Streptococcus agalactiae or Neisseria gonorrhoeae electron tube automatic detection system and detection method

Abstract

The present invention provides an automatic detection system and method for Streptococcus agalactiae or Neisseria gonorrhoeae in an electron tube form. Through a "one-tube" visual detection system for Streptococcus agalactiae or Neisseria gonorrhoeae jointly mediated by LAMP and CRISPR-Cas12a, combined with electronic automation technology, the LAMP reaction mixture and the CRISPR-Cas12a reaction mixture are mixed to form a "one-tube" reaction system, realizing the automatic mixing, preheating reaction of various detection reagents and samples, and performing fluorescence detection. A visualization device is used to visually display the detection results. The visual detection of bacteria is achieved by detecting fluorescence signals, which can provide rapid positive or quantitative detection results for users such as patients and improve the efficiency of clinical detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly to an automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae in an all-in-one tube, a method for detecting Streptococcus agalactiae or Neisseria gonorrhoeae, an electronic device, and a computer-readable storage medium. Background Art

[0002] ‌LAMP (Loop-mediated Isothermal Amplification) and CRISPR-Cas12a together constitute a highly sensitive and specific detection method in bacterial detection.

[0003] ‌Role of LAMP (Loop-mediated Isothermal Amplification) in bacterial detection:

[0004] LAMP is a simple, rapid, accurate, and inexpensive gene amplification method, especially suitable for pathogen detection.

[0005] It uses 4 - 6 primers that recognize specific regions of the target DNA and amplifies under isothermal conditions to produce a large amount of DNA products, thus enabling rapid detection of bacteria.

[0006] The LAMP technology is characterized by high sensitivity and specificity, and can amplify extremely trace amounts of DNA within a short time, making the detection more accurate.

[0007] ‌Role of CRISPR-Cas12a in bacterial detection:

[0008] CRISPR-Cas12a is a powerful detection and recognition tool that originated from the adaptive immune systems of bacteria and archaea and can combat attacks from foreign mobile genetic elements. In bacterial detection, CRISPR-Cas12a can bind to specific crRNA, recognize and cleave the target DNA, thereby achieving accurate detection of bacteria.

[0009] ‌Combined application of LAMP and CRISPR-Cas12a:

[0010] Combining the LAMP and CRISPR-Cas12a technologies can achieve highly sensitive and specific detection of bacteria. For example, by amplifying the target DNA of bacteria using LAMP and then using CRISPR-Cas12a for cleavage and recognition, accurate detection of bacteria can be achieved.

[0011] This method can not only detect highly pathogenic Helicobacter pylori in saliva samples but also be applied to the detection of other bacteria, such as African swine fever virus, etc. The combined use can significantly improve the sensitivity and accuracy of detection, and at the same time achieve rapid and on-site detection, which is of great significance for the early detection and prevention and control of diseases.

[0012] However, Streptococcus agalactiae or Neisseria gonorrhoeae plays an important role in reproduction. Currently, the detection of Streptococcus agalactiae or Neisseria gonorrhoeae mainly relies on blood or secretion culture, and biochemical tests such as catalase test and CAMP test are also helpful for differentiating them from other streptococci. However, clinical laboratory cultivation takes a lot of time and involves numerous steps, as well as the circulation management of various culture and experimental reaction equipment. Therefore, it is inconvenient to use, and each operation is relatively scattered and difficult to centrally manage.

[0013] LAMP and CRISPR-Cas12a play important roles in bacterial detection. Their combined application provides a new technical means for the rapid and accurate detection of bacteria, enabling new and rapid detection methods for Streptococcus agalactiae or Neisseria gonorrhoeae, achieving rapid detection effects for negative detection or quantity distribution. However, there is currently no mature reaction system indicating its application in the actual clinical detection of Streptococcus agalactiae or Neisseria gonorrhoeae. Summary of the Invention

[0014] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0015] On the one hand, an electronic tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae is provided. The system includes a bracket 1, and the bracket 1 is provided with:

[0016] A controller 3 for logical calculation and control;

[0017] A LAMP primer module for providing a LAMP primer mixed solution to the bacterial reaction module;

[0018] A CRISPR-Cas12a primer module for providing a CRISPR-Cas12a primer mixed solution to the bacterial reaction module;

[0019] A bacterial reaction module for preparing a LAMP-CRISPR-Cas12a reaction system according to the LAMP primer mixed solution and the CRISPR-Cas12a primer mixed solution; and, after injecting Streptococcus agalactiae or Neisseria gonorrhoeae, providing a mixed reaction environment for Streptococcus agalactiae or Neisseria gonorrhoeae and the LAMP-CRISPR-Cas12a reaction system, and outputting the corresponding bacterial reaction solution to the detection dish 17;

[0020] A fluorescence detector 12 for detecting the fluorescence signal of the bacterial reaction solution in the detection dish 17 and feeding it back to the controller 3. The controller 3 processes the fluorescence signal to generate a detection value corresponding to Streptococcus agalactiae or Neisseria gonorrhoeae and sends and displays the detection value on the electronic screen;

[0021] A power supply for power supply;

[0022] The LAMP primer module, the CRISPR-Cas12a primer module, the bacterial reaction module, the fluorescence detector 12, and the power supply are respectively electrically connected to the controller 3.

