first convex lens, and the first convex lens converges the excitation light into the optical fiber.

[0020] The excitation light signal in the optical fiber is transmitted to the bottom of the microbial sample, and the microbial sample generates an emission light signal, which is transmitted to the first convex lens through the optical fiber; the emission light signal is scattered by the first convex lens and reaches the dichroic mirror; the emission light signal passes through the dichroic mirror and reaches the second filter; the emission light signal is filtered by the second filter and reaches the second convex lens for focusing, and the emission light signal after focusing is received by the photodetector.

[0021] Specifically, the detection device is electrically connected to the control mainboard via a wire, thereby transmitting the fluorescence detection data.

[0022] Specifically, the temperature control device also includes a Peltier module and a vacuum pump.

[0023] The Peltier module, the vacuum pump and the control mainboard are connected by wired electricity, and the heat-conducting copper tube, the vacuum pump and the Peltier module are connected by a silicone tube containing a heat-conducting liquid.

[0024] The Peltier module performs cooling or heating under the control of the control mainboard; the vacuum pump promotes the circulation of the heat-conducting liquid between the Peltier module and the heat-conducting copper tube under the control of the control mainboard, thereby realizing the temperature control function.

[0025] Compared with the prior art, the present invention has the following advantages: 1. Through the specially designed fluorescence detection optical system, it can be used to detect and analyze the interactions between various microorganisms.

[0026] 2. An external computer can access the real-time analysis system for multiple microbial interactions and obtain detection data for further analysis.

[0027] 3. By introducing a temperature control device, real-time online analysis of microbial samples can be achieved.

[0028] 4. By introducing sample movement, the detection throughput of the system is increased and the detection efficiency is improved.

[0029] The above features and advantages are described below with reference to specific embodiments and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram of a real-time analysis system for multiple microbial interactions provided in this embodiment.

[0031] Figure 2 A top view of the internal structure of a real-time analysis system for multiple microbial interactions provided in this embodiment.

[0032] Figure 3 A schematic diagram of the bottom structure of a real-time analysis system for multiple microbial interactions provided in this embodiment.

[0033] Figure 4 Schematic diagram of the sample loading device of this embodiment.

[0034] Figure 5 It is a schematic diagram of the bottom exploded view of the sample loading device of this embodiment.

[0035] Figure 6 FIG. 4 is a schematic diagram of a sample moving device according to the present embodiment.

[0036] Figure 7 FIG. 4 is a schematic diagram of a fluorescence monitoring device according to the present embodiment.

[0037] Figure 8 Schematic diagram of the fluorescence detection optical path structure of this embodiment.

[0038] Fig. 9 Schematic diagram of the temperature control device of this embodiment.

[0039] Fig.10 Schematic diagram of the control logic of this embodiment.

[0040] Fig.11 The fluorescence curve shows the interspecies relationship between microorganisms. Implementation

[0041] The present embodiment will be described clearly and completely below in conjunction with the accompanying drawings. Based on the embodiments of this invention, other embodiments obtained by those skilled in the art without implementing innovative work are all within the scope of protection of this invention.

[0042] It should be noted that the terms "first", "second", etc. in the specification and claims of this document and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence.

[0043] like Figures 1 to 3 as well as Fig.10 As shown, the present invention discloses a real-time analysis system 1 for the interaction of multiple microorganisms, including a shell 3, and a connecting plate 100, an upper cover 8, a control main board 9, a sample moving device 20, a sample loading device 30, a temperature control device 40, a fluorescence detection device 50 and a U-shaped fixing frame 60 arranged in the shell; the shell 3 is provided with a hatch 2.

[0044] The connection board 100 includes a serial cable interface 6 and a power interface 7 and realizes a wired electrical connection with a control mainboard 9 with a microprocessor (MCU). An external computer 5 can be connected to the serial cable interface 6 via a serial cable 4, and then connected to the control mainboard 9 to obtain detection data.

