Constant-temperature real-time fluorescent quantitative multichannel detection equipment based on LAMP (loop-mediated isothermal amplification) method

Abstract

The invention relates to constant-temperature real-time fluorescent quantitative multichannel detection equipment based on an LAMP (loop-mediated isothermal amplification) method, and belongs to the field of molecular biology and medical detection instruments, as shown in Figure 1, the constant-temperature real-time fluorescent quantitative multichannel detection equipment comprises a central processing control system used for controlling all modules to work orderly, (1) a multichannel modularized plug-and-play detection system used for placing reaction tubes, and (2) a constant-temperature real-time fluorescent quantitative detection system used for placing reaction tubes, (2) converting a real-time fluorescence signal in the LAMP reaction process into an electric signal and sending the electric signal to a fluorescence signal sensing system of the central processing control system; (3) sending a real-time fluorescence signal to an upper computer or a handheld terminal data communication system (5) and a handheld terminal data communication system (6); the local display controls a touch screen; (4) a power management and storage system (8); the state of LAMP fluorescent quantitative reaction can be observed in real time by an upper computer or a handheld terminal through USB external equipment, WiFi or a Bluetooth system local machine, and LAMP detection results are analyzed and judged at the same time. According to the invention, a highly integrated real-time fluorescent quantitative reaction system based on an LAMP method and corresponding sensing, control and communication systems are adopted, a nucleic acid extraction function is integrated, and each detection unit can operate independently, so that multiple purposes of one machine are ensured, the universality and flexibility of the instrument are improved, certain portability is ensured, and the cost is reduced. The upper computer or the handheld terminal is adopted for real-time data analysis, unnecessary resource and time waste is avoided, and the equipment and experiment cost is effectively reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and medical detection instruments, and relates to a constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method. Background Art

[0002] LAMP (Loop-mediated isothermal amplification) is a new type of nucleic acid amplification method, which is characterized by designing 4 specific primers for 6 regions of the target gene, and performing constant temperature amplification at 60-65° under the action of chain displacement DNA polymerase. DNA can be amplified more than 1 million times in about 5-30 minutes. Its method is similar to PCR (Polymerase Chain Reaction). The LAMP method is relatively simple and can provide fast, efficient, and high-precision detection. It is suitable for field detectors in any scenario.

[0003] The real-time fluorescent LAMP technology based on Taqman probe is a method of fluorescence detection using oligonucleotides modified with fluorescent substances (such as FAM, etc.) at the 5' end and quenching substances (such as TAMRA, etc.) at the 3' end. When the probe is intact, the fluorescent substance at the 5' end is restricted by the quenching substance at the 3' end and cannot emit fluorescence. When the probe is decomposed, the fluorescent substance at the 5' end will be freed and emit fluorescence. When the fluorescent probe is added to the real-time fluorescent reaction solution, the fluorescent probe will hybridize with the template during the annealing process of the real-time fluorescent reaction. Further, during the extension process of the real-time fluorescent reaction, the 5'→3'Exonuclease activity of the heat-resistant DNA polymerase can decompose the fluorescent probe hybridized with the template, and the free fluorescent substance emits fluorescence. By detecting the fluorescence intensity in the reaction system, the purpose of detecting the amount of microbial DNA amplification can be achieved.

[0004] The instrument that implements the real-time fluorescence quantitative detection technology of the LAMP method is called a constant temperature real-time fluorescence quantitative analyzer, which is essentially a high-precision fluorescence detection and temperature control device. The basic principle is: the LED light source emits excitation light, which reaches the reaction tube with the sample through the optical component, and the sample emits fluorescence after being excited. The excited fluorescence then passes through the optical component to reach the detector. The filter is a component of the optical component that allows light of a specific wavelength to pass through, thereby achieving the purpose of distinguishing the product. The fluorescence that passes through the filter is captured by the detector after passing through the optical component. The captured fluorescence is input into the computer for calculation and analysis after a series of photoelectric conversions.