[0023] Preferably, the LAMP primer module includes a first reaction tube 2, a heater A, and an electronic valve A, where:

[0024] The first reaction tube 2 is vertically arranged on the bracket 1, and a LAMP primer mixed solution is prepared therein;

[0025] The heater A is arranged on the outer side surface of the first reaction tube 2 to provide preheating service;

[0026] The electronic valve A is arranged at the bottom of the first reaction tube 2 to control the dosage of the LAMP primer mixed solution delivered to the bacterial reaction module;

[0027] The heater A and the electronic valve A are respectively electrically connected to the controller 3.

[0028] Preferably, the CRISPR-Cas12a primer module includes a second reaction tube, a heater B9, and an electronic valve B10, where:

[0029] The second reaction tube is vertically arranged on the bracket 1, and a CRISPR-Cas12a primer mixed solution is prepared therein;

[0030] The heater B9 is arranged on the outer side surface of the second reaction tube to provide preheating service;

[0031] The electronic valve B10 is arranged at the bottom of the second reaction tube to control the dosage of the CRISPR-Cas12a primer mixed solution delivered to the bacterial reaction module;

[0032] The heater B9 and the electronic valve B10 are respectively electrically connected to the controller 3.

[0033] Preferably, the bacterial reaction module includes a third reaction tube 7, an injection port 8, a heater C13, and an electronic valve C14, where:

[0034] The injection port 8 is arranged at the top of the third reaction tube 7 for injecting the extract of Streptococcus agalactiae or Neisseria gonorrhoeae;

[0035] The third reaction tube 7 is vertically arranged on the bracket 1, and is pre-filled with sterile sodium chloride-peptone buffer solution at pH 7.0, 0.9% sterile sodium chloride solution or sterile phosphate buffer solution at pH 7.0; and the installation height of the third reaction tube 7 is lower than that of the LAMP primer module and the CRISPR-Cas12a primer module;

[0036] The heater C13 is cooperatively arranged on the outer side surface of the third reaction tube 7 for providing preheating service;

[0037] The solenoid valve C14 is arranged at the bottom of the third reaction tube 7 for controlling the dosage of the bacterial reaction solution transported to the test dish 17;

[0038] The heater C13 and the solenoid valve C14 are respectively electrically connected to the controller 3.

[0039] Preferably, a first sterile test platform 15 and a second sterile test platform 16 are arranged on the test dish 17 for containing the bacterial reaction solution and the contrast solution respectively.

[0040] Preferably, the fluorescence detector 12 is arranged on the bracket 1 through a flexible bracket 11 and is located above the test dish 17.

[0041] Preferably, there are two fluorescence detectors 12, which are respectively arranged directly above the first sterile test platform 15 and the second sterile test platform 16, and the two fluorescence detectors 12 are isolated by a light-shielding plate.

[0042] On the other hand, a method for detecting Streptococcus agalactiae or Neisseria gonorrhoeae is provided, which is implemented based on the above-mentioned automatic electronic one-tube detection system for Streptococcus agalactiae or Neisseria gonorrhoeae, and the method includes:

[0043] Pre-set the working parameters of each heater and solenoid valve in the controller 3 in advance;

[0044] After injecting the extract of Streptococcus agalactiae or Neisseria gonorrhoeae, activate the system through the controller 3 and control the system reaction;

[0045] When the preset time is reached, control the fluorescence detector 12 to detect the fluorescence signal of the bacterial reaction solution and feedback it to the controller 3, and the controller 3 processes the fluorescence signal and generates a detection value corresponding to Streptococcus agalactiae or Neisseria gonorrhoeae;

[0046] Send the detection value and display it on the electronic screen.

[0047] On the other hand, an electronic device is provided, which includes: a processor; a memory storing computer-readable instructions, and when the computer-readable instructions are executed by the processor, any one of the methods in the above-mentioned Streptococcus agalactiae or Neisseria gonorrhoeae electronic one-tube automatic detection system and detection method is implemented.

[0048] On the other hand, a computer-readable storage medium is provided, in which at least one instruction is stored, and the at least one instruction is loaded and executed by a processor to implement any one of the methods in the above-mentioned Streptococcus agalactiae or Neisseria gonorrhoeae electronic one-tube automatic detection system and detection method.

[0049] The beneficial effects brought by the technical solution provided by the embodiments of the present invention at least include:

[0050] In the present invention, a "one-tube" visual detection system for Streptococcus agalactiae or Neisseria gonorrhoeae mediated by the combination of LAMP and CRISPR-Cas12a is used. Combining with electronic automation technology, the LAMP reaction mixture and the CRISPR-Cas12a reaction mixture are mixed to form a "one-tube" reaction system. Through the designed "one-tube" electronic automation reaction system, through electronic automation control, the automatic mixing, preheating reaction of various detection reagents and samples, and fluorescence detection are realized, and a visualization device is used to visually display the detection results. The whole process can achieve electronic automation control, and can automatically and real-time monitor the change of fluorescence signal, and judge the presence or absence of Streptococcus agalactiae and / or Neisseria gonorrhoeae according to the change of fluorescence intensity. The analysis result is realized. If the fluorescence signal is significantly enhanced, it indicates that the sample contains Streptococcus agalactiae and / or Neisseria gonorrhoeae; if the fluorescence signal has no obvious change, it indicates that the sample does not contain the target bacteria. The system automatically records and reports the detection results.

