Intelligent PCR (polymerase chain reaction) and sample treatment system for agricultural breeding and liquid level detection method

Abstract

The invention discloses an intelligent PCR and sample treatment system for agricultural breeding and a liquid level detection method, and relates to the technical field of agricultural breeding. The pipette body, the air pressure sensor, the Z-axis driving unit, the material storage module, the refrigeration module, the material transfer module, the film sealing module, the PCR reaction module, the pneumatic adjusting unit and the processor are arranged on the operation table. The pipette body, the material storage module, the refrigeration module, the material transfer module, the film sealing module, the PCR reaction module and the processor are integrated through the operation table to form a full-process automatic system, so that the problems of large personal error and interference of environment and temperature factors in the market are solved.

Description

Technical Field

[0001] This application relates to the field of agricultural breeding technology, and more specifically, to an intelligent PCR and sample processing system and a liquid level detection method for agricultural breeding. Background Technology

[0002] As a core technology for genotyping in modern agricultural breeding, the construction of PCR systems, with its high throughput and high precision, directly determines breeding efficiency and the progress of large-scale application. With the breeding industry developing towards large-scale and precision, the demand for sample testing in large-scale breeding populations is becoming increasingly urgent, placing higher demands on the automation and integration of PCR system construction and sample processing. Traditional operating methods are no longer adequate for the efficient advancement requirements of modern breeding.

[0003] Currently, PCR-related sample processing and system construction in agricultural breeding mainly rely on manual operation or general-purpose automated liquid handling workstations. Manual operation requires repetitive dispensing, transfer, and mixing using pipettes, while general-purpose automated equipment can achieve partial automation of pipetting. However, both methods revolve around the core PCR operation and have not yet formed an integrated system that coordinates the entire process. Furthermore, as a core operating component, the reliability of the liquid level detection function of the pipette directly affects the pipetting accuracy and the accuracy of experimental results.

[0004] However, existing technologies have significant shortcomings: Firstly, they lack integration, resulting in extremely low throughput for manual operation, which cannot meet the needs of large-scale sample testing. Furthermore, they are prone to human error, leading to inaccurate pipetting volumes, cross-contamination, and other problems, resulting in high labor intensity and long breeding cycles. General-purpose automated equipment has limited functionality, only capable of pipetting operations. Steps such as sealing after PCR system construction and sample transfer to the PCR reaction module still require manual intervention, creating a fragmented process. Moreover, there is a lack of systematic recording of sample processing procedures and reagent consumption information, failing to meet the data traceability requirements of smart breeding. Secondly, the liquid level detection accuracy of pipettes is insufficient. Existing detection methods based on pressure sensors are easily affected by factors such as the physical properties of the liquid itself, ambient temperature and pressure, and the pipette's lifting and lowering speed. This requires manual adjustment of the pressure threshold by experimenters, making the operation complex and prone to human error. This makes it difficult to adapt to the precise detection needs of different types of reagents in agricultural breeding, thus affecting the accuracy of PCR system construction.

[0005] Therefore, it is necessary for the inventors to design a new intelligent PCR and sample processing system and liquid level detection method for agricultural breeding, in order to overcome the above problems by reducing pipetting errors and sample cross-contamination caused by human operation, and by incorporating a pressure sensor with temperature detection function into the pipette body and adding a pneumatic adjustment unit. Summary of the Invention

[0006] The main purpose of this application is to provide an intelligent PCR and sample processing system and liquid level detection method for agricultural breeding, so as to solve the problems of large human error and interference from environmental and temperature factors in the market.