[0005] At present, the molecular detection equipment on the market is mainly based on the PCR principle. It is large in size, heavy in weight, and complex to operate. It is very easy to cause sample contamination, and the average test time is 1-2 hours. It is not suitable for use in some special scenarios, which makes these instruments have a single application scenario and low applicability. Therefore, they do not meet the needs of rapid analysis, miniaturization, on-site real-time detection and diagnosis. It is only suitable for use in laboratories or laboratories of large and medium-sized medical institutions, and testing personnel generally need to undergo corresponding training. The LAMP-based detection method is an emerging genetic detection technology developed in recent years. Its characteristics are rapid, accurate, specific, and repeatable. The existing LAMP-based detectors on the market are still in the initial exploration stage. There are deficiencies in measurement accuracy and speed. Multi-channel instruments are not selective and practical, and most of them are in the exploration stage. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a multi-channel (96) constant temperature real-time fluorescence quantitative rapid molecular detection device based on the LAMP method, which uses a modular highly integrated fluorescence detection component, is equipped with efficient control software, and an optimized central processing control system and data communication system to increase the detection throughput while reducing the detection time to 5 to 15 minutes. In addition, the present invention integrates the DNA / RNA extraction and microbial detection processes into one on the basis of improving portability, realizes the miniaturization of the instrument and the multi-purpose of one machine, reduces the detection steps, and also reduces the probability of sample contamination, saving the capital investment of the end user. The system detection module can be plug-and-play, and online replacement of modules can greatly improve the flexibility and application environment of the equipment. The optimized local equipment does not need to be connected to any external control system and can achieve big data sharing. The simple detection process is: during the polymerase chain reaction process, the central processing controller real-time regulates and monitors the fluorescence reaction system of each module, and cooperates with the data communication system to realize real-time data transmission between the host computer or handheld terminal. The host computer or handheld terminal can monitor the reaction process in real time through the corresponding communication system, and obtain the specificity of the reaction and judge the test results by analyzing the reaction products and melting curves after the polymerase chain reaction. The test report and data can be directly uploaded to the remote big data system.

[0007] Furthermore, the real-time fluorescence quantitative multi-channel (96) detector based on the LAMP method described in the present invention includes: a modular fluorescence reaction system, a fluorescence signal conversion system, a central processing control system, a data communication system, and a data storage system.

[0008] Furthermore, the real-time fluorescence quantitative multi-channel (96) detector based on the LAMP method described in the present invention has the following system relationships: the modular polymerase chain reaction station irradiates the experimental sample through the built-in fluorescence exciter according to the requirements of the central control processor, and the generated fluorescence signal is collected by the fluorescence sensor and converted into an electrical signal by the photoelectric converter through the signal amplification and filtering device; the central processor receives and post-processes the fluorescence electrical signal and controls the orderly operation of each module; the data communication system sends the real-time fluorescence signal to the host computer or handheld terminal according to the instructions of the central processor, and the host computer or handheld terminal observes the status of the polymerase chain reaction in real time and analyzes and interprets the detection results.

[0009] Furthermore, the real-time fluorescence quantitative multi-channel (96) reaction system described in the present invention includes a polymerase chain reaction table, a temperature control system and a temperature sensing device, wherein: the polymerase chain reaction table is made of metal with excellent thermal conductivity, and includes a reaction tank for placing 8 rows of reaction tubes, each reaction tank having 8 reaction holes.

[0010] Furthermore, the temperature control system of the present invention includes a heating module, a thermal sensor and a heat dissipation module, wherein the heating module is embedded in the polymerase chain reaction table. When current passes through, the heating module generates heat to evenly increase the temperature of the reaction table. The heat dissipation module is a small fan located directly below the reaction table. When the central processing control system sends a cooling signal, the small fan is powered on and dissipates the heat, thereby reducing the temperature. The temperature sensing device is embedded in each reaction hole inside the metal workbench, and the temperature of the reaction table (hole) is collected in real time and fed back to the central processing control system.

[0011] Furthermore, the fluorescence signal sensing system of the present invention is composed of a fluorescence sensor, a signal amplifier and a filter. The fluorescence sensor is built-in at a 90° position on the side of each reaction well to obtain the real-time fluorescence signal during the reaction process in the polymerase chain reaction tube and finally convert it into an electrical signal.