[0051] The "one-tube" visual bacterial detection system mediated by the combination of LAMP (Loop-mediated isothermal amplification) and CRISPR-Cas12a (CRISPR-associated endonuclease) used in the present invention is an efficient and rapid bacterial detection method. This system combines the isothermal amplification ability of LAMP and the specific cleavage activity of CRISPR-Cas12a, and realizes the visual detection of bacteria by detecting fluorescence signals, and can provide rapid positive or quantitative detection results for users such as patients, improving the clinical detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0053] Figure 1 It is the application structure diagram of an automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae with electron tube conversion provided by an embodiment of the present invention;

[0054] Figure 2 It is the schematic diagram of the system control circuit provided by an embodiment of the present invention;

[0055] Figure 3 It is the flowchart of a detection method provided by an embodiment of the present invention;

[0056] Figure 4 It is the schematic diagram of the structure of an electronic device provided by an embodiment of the present invention. Detailed implementation manners

[0057] Next, the technical solutions in the present invention will be described with reference to the accompanying drawings.

[0058] In the embodiments of the present invention, words such as "exemplarily" and "for example" are used to represent examples, illustrations or explanations. Any embodiment or design solution described as an "example" in the present invention should not be construed as being more preferred or having more advantages than other embodiments or design solutions. Exactly speaking, the use of the word "example" is intended to present concepts in a specific way. In addition, in the embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one of the two can be selected.

[0059] In the embodiments of the present invention, "image" and "picture" can sometimes be used interchangeably. It should be noted that when the difference is not emphasized, the meanings they express are the same. "(of)", "corresponding", and "corresponding" can sometimes be used interchangeably. It should be noted that when the difference is not emphasized, the meanings they express are the same.

[0060] In the embodiments of the present invention, sometimes subscripts such as W 1 may be miswritten as non-subscript forms such as W1. When the difference is not emphasized, the meanings they express are the same.

[0061] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0062] In this embodiment, the experimental reagents, equipment, etc. involved can all be provided by a biological laboratory or purchased through the market, and this embodiment does not make any limitations.

[0063] Regarding the design of LAMP primers and CRISPR-Cas12a LAMP primers and the preparation of corresponding reagents, users can design and prepare them according to experimental requirements and conventional biochemical reaction steps. Regarding the primer sequences for the detection of Streptococcus agalactiae or Neisseria gonorrhoeae, they can be prepared by a biochemical laboratory. This solution does not involve the description of biological primer probes, but only presents a biological detection system and method.

[0064] The extraction and preparation of Streptococcus agalactiae or Neisseria gonorrhoeae can be collected from animal models, etc., or corresponding bacterial cultures can be purchased.

[0065] An embodiment of the present invention provides an automated detection system and method for Streptococcus agalactiae or Neisseria gonorrhoeae using an electron tube. This method can be implemented by an electronic device, which can be a terminal or a server. As Figure 1 shown in the composition structure diagram of the automated detection system for Streptococcus agalactiae or Neisseria gonorrhoeae using an electron tube, the system includes a bracket 1, and the following are provided on the bracket 1:

[0066] A controller 3 for logical calculation and control;

[0067] A LAMP primer module for providing a LAMP primer mixed solution to the bacterial reaction module;

[0068] A CRISPR-Cas12a primer module for providing a CRISPR-Cas12a primer mixed solution to the bacterial reaction module;

[0069] A bacterial reaction module for preparing a LAMP-CRISPR-Cas12a reaction system according to the LAMP primer mixed solution and the CRISPR-Cas12a primer mixed solution; and, after injecting Streptococcus agalactiae or Neisseria gonorrhoeae, providing a mixed reaction environment for Streptococcus agalactiae or Neisseria gonorrhoeae and the LAMP-CRISPR-Cas12a reaction system, and outputting the corresponding bacterial reaction solution to the detection dish 17;

[0070] A fluorescence detector 12 for detecting the fluorescence signal of the bacterial reaction solution in the detection dish 17 and feeding it back to the controller 3. The controller 3 processes the fluorescence signal to generate a detection value corresponding to Streptococcus agalactiae or Neisseria gonorrhoeae and sends and displays the detection value on an electronic screen;

[0071] A power supply for power supply;

[0072] The LAMP primer module, the CRISPR-Cas12a primer module, the bacterial reaction module, the fluorescence detector 12, and the power supply are respectively electrically connected to the controller 3.

[0073] The present invention is provided with a plurality of reaction detection modules on the bracket, which can realize the mixing reaction of Streptococcus agalactiae or Neisseria gonorrhoeae bacteria and their primer solutions, and output the reaction solution. The fluorescence detector is used to detect the fluorescence signal after the bacteria undergo amplification reaction, and then the fluorescence signal is processed by the chip in the controller, such as signal A / D conversion, filtering, and operational amplification (these circuits are common integrated circuits on the control circuit board, so the processing circuit in the controller will not be described in detail in this embodiment), etc., to obtain the corresponding fluorescence value, and the concentration or quantity of the corresponding bacteria is reflected through the fluorescence value.

[0074] The fixing method of each reaction tube on the bracket can be fixed by using a buckle or a hoop or a strap. Its installation position, quantity, etc. can be configured according to the requirements of this experiment or the laboratory. The controller adopts a single-chip microcomputer MCU chip of the STM32 / 64-bit series, etc.

[0075] The specific system design is as follows:

[0076] 1. Design primers:

[0077] According to the specific gene sequence of the target bacteria, the inner and outer primers required for LAMP amplification are designed.

[0078] At the same time, the crRNA of CRISPR-Cas12a is designed so that it can specifically recognize and cut a specific sequence in the LAMP amplification product.