[0007] To achieve the above objectives, this application provides an intelligent PCR and sample processing system for agricultural breeding, including an operating table, and a pipette body, a pressure sensor, a Z-axis drive unit, a material storage module, a refrigeration module, a material transfer module, a sealing module, a PCR reaction module, a pneumatic adjustment unit, and a processor disposed on the operating table. The pressure sensor is located inside the pipette body and has a temperature detection function, used to simultaneously collect pressure and temperature data inside the sealed pipette body; the pipette body is located on the output end of the Z-axis drive unit for lifting and lowering; the pneumatic adjustment unit includes a miniature air pump, a solenoid valve and a pressure adjustment pipeline, one end of which is connected to the sealed pipette body and the other end is connected to an external clean air source, used to receive control signals from the processor to adjust the pressure inside the pipette body; The processor is communicatively connected to the pipette body, pressure sensor, Z-axis drive unit, material transfer module, sealing module, PCR reaction module, and pneumatic adjustment unit. The processor is configured to: The control material transfer module removes the pipette tip box, PCR plate and reagents from the material storage module in the refrigeration module and transfers them to the operating table work area; The pipette body is controlled to load the pipette tip. Based on the detection data of the pressure sensor, the pressure is calibrated by the pneumatic adjustment unit. The Z-axis drive unit drives the pipette body to perform liquid dosing, aspiration and discharge operations to complete the construction of the PCR system. The material transfer module controls the transfer of the PCR plate containing the PCR system to the sealing module for sealing, and then to the PCR reaction module to perform the PCR reaction; after the reaction is completed, the material transfer module controls the return of the PCR plate and related materials to the material storage module. At the same time, the processing procedures, reagent consumption, and equipment operating parameters of each breeding sample are recorded to form a traceable data archive.

[0008] Optionally, the pipette body includes a single-channel spraying unit for precise spot spraying of liquid, a multi-channel pipetting unit for bulk liquid transfer, and a transport gripper for gripping and transporting PCR plates.

[0009] Optionally, the material transfer module includes a lifting and conveying shaft for transporting materials from the material storage module to the operating table, a material handling shaft for transporting the sealed PCR plates to the PCR reaction area, and a six-axis robotic arm for transporting the PCR plates to the PCR reaction module and returning them after the reaction; the refrigeration module is a refrigerator, and the material storage module is a material stack located inside the refrigerator.

[0010] Optionally, the processor is further configured to control the pipette body to perform a cleaning action after each liquid dosing, aspiration and discharge operation. The cleaning method is deionized water rinsing + nitrogen drying, wherein the deionized water rinsing time is 3-5 seconds, the nitrogen drying time is 2-3 seconds, and the nitrogen pressure is 0.1-0.3 MPa.

[0011] A method for pipette level detection in an integrated intelligent PCR device for agricultural breeding includes the following steps: Step 1: Set up the detection system through the operating table of the integrated device, use the processor to connect the pipette body, air pressure sensor, Z-axis drive unit and pneumatic adjustment unit, complete the initial calibration of the reagent liquid level through the host computer software, and prepare the reagents, pipette tips, pipette tip box and waste pipette tip box required for the experiment. Step 2: Initiate the air pressure calibration process via the processor, and control the air pressure sensor to synchronously acquire the initial air pressure value inside the sealed pipette body. Compared with the initial temperature value Simultaneously, the air pressure value P of the external environment is collected by a barometric pressure sensor; calibration is based on the formula P = Calculate the calibration pressure value, and adjust the internal pressure of the pipette body to the calibration value P using the pneumatic adjustment unit, ensuring that |Pcalibration - Pexternal| ≤ 3 Pa; where... Standard ambient temperature; Step 3: The processor plans the motion parameters of the Z-axis drive unit. These parameters include an initial velocity of 5-15 mm / s, acceleration parameters of 50-200 mm / s², acceleration time of 10-50 ms, constant speed parameters, deceleration parameters, and stopping parameters. The pipette body is controlled to perform lifting and lowering tests according to these motion parameters. The air pressure waveform uploaded by the air pressure sensor is acquired in real time. The stability of the air pressure waveform is judged by the standard that the difference between the maximum and minimum air pressure values ​​within 500 ms is ≤5 Pa. If the difference is >5 Pa, the motion parameters are adjusted and the test is repeated until the air pressure waveform meets the stability standard. This motion parameter is then determined as the fixed motion parameter for liquid level detection. When the constant speed is >11 mm / s, the deceleration value of the deceleration stage is adjusted to 150-200 mm / s² and the deceleration time is 30-50 ms to ensure that the final velocity is ≤1 mm / s. Step 4: The processor controls the pipette body to load the pipette tip, move it to a preset position above the reagent liquid surface, and execute the air pressure calibration process of Step 2 again. Then, the Z-axis drive unit is driven to lower the pipette body according to the motion parameters determined in Step 3, so that the pipette tip contacts the reagent liquid surface. At the same time, the air pressure sensor is controlled to collect air pressure waveform data throughout the liquid contact process. After the collection is completed, the pipette body is controlled to unload the pipette tip into the waste pipette tip box, replace it with a new pipette tip, and repeat the above collection process 3-5 times to obtain multiple collections of air pressure waveform data. Step 5: Analyze the collected air pressure waveform data through the host computer software, extract feature information such as air pressure step increment range, step waveform acquisition time range, maximum step value range, minimum step value range and step threshold band, assign a unique identifier to the current reagent, associate the unique identifier with the corresponding feature information and store it to establish a reagent air pressure waveform database. Step 6: When performing liquid level detection, the target reagent is selected through the host computer software. The processor calls the feature information corresponding to the reagent in the database, controls the pipette body to load the pipette tip and completes the air pressure calibration in step 2, and drives the Z-axis drive unit to descend according to the motion parameters determined in step 3. The air pressure waveform characteristics during the pipette tip's contact with the liquid are collected in real time and matched item by item with the feature information in the database. If the matching degree of all feature information is ≥95%, it is determined that the liquid level has been detected. The processor immediately controls the Z-axis drive unit to stop moving and sends a successful detection signal back to the host computer software.