[0012] Furthermore, the central processing control system is connected to the system detection module (up to 12 modules), temperature control system, optoelectronic system, and signal transmission system; the central processing unit directly controls the temperature change of the heating module and adjusts the temperature in the instrument cavity through the heat dissipation module; the central processing control system adopts the PID control principle to accurately control the reaction temperature. When the temperature collected by the temperature sensor deviates from the given value, the PID control performs proportional, integral, and differential operations based on the deviation between the feedback measurement signal and the given value, thereby outputting an appropriate control signal to adjust the heating module or the heat dissipation module, causing the measurement value to return to the given value, thereby achieving the effect of automatic control.

[0013] Further, in the data communication system, before the reaction starts, the required parameters of the polymerase chain reaction are set by a host computer or a handheld terminal and transmitted to the central processor through the data communication system to control the reaction process, and the parameters include CT threshold, temperature, time, and sample name.

[0014] Further to the function of the device, each detection module of the system integrates DNA / RNA extraction and polymerase chain reaction, which improves the versatility of the device and reduces the probability of cross contamination and the cost of use for the end user.

[0015] Furthermore, the heating module and the heat dissipation module can ensure the uniformity of the polymerase chain reaction temperature to be ± 0.1℃ .

[0016] According to further functions, after the polymerase chain reaction is completed, the central processing control system controls the temperature of the reaction table of the detection module to rise, so that the DNA double helix structure is degraded; during this process, the fluorescent signal sensing system (2) collects the fluorescent signal and sends the signal to the host computer or handheld device through the data communication system; the host computer or handheld terminal completes the specific analysis of the reaction product by analyzing the melting curve and melting temperature, and then obtains the detection result. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the architecture diagram of the Genie-HT96 system of the present invention.

[0018] Figure 2 Modular embedded detection unit - base and cooling.

[0019] Figure 3 Modular embedded detection unit - signal control and transmission.

[0020] Figure 4 Modular embedded detection unit - heating module and temperature sensing.

[0021] Figure 5 Modular embedded detection unit - fluorescence signal processing.

[0022] Figure 6 Genie-HT96 software flow. Figure 7 Genie-HT Overall Appearance DETAILED DESCRIPTION

[0023] 1. The present invention will be further described in detail below based on the accompanying drawings and embodiments.

[0024] like Figure 1As shown, a constant temperature real-time fluorescence quantitative multi-channel (96) rapid portable molecular detection device based on LAMP method includes: a central processing unit (1), a modular real-time fluorescence quantitative reaction and detection system, a maximum of 12 modules with 96 channels (2), a detection module control instruction bus set (3), an LCD touch screen controller (4), a wireless communication system (5), a USB external system (6), a detection verification and analysis unit (7), a memory management system (8), and a modular fluorescence signal detection and processing system ( Figure 5 ).

[0025] The central processor receives the test instruction program through the local human-machine interactive interface LCD touch screen (4), or the host computer and handheld device terminal equipment through the wireless connection system (5 or 6); according to different experimental requirements, the central controller sends a detection signal to the specified polymerase chain reaction fluorescence system control module (2), and controls the heating system in the module according to the instruction detection module, and fine-tunes the temperature according to the temperature information fed back in real time from each test hole, and gives an alarm for the situation that exceeds the temperature control range; the processor turns on the excitation light source at an appropriate time according to the pre-set, and the fluorescence reaction is detected by the photoelectric conversion system ( Figure 5 ) is converted into a high-precision electrical signal; the central processor analyzes and compares the fluorescent electrical signal, and outputs the test results to the multi-function touch screen display (4), or transmits the test results to the user-specified terminal device or large database through a wireless transmission system (5) or a USB external device. This instrument does not need to be connected to a computer to achieve complete device operation and data transmission, and can also be connected to a local computer via USB for operation and data transmission; all detailed experimental data will be backed up and retained in the instrument's internal storage system (8); based on the requirements of portability, the machine is compact in design, light in weight, and of moderate size. It can work in any voltage environment from 110V to 220V, and is completely suitable for mobile vehicle-mounted experimental platforms and temporary testing laboratories.