[0079] 2. Prepare the reaction system:

[0080] The components required for LAMP amplification, such as enzymes, primers, dNTPs, buffer solutions, etc., are mixed together.

[0081] CRISPR-Cas12a protein, crRNA and a fluorescence reporting system (such as a DNA probe or ssDNA-FQ labeled with a fluorescent dye) are added.

[0082] The conversion of the number of bacteria according to the detection value of the fluorescent label mainly depends on the relationship between the fluorescence signal intensity and the ATP content in the bacteria. The following is the conversion description:

[0083] Relationship between fluorescence signal and ATP content:

[0084] The fluorescent label (such as luciferase) can react with ATP in the bacteria to form a luciferase complex and emit a fluorescence signal.

[0085] The intensity of the fluorescence signal is proportional to the content of ATP, that is, the more ATP, the stronger the fluorescence signal.

[0086] Conversion steps:

[0087] First, use a fluorescence detector to measure the fluorescence signal intensity of the sample.

[0088] Next, according to the known relationship between the fluorescence signal intensity and the ATP content (usually established through a standard curve or a mathematical model), convert the fluorescence signal intensity into the ATP content.

[0089] Finally, since living cells such as bacteria contain an equal amount of ATP, the number of bacteria can be indirectly estimated through the ATP content.

[0090] 3. Optimize the reaction conditions:

[0091] Determine the optimal temperature and time for LAMP amplification and CRISPR-Cas12a cleavage.

[0092] Ensure that the reaction system can proceed stably under the "one-tube" condition.

[0093] 4. Detection process

[0094] 4.1 Sample treatment:

[0095] Extract bacterial DNA or RNA from the sample to be detected.

[0096] Directly add the extracted nucleic acid to the prepared reaction system.

[0097] 4.2 Isothermal amplification and cleavage:

[0098] At a constant temperature (such as 60 - 65 °C), the LAMP primers guide the nucleic acid for isothermal amplification.

[0099] The amplification product is recognized and cleaved by CRISPR-Cas12a, releasing the signal of the fluorescence reporting system.

[0100] 4.3 Fluorescence detection:

[0101] Use a fluorescence detector (such as a fluorescence spectrophotometer, a fluorescence microscope, or a portable fluorescence detector) to detect the fluorescence signal in the reaction system.

[0102] The intensity of the fluorescence signal is proportional to the number of target bacteria in the sample, thus enabling quantitative detection.

[0103] 4.4 Result analysis:

[0104] Judge whether the target bacteria exist in the sample and the number of bacteria according to the strength of the fluorescence signal.

[0105] A threshold can be set to distinguish positive and negative results, improving the accuracy of detection (in this invention, a control group (the fluorescence value of the control solution) is set).

[0106] An electronic screen is integrated on the controller, which can be used to display the detection values. The contrast coefficient between the intensity of the corresponding fluorescence signal and the number of target bacteria in the sample, the bacterial quantity threshold for distinguishing positive and negative results, etc. can be determined according to clinical diagnostic criteria. A (wired or wireless) communication module or interface can also be configured to transmit the calculation results of the MCU to the laboratory PC (terminal), report and record the corresponding bacterial detection results, which can be used for subsequent bacterial negative analysis, quantity comparison analysis, etc.

[0107] Therefore, through the "one-tube" visual Streptococcus agalactiae or Neisseria gonorrhoeae detection system jointly mediated by LAMP and CRISPR-Cas12a, combined with electronic automation technology, the LAMP reaction mixture and the CRISPR-Cas12a reaction mixture are mixed to form a "one-tube" reaction system. Through the designed "one-tube" electronic automation reaction system, through electronic automation control, the automatic mixing, preheating reaction of various detection reagents and samples, and fluorescence detection are realized, and a visualization device is used to visually display the detection results. The whole process can achieve electronic automation control, and can automatically and real-time monitor the change of fluorescence signal, and judge the presence or absence of Streptococcus agalactiae and / or Neisseria gonorrhoeae according to the change of fluorescence intensity. The analysis result is realized. If the fluorescence signal is significantly enhanced, it indicates that the sample contains Streptococcus agalactiae and / or Neisseria gonorrhoeae; if the fluorescence signal has no obvious change, it indicates that the sample does not contain the target bacteria. The system automatically records and reports the detection results.

[0108] The composition structure and principle of the present invention will be further described below.

[0109] Each of the following modules is basically composed of a reaction tube, a heater and a solenoid valve. Corresponding solutions can be injected into the reaction tube by injection or other means; and a solenoid valve is arranged at the bottom of the reaction tube, and the on-off control is carried out by the controller to control the output volume. Heating and preheating services are also provided. On the outer side surface of the reaction tube near the bottom, a corresponding heater is installed and fitted, and the heater is deployed in a ring structure and can be heated under the control of the controller to preheat the solution in the reaction tube.

[0110] The dimensions and structures of the respective reaction tubes are not limited in this embodiment. The input and output ports, etc. thereon can be designed or selected according to requirements. For the specific models and installation structures of the heater and the solenoid valve, etc., they can be selected and matched by oneself. For the corresponding sealing and fixed installation methods, they can be provided by the equipment provider or the manufacturer.

[0111] There are various heating modules that can provide heating. The following are some common types of heating modules:

[0112] Instant heating module: This kind of heating module is usually used in devices such as water dispensers and pipeline machines. It adopts a flat spiral heating element or rare earth thick film spiral rapid heating technology, and can quickly heat water or fluid.