[0012] Optionally, the initial velocity in step 3 is in the range of 5-15 mm / s; the relationship between the acceleration value and acceleration time during the acceleration phase is as follows: when the acceleration value is 50-100 mm / s², the acceleration time is 30-50 ms; when the acceleration value is 101-200 mm / s², the acceleration time is 10-29 ms; the relationship is fixed in the processor by a preset algorithm, which is used to maintain the relationship between acceleration and acceleration time according to the real-time motion feedback of the Z-axis drive unit, and ensure that the stability of the air pressure waveform during the movement of the Z-axis drive unit meets the judgment criterion that the difference between the maximum and minimum air pressure values ​​within 500 ms is ≤5 Pa in step 3.

[0013] Optionally, the reagent pressure waveform database established in step 5 is deployed on a cloud server, supporting multiple integrated intelligent PCR devices for agricultural breeding to access and share it through the following methods: Access Authentication: Devices complete registration and authentication on the cloud server using their unique hardware identifier. Once authentication is successful, data access permissions are granted. Data Acquisition: For integrated devices that do not store target reagent information, a data acquisition request is initiated by inputting the target reagent name or unique identifier through the host computer software. After the cloud server verifies the permissions, it transmits the unique identifier and characteristic information corresponding to the reagent to the requesting device in an encrypted format. Data application: After the requesting device receives the data, it automatically stores it in the local database and directly uses it for the feature matching process of subsequent liquid level detection, without having to repeat the data acquisition and feature extraction process in steps 4-5.

[0014] Optionally, during the air pressure calibration process in step 2, if the difference between the internal air pressure of the pipette body and the external air pressure is greater than 3Pa after the first adjustment, the acquisition-calculation-adjustment process is repeated until the difference is less than or equal to 3Pa, and the time interval between each repeated adjustment is 100ms.

[0015] Optionally, the specific height of the preset position mentioned in step 4 can be adjusted by the host computer software according to the reagent type, with an adjustment accuracy of ±0.5mm.

[0016] Optionally, in step 4, when the pipette tip contacts the reagent liquid surface, the step phenomenon of the gas pressure waveform collected by the gas pressure sensor meets the following requirements: the step increment is ≥10Pa and the step duration is 5-20ms. This step waveform data is the core basis for feature information extraction.

[0017] The present invention provides an intelligent PCR and sample processing system and liquid level detection method for agricultural breeding, which, compared with the prior art, has the following advantages: The system integrates a pipette body, material storage module, refrigeration module, material transfer module, sealing module, PCR reaction module, pneumatic control unit, and processor into a fully automated system. The processor coordinates the operation of each module, enabling a fully automated process from material extraction, PCR system construction, sealing, PCR reaction to material return without human intervention. This effectively increases the throughput of large-scale breeding samples and reduces pipetting errors and cross-contamination caused by human operation. Simultaneously, the processor records the processing flow, reagent consumption, and equipment operating parameters for each sample, creating a traceable data archive. This meets the data standardization management requirements of smart breeding and solves the problems of fragmented processes and untraceable data.