[0026] 2. If Figure 2 , 3, 4, 5, a core component of a constant temperature real-time fluorescence quantitative multi-channel (96) rapid portable molecular detection device based on the LAMP method - a modular embedded polymerase chain reaction table, including: a cooling fan, a temperature balancer, a heating module, a built-in temperature acquisition sensor, a built-in fluorescence signal exciter, a built-in fluorescence signal collector, a physical filter, in order to reduce signal loss, a fluorescence signal analysis conversion circuit ( Figure 3 ) is also attached to the reaction table.

[0027] The modular reaction table is made of metal with excellent thermal conductivity. The heating module is embedded in the workbench and is close to the test tube reaction tank to achieve high-precision temperature control of the reaction tank (±0.1°). The workbench includes 1 Figure 4 The reaction tank shown is used to place polymerase chain reaction tubes. The reaction tank has 8 reaction holes to match the specially designed reaction tube row. According to the instructions of the central controller, when the current passes through, the heating module heats up to increase the overall temperature of the reaction table to prompt the test sample to undergo a DNA chain reaction. The embedded fluorescence stimulator located on the side of each reaction hole stimulates the fluorescent substance in the test tube at an appropriate time, and the embedded fluorescence sensor at 90° on the other side of the reaction hole collects the fluorescence signal. The fluorescence signal undergoes complex amplification, filtering and photoelectric converter ( Figure 5 ) is converted into a weak electrical signal, which is further amplified and filtered, and then transmitted to the central processor for data analysis to finally give the experimental results. The modular detection platform is cooled by two small fans. The air cooling system is located under the polymerase chain reaction table. When the central processing control system sends a cooling signal, the small fan is powered on to dissipate the heat and reduce the temperature; the temperature sensor device is built into each reaction well on the reaction table, and the temperature of the reaction tube is collected in real time and fed back to the central processing control system.

[0028] 3. This example describes a constant temperature real-time fluorescence quantitative multi-channel (96) rapid portable molecular detection device based on the LAMP method. The main method of use is as follows:

[0029] like Figure 6 As shown, the device of the present invention has five main preset functional modules, and the functional modules can be increased or decreased according to the needs of the user. Functional module 1 is: virus detection (COVID detection); select this function key, the device will perform DNA detection on the new crown sample for the specified detection module according to the system preset settings, and this function meets the requirements of fast and simple operation. The operator can issue a detection or stop instruction to any module as required without affecting the detection process of other modules on the device. Each module can be independently programmed to achieve different detection requirements. This function is executed with one button, and the preset program includes amplification, annealing and cooling steps. The setting of the detection template is a subsystem under this functional module.

[0030] Functional module 2 is: System settings; entering this module is protected by a first-level password, and can reset the device's operating system, set new users, store data, change the device date, adjust the display brightness, and set the color of the indicator; this module can also calibrate the device screen and set the display background. This function setting needs to be changed and set by relevant technical personnel.

[0031] Function module 3 is: external device settings; here you can set the device's WiFi, USB usage functions, and you can also set printing, data transmission and storage methods here.

[0032] Functional module 4 is: device guidance; entering this mode allows you to set the display content of the main screen, and the instructions for use that come with the machine can also be indexed here.

Claims

1. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim, characterized in that: A modular multi-channel constant temperature real-time fluorescence quantitative reaction system is used to place reaction tubes; a fluorescence signal detection system converts the real-time fluorescence signal in the polymerase chain reaction process into an electrical signal and sends it to the central processing control system through the data bus; the detection verification and data analysis unit analyzes the data according to the system settings and gives a test result report; the central processing control system controls the orderly operation of each module; the data communication system sends the real-time fluorescence signal to the host computer or handheld terminal, which can observe the status of the polymerase chain reaction in real time and perform personalized analysis and judgment on the test results; the memory management system stores or uploads the data according to the internal settings.

2. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The modular independently operated constant temperature real-time fluorescence quantitative reaction system comprises a polymerase chain reaction table, a temperature control system and a temperature sensing device, wherein: The modular polymerase chain reaction table (Figure 2, Figure 4) is made of metal with excellent thermal conductivity. The plug-and-play modular reaction table can be replaced online, and up to 12 modules (96 channels) can be installed. Each detection module consists of a reaction tank and attached temperature control and fluorescence detection equipment, and each reaction tank has 8 reaction holes; the temperature control system includes a heating module, a temperature sensor and a heat dissipation module, wherein the heating module is built into the metal reaction table (Figure 4) and is close to the reaction tank. When the current passes through, the heating module generates heat to increase the temperature of the polymerase chain reaction table to prompt the test sample to perform a DNA chain reaction; the heat dissipation module is built into the reaction table, which is two air-cooled fans located directly below the reaction table. When the central processing control system sends a cooling signal, the small fan is powered on and the heat is dissipated, and the temperature is reduced; the temperature sensor device is built into each reaction hole on the polymerase chain reaction table, and the temperature of each reaction tube is collected in real time and fed back to the central processing control system. The entire reaction table is built into a closed black metal box, and the metal box is sprayed with a high-efficiency light-absorbing layer to reduce interference from light sources.

3. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The fluorescent signal sensing system is composed of a microporous optical fiber sensor, which is arranged at a 90° position on the side of each reaction tank of the polymerase chain reaction table. The optical fiber sensor is modularly embedded to minimize interference with the light source. The sensor obtains the real-time fluorescent signal during the polymerase chain reaction process in each reaction tube and converts it into an electrical signal.

4. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The fluorescent signal stimulator adopts a 470nm and 590nm dual-color LED laser stimulator, which can detect all dyes that can be excited by a blue light source and have an emission wavelength between 510 and 560nm, and all dyes that are excited by a yellow light source and have an emission wavelength higher than 620nm. The fluorescent stimulator is set on the side of each reaction tube of the polymerase chain reaction table and close to the test tube in the reaction tank to minimize light source loss and interference with the light source.

5. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The specially designed reaction tube uses the material as one of the specific wavelength fluorescence filters and is used in conjunction with the fluorescence signal analysis system to filter out the fluorescence background noise, thereby reducing the fluorescence filtering devices and improving the photosensitivity.

6. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: In order to improve the sensing sensitivity of weak fluorescence signals, this detection equipment systematically optimizes the fluorescence exciter, test tube material, photoelectric converter, signal amplifier, physical and digital filters; the entire fluorescence excitation, detection, and signal pre-processing are miniaturized and integrated, and the overall photoelectric system is placed inside a sealed black box with an attached light-absorbing layer, making the best possible use of the low noise advantage of the high-integration system, Figure 5.

7. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The polymerase chain reaction detection device integrates the nucleic acid extraction process, reduces the operation steps of the operator, reduces the risk of cross contamination, reduces the equipment purchase cost of the end user, and improves the cost performance and use efficiency of the equipment.

8. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The central processing control system is connected to the temperature control system, optoelectronic system, wireless and wired transmission systems in the detection module; the central processing unit controls the heating and heat dissipation module according to the preset program to adjust the temperature of the molecular detection equipment in real time; the central processing system adopts the PID control principle to accurately control the reaction temperature. When the measured temperature collected by the temperature sensor device deviates from the desired given value, the PID control performs proportional, integral and differential operations based on the deviation between the feedback measurement signal and the given value, thereby outputting a proper control signal to the electric heating block and the small fan, prompting the measurement value to return to the given value, thereby achieving the effect of automatic control.

9. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: Before the reaction starts, the parameters required for the polymerase chain reaction are set by a host computer, a computer or a handheld terminal, and transmitted to the central processing control system through a data communication system to control the reaction process. The parameters include temperature, time and data transmission.

10. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: The heating module and the heat dissipation module can adjust the uniformity of the polymerase chain reaction temperature to ±0.1°C through the central processing control system.

11. The constant temperature real-time fluorescence quantitative multi-channel (96) rapid molecular detection device based on the LAMP method according to claim 1, characterized in that: After the polymerase chain reaction is completed, the central processing control system controls the temperature of the reaction table to rise, causing the DNA double helix structure to degrade; during this process, the fluorescent signal sensing system (Figure 5) collects the fluorescent signal, and the filtered fluorescent signal is converted into an electrical signal by the photoelectric converter and transmitted to the central processing unit. According to the pre-set program, the central processing unit can send the signal to the host computer or handheld device through the data communication system; the host computer or handheld terminal completes the specific analysis of the polymerase chain reaction product by analyzing the melting curve and melting temperature, and then obtains the test result.

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