[0113] Infrared heating module: The infrared heating module uses the thermal effect of infrared rays for heating, and is widely used in various occasions that require rapid and uniform heating, such as drying, curing, heat treatment, etc.

[0114] Industrial heating module: This kind of heating module is usually used in industrial production equipment, such as kilns, heating furnaces, etc. It is made of high-temperature resistant materials such as zirconium-containing aluminosilicate fiber, and can withstand high-temperature environments and provide stable heating effects.

[0115] PTC heating module: The PTC heating module is a heating module that uses a PTC thermistor as a heating element. It has the characteristics of automatic temperature control, safety and reliability, energy saving and high efficiency, and is widely used in fields such as automobiles, household appliances, and industrial equipment.

[0116] Induction heating module: Induction heating technology is a non-contact heating method that converts electrical energy into heat energy through the principle of electromagnetic induction. It is usually used in processes such as heating, melting, and welding of metal materials. The induction heating module is the key component to realize this technology.

[0117] The controller can control the heaters and solenoid valves of each reaction tube to work for corresponding times according to preset program instructions. For example, at the start of work, the controller controls the heaters of the two primer modules to work for 1 - 3 minutes first, preheat the primer mixed solution therein, and then control the solenoid valves to work for 3 - 5 seconds respectively before and after, so as to output a corresponding dose of primer mixed solution into the total reaction tube. In the total reaction tube, preheating can also be controlled by the controller in the above way. The preheating temperature, time, etc. can be confirmed according to the working parameters of the selected heater. The specific reaction amount to be added can be calculated according to the requirements, and the controller controls the opening and closing of the solenoid valve. Finally, a part of the bacterial culture solution after the amplification reaction in the total reaction tube is discharged onto the detection dish. After a certain time (such as 3 minutes for setting), the fluorescence detector is then controlled to start for fluorescence detection and feedback the corresponding detection signal.

[0118] In the above manner, the solution in this system can also be divided into several batches. By detecting the fluorescence detection values of each batch and finally taking the average value as the final bacterial detection value, the accuracy can be improved. For example, if 10 ml of solution is configured in the reaction tube of the primer module, it can be divided into 5 batches, and each time the electronic valve is controlled to output 2 ml of solution. After reaction and detection, a value is recorded by the MCU controller. After receiving the fluorescence signal, the administrator can clean or replace the test dish, and then after a 5-minute delay by the controller, the next batch of detection can be carried out, during which the administrator replaces or cleans the test dish.

[0119] Preferably, the LAMP primer module includes a first reaction tube 2, a heater A, and an electronic valve A, where:

[0120] The first reaction tube 2 is vertically arranged on the bracket 1, and a LAMP primer mixed solution is prepared therein;

[0121] The heater A is cooperatively arranged on the outer side surface of the first reaction tube 2 for providing preheating service;

[0122] The electronic valve A is arranged at the bottom of the first reaction tube 2 for controlling the dosage of the LAMP primer mixed solution delivered to the bacterial reaction module;

[0123] The heater A and the electronic valve A are respectively electrically connected to the controller 3.

[0124] Combined with the attached Figure 2 Shown is a schematic diagram of the control circuit of this system (the reference numerals of each electronic device in Figure 1 are not marked here).

[0125] Preferably, the CRISPR-Cas12a primer module includes a second reaction tube, a heater B9, and an electronic valve B10, where:

[0126] The second reaction tube is vertically arranged on the bracket 1, and a CRISPR-Cas12a primer mixed solution is prepared therein;

[0127] The heater B9 is cooperatively arranged on the outer side surface of the second reaction tube for providing preheating service;

[0128] The electronic valve B10 is arranged at the bottom of the second reaction tube for controlling the dosage of the CRISPR-Cas12a primer mixed solution delivered to the bacterial reaction module;

[0129] The heater B9 and the electronic valve B10 are respectively electrically connected to the controller 3.

[0130] Preferably, it is characterized in that the bacterial reaction module includes a third reaction tube 7, an injection port 8, a heater C13 and an electronic valve C14, where:

[0131] The injection port 8 is provided at the top of the third reaction tube 7 for injecting the extract of Streptococcus agalactiae or Neisseria gonorrhoeae;

[0132] The third reaction tube 7 is vertically arranged on the bracket 1, and is pre-filled with a pH 7.0 sterile sodium chloride-peptone buffer solution, a 0.9% sterile sodium chloride solution or a pH 7.0 sterile phosphate buffer solution; and the installation height of the third reaction tube 7 is lower than that of the LAMP primer module and the CRISPR-Cas12a primer module;

[0133] The heater C13 is cooperatively arranged on the outer side surface of the third reaction tube 7 for providing preheating service;

[0134] The electronic valve C14 is provided at the bottom of the third reaction tube 7 for controlling the dosage of the bacterial reaction solution delivered to the test dish 17;

[0135] The heater C13 and the electronic valve C14 are respectively electrically connected to the controller 3.

[0136] During bacterial detection, the preparation solution before mixing the bacterial extract can be a pH 7.0 sterile sodium chloride-peptone buffer solution, a 0.9% sterile sodium chloride solution or a pH 7.0 sterile phosphate buffer solution, etc. These solutions are usually used as diluents. In the microbial limit test, they can help dilute the antibacterial products by an appropriate multiple, reduce the concentration of antibacterial components in the products, thereby weakening or eliminating their inhibitory effect on microorganisms, making the subsequent bacterial detection more accurate and reliable. Specifically, which preparation solution to choose may need to be determined according to the specific requirements of the experiment, the type of bacteria and the characteristics of the antibacterial product.