[0045] like Figure 4 , Figure 5 and Fig.10 As shown, the sample loading device 30 also includes a heat-conducting copper tube 31 , a temperature sensor 32 , a detection baffle 34 , an exit baffle 33 , a cover plate 35 , and a sample loading base 37 .

[0046] A 96-well plate 36 can be placed in the sample loading device 30 , and the temperature sensor 32 is connected to the control main board 9 for reading the real-time temperature of the sample loading base 37 .

[0047] like Figure 4 , Figure 6 ,and Fig.10 As shown, the sample moving device 20 further includes a first U-shaped photoelectric switch 21 , a second U-shaped photoelectric switch 22 , a third U-shaped photoelectric switch 23 , a fixed shaft 24 , a moving base 26 , a transmission wheel 28 , a first motor 29 , a driven wheel 27 and a slide rail 210 .

[0048] The first motor 29 is electrically connected to the control main board 9 by wire. A transmission wheel 28 is mounted on the first motor 29. The transmission wheel 28 and the driven wheel 27 are connected to the sample loading device 30 by a crawler belt.

[0049] The slide rail 210 includes a loading rail 212 and a fixed rail 211 . The loading device 30 is fixed on the loading rail 212 . The loading rail 212 can slide on the fixed rail 211 , thereby realizing the movement of the sample driven by the first motor 29 .

[0050] The first U-shaped photoelectric switch 21, the second U-shaped photoelectric switch 22, and the third U-shaped photoelectric switch 23 are all electrically connected to the control main board 9. The first U-shaped photoelectric switch 21 and the exit baffle 33 provide logic for the sample loading device 30 to enter and exit the cabin door 2; the second U-shaped photoelectric switch 22 and the detection baffle 34 provide initial position information for the sample loading device 30; the third U-shaped photoelectric switch 23 and the detection baffle 34 provide precise positioning for sample detection.

[0051] like Figure 7 and Fig.10 As shown, the fluorescence detection device 50 also includes a second motor 51 , a transmission belt 52 , a first optical fiber holder 53 , a detection rail 54 , a slider 59 , a detection device 57 , an optical fiber 58 and a second optical fiber holder 25 .

[0052] The first optical fiber holder 53 and the second optical fiber holder 25 can install eight optical fibers 58 . The second optical fiber holder 25 is installed under the movable base 26 . The detection slide rail 54 and the first optical fiber holder 53 are installed on the U-shaped fixing frame 60 . The second motor 51 is installed on the first optical fiber holder 53 .

[0053] The detection device 57 is installed on the slider 59, and the slider 59 is connected to the second motor 51 through the transmission belt 52. The second motor 51 is electrically connected to the control main board 9 by wires. Under the control of the control main board 9, the detection device 57 is driven to detect the fluorescent signals in the eight optical fibers 58.

[0054] The detection device 57 includes two sets of optical path systems, two light source installation ports 55 and two photoelectric sensor installation ports 56, which can realize the simultaneous monitoring of the interaction between two microorganisms.

[0055] like Figure 8 As shown, a single optical path principle in the detection device 57 includes a light source 71 , a first filter 72 , a dichroic mirror 73 , a first convex lens 74 , an optical fiber 58 , a second filter 76 , a second convex lens 77 and a photodetector 78 .

[0056] The light source 71 is aligned with the center of the first filter 72, thereby generating an excitation light signal of the required wavelength, which reaches the dichroic mirror 73; the dichroic mirror 73 is installed at a 45-degree angle to the end of the light source 71, thereby reflecting the excitation light signal to the first convex lens 74, and the first convex lens 74 converges the excitation light into the optical fiber 58.

[0057] The excitation light signal in the optical fiber is transmitted to the bottom of the microbial sample 75, and the microbial sample 75 generates an emission light signal, which is transmitted to the first convex lens 74 via the optical fiber 58; the emission light signal is scattered by the first convex lens 74 and reaches the dichroic mirror 73; the emission light signal passes through the dichroic mirror 73 and reaches the second filter 76; the emission light signal is filtered by the second filter 76 and reaches the second convex lens 77 for focusing, and the emission light signal after focusing is received by the photodetector 78.