[0018] To address the shortcomings of existing pipettes in terms of insufficient accuracy and poor adaptability in liquid level detection, this invention incorporates a pressure sensor with temperature detection within the pipette body. A pneumatic adjustment unit is added, and the processor, based on the ideal gas law, performs pressure calibration by combining temperature and pressure data synchronously collected by the pressure sensor, eliminating interference from environmental and temperature factors. By planning the motion parameters of the Z-axis drive unit and clarifying the definitions of each parameter and closed-loop control logic, the pressure waveform is ensured to be stable during the pipette body's ascent and descent, providing a stable motion basis for detection. A reagent pressure waveform database is established using host computer software. Liquid level detection is achieved by matching the real-time collected pressure waveform characteristics with the database information, eliminating the need for manual threshold adjustment. This adapts to reagents with different physical properties, significantly improving the accuracy and adaptability of liquid level detection, thereby ensuring the precision of liquid transfer in PCR system construction. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the application and to make other features, objects, and advantages of the application more apparent. The illustrative embodiments and descriptions of this application are used to explain the application and do not constitute an undue limitation of the application. In the drawings: Figure 1 This is a schematic diagram of the overall structure of the present invention; Figure 2 This is a schematic diagram of the liquid level detection method of the present invention.

[0020] The components include: 1. Material storage module; 2. Pipette body; 3. Z-axis drive unit; 4. Lifting and transfer axis; 5. Material handling axis; 6. Sealing module; 7. Six-axis robotic arm; 8. PCR reaction module; 9. Refrigeration module; and 10. Pressure sensor. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0022] It should be noted that the terms "first," "second," etc., in the specification and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be used interchangeably where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0023] In this application, the terms "upper," "lower," "left," "right," "front," "rear," "top," "bottom," "inner," "outer," "middle," "vertical," "horizontal," "lateral," and "longitudinal" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for the purpose of better describing this application and its embodiments, and are not intended to limit the indicated devices, elements, or components to having a specific orientation, or to be constructed and operated in a specific orientation.

[0024] Furthermore, some of the aforementioned terms, besides indicating location or positional relationships, may also have other meanings. For example, the term "above" may, in certain circumstances, indicate a dependency or connection. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0025] In addition, the term "multiple" should mean two or more.

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0027] Example 1, as Figures 1 to 2 As shown, an intelligent PCR and sample processing system for agricultural breeding includes: an operating table, and a pipette body 2, a pressure sensor 10, a Z-axis drive unit 3, a material storage module 1, a refrigeration module 9, a material transfer module, a sealing module 6, a PCR reaction module 8, a pneumatic adjustment unit, and a processor, all mounted on the operating table.

[0028] The pipette body 2 is the core operating component of the system. It integrates a single-channel spray unit, a multi-channel pipetting unit, and a transport gripper. The single-channel spray unit is used to achieve precise spot spraying of liquids with an accuracy of ±0.1μL, meeting the precise addition requirements of specific reagents in PCR system construction. The multi-channel pipetting unit can realize batch liquid transfer, adapting to the high-throughput requirements of large-scale sample processing in agricultural breeding. The transport gripper is used for gripping and transporting PCR plates, with a clamping force of 5-10N, ensuring the stable transfer of PCR plates between various processes.

[0029] The pressure sensor 10 is built into the pipette body 2. It is a high-precision MEMS pressure sensor with a measurement range of 0-200kPa and an accuracy of ±0.1Pa. The temperature measurement range is -40℃-85℃ with an accuracy of ±0.1℃. It can simultaneously collect pressure and temperature data inside the sealed pipette body 2, providing basic test data for subsequent pressure calibration and avoiding the influence of ambient temperature and pressure fluctuations on the accuracy of liquid level detection.