[0137] Preferably, a first sterile detection platform 15 and a second sterile detection platform 16 are arranged on the test dish 17 for containing the bacterial reaction solution and the comparison solution respectively.

[0138] The way to set the comparison solution can be through fluorescence signal detection, calculation and comparison, and referring to the detection value of the comparison solution to quickly distinguish the detection and analysis results of the current bacteria. For example, if the comparison solution uses a sterile solution with a standard concentration or a bacterial solution with a known positive structure, the detection results can be quickly compared to distinguish between positive and negative.

[0139] When analyzing the number of bacteria, the following steps can also be taken:

[0140] Establish a standard curve: Before the experiment, it is necessary to establish a standard curve of fluorescence signal intensity versus the number of bacteria. This is usually done by amplifying and detecting bacterial samples with known concentrations, and then plotting a graph of the relationship between fluorescence signal intensity and the number of bacteria.

[0141] Calculate the number of bacteria: Based on the measured fluorescence signal intensity, find the corresponding number of bacteria on the standard curve. This usually involves converting the fluorescence signal intensity to ATP content, and then calculating the number of bacteria according to the relationship between ATP content and the number of bacteria.

[0142] Calibration and verification: Regularly calibrate the fluorescence detector to ensure the accuracy of the detection results. At the same time, use bacterial samples with known concentrations for verification to evaluate the reliability of the detection method.

[0143] Preferably, the fluorescence detector 12 is arranged on the bracket 1 through a flexible bracket 11 and is located above the detection dish 17.

[0144] Preferably, there are two fluorescence detectors 12, which are respectively arranged directly above the first sterile detection platform 15 and the second sterile detection platform 16, and the two fluorescence detectors 12 are isolated by a light-shielding plate.

[0145] Using two fluorescence detectors 12 with separate shielding settings here can avoid light interference and improve accuracy. The flexible bracket can be a multi-axis connecting rod, which can rotate in three-dimensional space to adjust the height, angle, etc. of the fluorescence detector 12, facilitating operation. The flexible bracket can be hinged on the bracket 1.

[0146] Therefore, the present invention utilizes a "one-tube" visual bacterial detection system mediated by the combination of LAMP (Loop-mediated Isothermal Amplification) and CRISPR-Cas12a (CRISPR-associated endonuclease), which is an efficient and rapid bacterial detection method. This system combines the isothermal amplification ability of LAMP and the specific cleavage activity of CRISPR-Cas12a, and realizes the visual detection of bacteria by detecting fluorescence signals, which can provide rapid positive or quantity detection results for users such as patients and improve the clinical detection efficiency.

[0147] As Figure 3 shown, on the other hand, a method for detecting Streptococcus agalactiae or Neisseria gonorrhoeae is provided, which is implemented based on the above-mentioned electronic one-tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae, and the method includes:

[0148] S1. Preset the working parameters of each heater and solenoid valve in the controller 3;

[0149] S2. After the extract of Streptococcus agalactiae or Neisseria gonorrhoeae is to be injected, activate the system through the controller 3 and control the system reaction;

[0150] S3. When the preset time is reached, control the fluorescence detector 12 to detect the fluorescence signal of the bacterial reaction solution and feedback it to the controller 3, and the controller 3 processes the fluorescence signal and generates a detection value corresponding to the Streptococcus agalactiae or Neisseria gonorrhoeae;

[0151] S4. Send the detection value and display it on the electronic screen.

[0152] The above steps should be implemented in combination with the above system description, which will not be elaborated here.

[0153] Figure 4 It is a schematic structural diagram of an electronic device provided by an embodiment of the present invention. As Figure 4 shown, the electronic device 410 may include a first processor 2001.

[0154] Optionally, the electronic device 410 may further include a memory 2002 and a transceiver 2003.

[0155] Among them, the first processor 2001 is connected to the memory 2002 and the transceiver 2003, such as through a communication bus.

[0156] Next, in combination with Figure 4 specific introductions will be made to the respective components of the electronic device 410:

[0157] Among them, the first processor 2001 is the control center of the electronic device 410, which can be a single processor or a collective term for multiple processing elements. For example, the first processor 2001 is one or more central processing units (CPUs), or can be an application specific integrated circuit (ASIC), or is an integrated circuit configured to implement the embodiments of the present invention, such as: one or more digital signal processors (DSPs), or, one or more field programmable gate arrays (FPGAs).

[0158] Optionally, the first processor 2001 can execute various functions of the electronic device 410 by running or executing software programs stored in the memory 2002 and calling data stored in the memory 2002.

[0159] In a specific implementation, as an example, the first processor 2001 may include one or more CPUs, such as Figure 4 the CPU0 and CPU1 shown in

[0160] In a specific implementation, as an example, the electronic device 410 may also include multiple processors, such as Figure 4 the first processor 2001 and the second processor 2004 shown in. Each of these processors may be a single-core processor (single-CPU) or a multi-core processor (multi-CPU). The processor here may refer to one or more devices, circuits, and / or processing cores for processing data (such as computer program instructions).

[0161] Among them, the memory 2002 is used to store the software program for implementing the solution of the present invention and is controlled by the first processor 2001 to execute. The specific implementation manner may refer to the above method embodiment and will not be elaborated here.