[0058] like Fig.10 As shown, the detection device 57 is electrically connected to the control main board 9 via a wire, thereby transmitting the fluorescence detection data.

[0059] like Fig. 9 As shown, the temperature control device 40 further includes a Peltier module 42 and a vacuum pump 41 .

[0060] The Peltier module 42, the vacuum pump 41 and the control mainboard 9 are connected by wired electricity, and the heat-conducting copper tube 31, the vacuum pump 41 and the Peltier module 42 are connected by a silicone tube containing a heat-conducting liquid.

[0061] The Peltier module 42 performs cooling or heating under the control of the control mainboard 9 ; the vacuum pump 41 promotes the circulation of the heat-conducting liquid between the Peltier module 42 and the heat-conducting copper tube 31 under the control of the control mainboard 9 , thereby realizing the temperature control function.

[0062] The detection process is set up as follows: first, the first column of 8 holes of the 96-well plate 36 are aligned with the second optical fiber holder 25, and the detection device 57 moves to detect the fluorescent signals in the 8 optical fibers 58; after the detection is completed, the second column of 8 holes of the 96-well plate 36 are switched to align with the second optical fiber holder 25, and so on, until all the samples of the entire 96-well plate are detected, and the sample loading device 30 returns to the initial position marked by the second U-shaped photoelectric switch 22.

[0063] Select a suitable fluorescent protein (such as green fluorescent protein-GFP, red fluorescent protein-RFP, blue fluorescent protein-BFP), and select it according to the wavelength and fluorescence intensity required by the experiment. Clone the gene of the fluorescent marker (such as GFP gene, RFP gene, or BFP gene) into an appropriate expression vector, usually by connecting it to the specific promoter and maintenance element of the target bacteria. Introduce the cloned expression vector into the target bacteria, usually by electroporation, hot zinc method or chemical method to introduce the fluorescent marker into the bacterial cells (Enterobacter, Pseudomonas aeruginosa, Salmonella, Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, etc.). These methods can express the gene of the fluorescent marker in the bacterial cells. Use selective culture medium or antibiotics and other screening methods to screen out bacteria that have been successfully transformed and express fluorescent markers. Then, culture the transformed bacteria under appropriate culture conditions to obtain a sufficient number of bacteria with fluorescent markers. Mix these fluorescent protein gene-tagged microorganisms in pairs to study the interaction between different microorganisms.

[0064] like Fig.11 As shown, the detection results showing the shape of curve 81 can be interpreted as the existence of interspecific coexistence, mutualism or symbiosis between the microorganisms; the detection results showing the shape of curve 82 can be interpreted as the existence of antagonistic interspecific relationship between the microorganisms; the detection results showing the shape of curve 83 can be interpreted as the existence of competitive interspecific relationship between the microorganisms; the detection results showing the shape of curve 84 can be interpreted as the existence of parasitic interspecific relationship between the microorganisms; the detection results showing the shape of curve 85 can be interpreted as the existence of predation interspecific relationship between the microorganisms.

[0065] This article uses specific embodiments to illustrate the principles and implementation methods of this article. This narrative style of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A real-time analysis system for multiple microbial interactions, characterized in that: The invention comprises a shell, and a connecting plate, an upper cover, a control main board, a sample moving device, a sample loading device, a temperature control device, a fluorescence detection device and a U-shaped fixing frame arranged in the shell; a hatch is arranged on the shell.

2. The system according to claim 1, characterized in that The connection board includes a serial cable interface and a power supply interface and realizes a wired electrical connection with the control main board with a microprocessor (MCU). An external computer can be connected to the serial cable interface via a serial cable, and then connected to the control main board to obtain detection data.

3. The system according to claim 1, characterized in that The sample loading device also includes a heat-conducting copper tube, a temperature sensor, a detection baffle, an exit baffle, a cover plate, and a sample loading base. A 96-well plate can be placed in the sample loading device, and the temperature sensor is connected to the control main board to read the real-time temperature of the sample loading base.