[0030] The pneumatic adjustment unit includes a miniature air pump with a working pressure of 0-0.5MPa and a flow rate of 1L / min, an electromagnetic reversing valve with a response time of ≤10ms, and a polytetrafluoroethylene (PTFE) air pressure regulating pipeline with an inner diameter of 2mm. One end of the pipeline is connected to the sealed body of the pipette 2 through a sealed interface, and the other end is connected to an external clean air source with a filtration accuracy of 0.1μm. The pipeline is used to receive PWM control signals from the processor and precisely adjust the air pressure inside the pipette through inflation and deflation actions.

[0031] The Z-axis drive unit 3 uses a ball screw slide, and its output end is fixedly connected to the pipette body 2 through a flange. Under the drive of the processor's pulse control signal, it realizes the vertical lifting and lowering movement of the pipette body 2, providing stable motion support for the liquid aspiration, discharge and liquid level detection of the pipette body 2.

[0032] The material storage module 1 is a layered material stack located inside the refrigeration module 9. It is used to classify and store the pipette tip boxes, PCR plates and various reagents required for the experiment. The refrigeration module 9 adopts a small embedded refrigerator, which can maintain the stability of the internal storage environment and ensure that the properties of the reagents are not affected during storage.

[0033] The material transfer module includes a lifting and conveying shaft 4, a material handling shaft 5, and a six-axis robotic arm 7. The lifting and conveying shaft 4 uses an electric push rod to transport materials from the material storage module 1 in the refrigeration module 9 to the work area of ​​the operating table. The material handling shaft 5 uses a belt conveyor to transport PCR plates sealed by the sealing module 6 to the PCR reaction area. The six-axis robotic arm 7 is responsible for transporting PCR plates from the PCR reaction area to the PCR reaction module 8, and returning the PCR plates to the material storage module 1 after the PCR reaction is completed. Through the collaboration of multiple components, the automated transfer of materials between various functional modules is realized.

[0034] The sealing module 6 uses a heat-sealing sealing machine to seal the PCR plate after the PCR system has been constructed, preventing liquid evaporation or cross-contamination between samples during the PCR reaction. The PCR reaction module 8 is a real-time fluorescence quantitative PCR instrument used to perform PCR amplification reactions. The processor is an industrial-grade microcontroller that communicates with the above functional modules via RS485 or Ethernet, serving as the control core of the system and coordinating the working sequence and action execution of each module.

[0035] The processor's control logic is as follows: First, it sends a command to the material transfer module via the RS485 bus to control the lifting and conveying shaft 4 to retrieve the pipette tip box, PCR plate, and reagents from the material storage module 1 within the refrigeration module 9, and precisely transfer them to the operating area of ​​the worktable with a positioning accuracy of ±0.5mm. Then, it controls the gripper mechanism of the pipette body 2 to load the pipette tip. The pressure sensor 10 collects the initial pressure inside the pipette, P1 = 101300Pa, the initial temperature T1 = 295.15K (22℃), and the external air pressure P_external = 101298Pa. According to the formula P_calibration = 101300 × (296.15 / 295.15) ≈ 101640Pa, it controls the micro-pump of the pneumatic adjustment unit to start, filling the pipette body with 340Pa of air pressure until the pressure sensor detects that the pressure inside the pipette body reaches 101640Pa. At this point, the internal and external pressure difference is... 2Pa≤3Pa, complete the gas pressure calibration; then drive the Z-axis drive unit 3 to drive the pipette body 2, and perform liquid spot spraying, aspiration and discharge operations through the single-channel spray unit and multi-channel pipetting unit to gradually complete the construction of the PCR system; after each liquid operation, the processor controls the pipette body 2 to open the cleaning channel, rinse with 0.2MPa deionized water for 4s, and then blow dry with 0.15MPa nitrogen for 3s to avoid cross-contamination.

[0036] After the PCR system is constructed, the material transport axis 5 transfers the PCR plate carrying the PCR system to the sealing module 6, where it is heat-sealed at 120°C for 2 seconds. Then, the six-axis robotic arm 7 transports the sealed PCR plate to the PCR reaction module 8, sets the PCR reaction program (pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, 40 cycles), and executes the reaction. After the PCR reaction, the six-axis robotic arm 7 returns the PCR plate and related experimental materials to the designated layer in the material storage module 1. Simultaneously, the processor records the processing flow of each breeding sample in real time via SD card throughout the experiment, including pipetting time, sealing parameters, reaction program, reagent consumption (e.g., volume of each reagent used, pipette tip consumption), and equipment operating parameters (e.g., voltage, current, temperature of each module), forming a traceable data archive, which is then exported to Excel format via the host computer software.