[0162] Optionally, the memory 2002 may be a read-only memory (ROM) or other types of static storage devices that can store static information and instructions, a random access memory (RAM) or other types of dynamic storage devices that can store information and instructions, or an electrically erasable programmable read-only memory (EEPROM), a compact disc read-only memory (CD-ROM), or other optical disc storage (including compact discs, laser discs, optical discs, digital versatile discs, Blu-ray discs, etc.), magnetic disk storage media, or any other medium that can be used to carry or store the desired program code in the form of instructions or data structures and can be accessed by a computer, but not limited thereto. The memory 2002 may be integrated with the first processor 2001 or exist independently and be coupled to the first processor 2001 through the interface circuit of the electronic device 410 ( Figure 4 not shown in). The embodiments of the present invention do not make specific limitations on this.

[0163] The transceiver 2003 is used to communicate with a network device or with a terminal device.

[0164] Optionally, the transceiver 2003 may include a receiver and a transmitter ( Figure 4 not separately shown in). Among them, the receiver is used to implement the receiving function, and the transmitter is used to implement the sending function.

[0165] Optionally, the transceiver 2003 may be integrated with the first processor 2001 or exist independently, and is coupled to the first processor 2001 through an interface circuit (not shown) of the electronic device 410. The embodiments of the present invention do not make specific limitations on this. Figure 4 It should be noted that the structure of the electronic device 410 shown in does not constitute a limitation on the router. The actual knowledge structure recognition device may include more or fewer components than shown in the figure, or combine certain components, or have different component arrangements.

[0166] It should be noted that Figure 4 The structure of the electronic device 410 shown in does not constitute a limitation on the router. The actual knowledge structure recognition device may include more or fewer components than shown in the figure, or combine certain components, or have different component arrangements.

[0167] In addition, the technical effects of the electronic device 410 can refer to the technical effects of the Streptococcus agalactiae or Neisseria gonorrhoeae electronic tube automatic detection system and detection method described in the above method embodiments, which will not be elaborated here.

[0168] It should be understood that the first processor 2001 in the embodiments of the present invention may be a central processing unit (CPU), and the processor may also be other general-purpose processors, digital signal processors (DSPs), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc.

[0169] It should also be understood that the memory in the embodiments of the present invention can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. Among them, the non-volatile memory can be a read-only memory (ROM), a programmable ROM (PROM), an erasable PROM (EPROM), an electrically erasable PROM (EEPROM), or a flash memory. The volatile memory can be a random access memory (RAM), which is used as an external cache. By way of example but not limitation, many forms of random access memory (RAM) are available, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), enhanced SDRAM (ESDRAM), synchlink DRAM (SLDRAM), and direct rambus RAM (DR RAM).

[0170] The above embodiments can be implemented in whole or in part by software, hardware (such as circuits), firmware, or any other combination. When implemented using software, the above embodiments can be implemented in whole or in part in the form of a computer program product. The computer program product includes one or more computer instructions or computer programs. When the computer instructions or computer programs are loaded or executed on a computer, the processes or functions described in the embodiments of the present invention are generated in whole or in part. The computer can be a general-purpose computer, a special-purpose computer, a computer network, or other programmable devices. The computer instructions can be stored in a computer-readable storage medium or transmitted from one computer-readable storage medium to another. For example, the computer instructions can be transmitted from one website, computer, server, or data center to another website, computer, server, or data center by wired (such as infrared, wireless, microwave, etc.) means. The computer-readable storage medium can be any available medium that can be accessed by a computer or a data storage device such as a server or a data center that contains one or more collections of available media. The available medium can be a magnetic medium (such as a floppy disk, a hard disk, or a magnetic tape), an optical medium (such as a DVD), or a semiconductor medium. The semiconductor medium can be a solid-state drive.

[0171] It should be understood that the term "and / or" in this document is merely a description of the association relationship between associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B can be singular or plural. In addition, the character " / " in this document generally represents an "or" relationship between the associated objects before and after, but it may also represent an "and / or" relationship, which can be specifically understood with reference to the context.

[0172] In the present invention, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, at least one of a, b, or c can represent: a, b, c, a - b, a - c, b - c, or a - b - c, where a, b, and c can be single or multiple.

[0173] It should be understood that in various embodiments of the present invention, the magnitudes of the sequence numbers of the above processes do not mean the order of execution. The order of execution of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present invention.

[0174] Those of ordinary skill in the art can realize that the units and algorithm steps of each example described in combination with the embodiments disclosed herein can be implemented by electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are executed in a hardware or software manner depends on the specific application and design constraints of the technical solution. Professional technicians can use different methods to implement the described functions for each specific application, but such implementation should not be considered to exceed the scope of the present invention.

[0175] Those skilled in the art can clearly understand that for the convenience and conciseness of description, the specific working processes of the above-described devices, apparatuses, and units can refer to the corresponding processes in the foregoing method embodiments and will not be elaborated herein.

[0176] In several embodiments provided by the present invention, it should be understood that the disclosed devices, apparatuses, and methods can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the units is only a logical function division. In actual implementation, there may be other division methods. For example, multiple units or components can be combined or integrated into another device, or some features can be ignored or not executed. Another point is that the displayed or discussed couplings or direct couplings or communication connections to each other can be through some interfaces. The indirect couplings or communication connections of the devices or units can be in electrical, mechanical, or other forms.

[0177] The units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they can be located in one place, or can be distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0178] In addition, the functional units in each embodiment of the present invention can be integrated into one processing unit, or each unit can exist physically alone, or two or more units can be integrated into one unit.