4. The system according to claim 1, characterized in that The sample moving device also includes a first U-shaped photoelectric switch, a second U-shaped photoelectric switch, a third U-shaped photoelectric switch, a fixed shaft, a movable base, a transmission wheel, a first motor, a driven wheel and a slide rail. The first motor is electrically connected to the control main board by wires, and a transmission wheel is installed on the first motor. The transmission wheel, the driven wheel and the sample loading device can be connected by tracks. The slide rail includes a loading rail and a fixed rail. The loading device is fixed on the loading rail, and the loading rail can slide on the fixed rail, thereby realizing the movement of the sample driven by the first motor.

5. The device according to claim 4, characterized in that The first U-shaped photoelectric switch, the second U-shaped photoelectric switch, and the third U-shaped photoelectric switch are all electrically connected to the control main board by wire; the first U-shaped photoelectric switch and the exit door provide logic for the sample loading device to enter and exit the cabin door; the second U-shaped photoelectric switch and the detection baffle provide initial position information for the sample loading device; the third U-shaped photoelectric switch and the detection baffle provide precise positioning for sample detection.

6. The system according to claim 1, characterized in that The fluorescence detection device also includes a second motor, a transmission belt, a first optical fiber holder, a detection slide rail, a slider, a detection device, an optical fiber and a second optical fiber holder. The first optical fiber holder and the second optical fiber holder can install 8 optical fibers, the second optical fiber holder is installed under the mobile base, the detection slide rail and the first optical fiber holder are installed on the U-shaped fixing frame, and the second motor is installed on the first optical fiber holder. The detection device is installed on the slider, and the slider is connected to the second motor through the transmission belt. The second motor is electrically connected to the control mainboard by wire, and under the control of the control mainboard, the detection device is driven to detect the fluorescence signals in the 8 optical fibers. The detection device contains multiple optical path systems, which can monitor the interactions between multiple microorganisms at the same time.

7. The device according to claim 6, characterized in that The single optical path principle in the detection device includes a light source, a first filter, a dichroic mirror, a first convex lens, an optical fiber, a second filter, a second convex lens and a photodetector. The detection device is electrically connected to the control mainboard via a wire, thereby transmitting fluorescence detection data.

8. The system according to claim 1, characterized in that The temperature control device also includes a Peltier module and a vacuum pump. The Peltier module, the vacuum pump and the control mainboard are connected by wired electricity. The heat-conducting copper tube, the vacuum pump and the Peltier module are connected by a silicone tube containing a heat-conducting liquid.

9. The device according to claim 8, characterized in that The Peltier module performs cooling or heating under the control of the control mainboard; the vacuum pump promotes the circulation of the heat-conducting liquid between the Peltier module and the heat-conducting copper tube under the control of the control mainboard, thereby realizing the temperature control function.

10. The system according to claim 1, characterized in that Select a suitable fluorescent protein (such as green fluorescent protein-GFP, red fluorescent protein-RFP, blue fluorescent protein-BFP), and select it according to the wavelength and fluorescence intensity required by the experiment. The gene of the fluorescent marker (such as GFP gene, RFP gene, or BFP gene) is cloned into an appropriate expression vector, usually by connecting it to the specific promoter and maintenance element of the target bacteria. The cloned expression vector is introduced into the target bacteria, and the fluorescent marker is usually introduced into the bacterial cell (Enterobacter, Pseudomonas aeruginosa, Salmonella, Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, etc.) by electroporation, hot zinc method or chemical method. These methods can express the gene of the fluorescent marker in the bacterial cell. Use selective culture medium or antibiotics and other screening methods to screen out bacteria that have been successfully transformed and express fluorescent markers. Then, culture the transformed bacteria under appropriate culture conditions to obtain a sufficient number of bacteria with fluorescent markers. Mix these fluorescent protein gene-tagged microorganisms in pairs, and the system monitors the changes of different fluorescent signals in real time to study the interaction relationship between different microorganisms.

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