[0179] When the above-mentioned function 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 such an understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or a part of the technical solution, can be embodied in the form of a software product. The computer software product is stored in a storage medium and includes several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the methods described in various embodiments of the present invention. The aforementioned storage medium includes: various media that can store program codes, such as USB flash drives, mobile hard disks, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical discs.

[0180] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. An electronic and automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae, characterized in that: The system comprises a support (1), on which is arranged: Controller (3), used for logic calculation and control; The LAMP primer module comprises a first reaction tube (2), a heater A and an electronic valve A, and is used to provide a LAMP primer mixed solution to the bacterial reaction module; A CRISPR-Cas12a primer module, comprising a second reaction tube, a heater B (9) and an electronic valve B (10), for providing a CRISPR-Cas12a primer mixed solution to the bacterial reaction module; A bacterial reaction module, comprising a third reaction tube (7), an injection port (8), a heater C (13) and an electronic valve C (14), for preparing a LAMP-CRISPR-Cas12a reaction system according to the LAMP primer mixed solution and the CRISPR-Cas12a primer mixed solution; and, after the Streptococcus agalactiae or gonococcus is injected, providing a mixed reaction environment for the Streptococcus agalactiae or gonococcus and the LAMP-CRISPR-Cas12a reaction system, and outputting the corresponding bacterial reaction solution to a detection dish (17); A fluorescence detector (12) for detecting the fluorescence signal of the bacterial reaction liquid in the detection dish (17) and feeding it back to the controller (3); the controller (3) processes the fluorescence signal and generates a detection value corresponding to the Streptococcus agalactiae or Neisseria gonorrhoeae, and transmits and displays the detection value on an electronic screen; Power supply, used for power supply; The LAMP primer module, the CRISPR-Cas12a primer module, the bacterial reaction module, the fluorescence detector (12) and the power supply are electrically connected to the controller (3) respectively.

2. The electronic-tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to claim 1, characterized in that: The first reaction tube (2) is vertically arranged on the support (1), and contains a LAMP primer mixed solution; The heater A is arranged on the outer side of the first reaction tube (2) to provide preheating service; The electronic valve A is arranged at the bottom of the first reaction tube (2) and is used to control the dosage of the LAMP primer mixed solution delivered to the bacterial reaction module; The heater A and the electronic valve A are electrically connected to the controller (3) respectively.

3. The electronic-tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to claim 1, characterized in that: The second reaction tube is vertically arranged on the support (1), and a CRISPR-Cas12a primer mixed solution is prepared therein; The heater B (9) is arranged on the outer side of the second reaction tube to provide preheating service; The electronic valve B (10) is disposed at the bottom of the second reaction tube and is used to control the dosage of the CRISPR-Cas12a primer mixture solution delivered to the bacterial reaction module; The heater B (9) and the electronic valve B (10) are electrically connected to the controller (3) respectively.

4. The electronic-tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to claim 1, characterized in that: The injection port (8) is disposed at the top of the third reaction tube (7) and is used for injecting the extract of Streptococcus agalactiae or Neisseria gonorrhoeae; The third reaction tube (7) is vertically arranged on the support (1), and contains a pH 7.0 sterile sodium chloride-peptone buffer, a 0.9% sterile sodium chloride solution, or a pH 7.0 sterile phosphate buffer; and the installation height of the third reaction tube (7) is lower than that of the LAMP primer module and the CRISPR-Cas12a primer module; The heater C (13) is arranged on the outer side of the third reaction tube (7) to provide preheating service; The electronic valve C (14) is arranged at the bottom of the third reaction tube (7) and is used to control the dosage of the bacterial reaction liquid delivered to the detection dish (17); The heater C (13) and the electronic valve C (14) are electrically connected to the controller (3) respectively.

5. The electronic-tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to claim 1, characterized in that: The detection dish (17) is provided with a first sterile detection platform (15) and a second sterile detection platform (16), which are used to hold the bacterial reaction solution and the comparison solution respectively.

6. The electronic-tube automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to claim 5, characterized in that: The fluorescence detector (12) is arranged on the support (1) via a flexible support (11) and is located above the detection dish (17).

7. The electronic and tube-based automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to claim 6, characterized in that: Two fluorescence detectors (12) are provided, which are respectively arranged directly above the first sterility detection platform (15) and the second sterility detection platform (16), and the two fluorescence detectors (12) are isolated by a light shielding plate.

8. A method for detecting Streptococcus agalactiae or Neisseria gonorrhoeae, which is implemented based on the electronic and automatic detection system for Streptococcus agalactiae or Neisseria gonorrhoeae according to any one of claims 1 to 7, characterized in that: The method comprises: The operating parameters of each heater and electronic valve are pre-set in the controller (3); After the extract of Streptococcus agalactiae or Neisseria gonorrhoeae is injected, the system is activated and the system reaction is controlled by the controller (3); When a preset time is reached, the fluorescence detector (12) is controlled to detect the fluorescence signal of the bacterial reaction liquid and feed it back to the controller (3), and the controller (3) processes the fluorescence signal and generates a detection value corresponding to the Streptococcus agalactiae or Neisseria gonorrhoeae; The detected value is sent and displayed on an electronic screen.

9. An electronic device, characterized in that: The electronic device comprises: processor; A memory having computer-readable instructions stored thereon, wherein when the computer-readable instructions are executed by the processor, the method according to claim 8 is implemented.

10. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores program codes, which can be called by a processor to execute the method according to claim 8.

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