[0037] Pipette liquid level detection method Step 1: Set up the detection system. Based on the operating table, establish communication between the pipette body 2, the pressure sensor 10, the Z-axis drive unit 3, and the pneumatic adjustment unit through the processor's GPIO interface to ensure that all components work together. Complete the initial calibration of the reagent liquid level through the host computer software, and use the CCD vision sensor to assist in positioning, with a calibration accuracy of ±0.1mm. At the same time, prepare the reagents required for the experiment, such as alcohol, PCR buffer, primer mixture, etc. Select pipette tips of 10μL, 100μL, or 1000μL according to the reagent volume, and prepare pipette tip boxes and waste pipette tip boxes. Place the pipette tip boxes in the pipette tip replenishment area of ​​the operating table, and place the waste pipette tip boxes in the waste recycling area.

[0038] Step 2: Initiate the pressure calibration process. The processor controls the pressure sensor 10 via the I2C bus to synchronously acquire the initial pressure value inside the sealed pipette body 2. Compared with the initial temperature value Simultaneously, the external air pressure value P is collected; the formula P calibration is derived based on the ideal gas law. Calculate the calibration pressure value, where The standard ambient temperature is 296.15K (23℃) by default, and can be calibrated according to the actual laboratory environment via host computer software. The processor sends a PWM control signal to the pneumatic adjustment unit to control the opening of the solenoid valve and the start of the micro air pump, which fills or evacuates air into the gun body through the air pressure adjustment pipeline until the air pressure sensor 10 detects that the air pressure inside the gun body reaches the P calibration value, and |P calibration - P external| ≤ 3Pa. For example: when the pressure sensor 10 detects that the air pressure inside the gun body reaches the P calibration value, |P calibration - P external| ≤ 3Pa. =100500Pa When the pressure is 300.15K (27℃) and P_external = 100497Pa, the calculated P_calibration = 100500 × (296.15 / 300.15) ≈ 99170Pa. The processor controls the pneumatic adjustment unit to extract 1330Pa of air pressure, so that the air pressure inside the gun is reduced to 99170Pa. At this time, the pressure difference = 3Pa, which meets the requirements. If the pressure difference is 5Pa after the first adjustment, the data is collected again and adjusted at 100ms intervals until the pressure difference ≤ 3Pa.

[0039] Step 3: Plan the motion parameters of the Z-axis drive unit 3 through the processor's motion control module. These parameters include: initial velocity (5-15 mm / s); acceleration phase parameters (acceleration value 50-200 mm / s², acceleration time 10-50 ms); constant velocity phase parameters (constant velocity 10-50 mm / s, constant velocity time 50-200 ms); deceleration phase parameters (deceleration value 50-200 mm / s², deceleration time 10-50 ms); and stopping parameters (final velocity ≤ 1 mm / s and displacement deviation ≤ ±0.1 mm upon stopping). The correspondence between acceleration value and acceleration time during the acceleration phase is as follows: when the acceleration value is 50-100 mm / s², the acceleration time is 30-50 ms; when the acceleration value is 101-200 mm / s², the acceleration time is 30-50 ms; when the acceleration value is 101-200 mm / s², the acceleration time is 30-50 ms; and when the acceleration value is 101-200 mm / s², the acceleration time is 30-50 ms. At that time, the acceleration time is 10-29ms. This correspondence is fixed in the processor's Flash memory through a preset algorithm. The algorithm dynamically adjusts the output pulse frequency by acquiring the encoder feedback signal of the Z-axis drive unit 3 in real time, so as to maintain the correspondence between acceleration and acceleration time.

[0040] The processor controls the pipette body 2 to perform lifting and lowering tests according to the initially set motion parameters, acquiring real-time pressure waveform data uploaded by the pressure sensor 10 at a sampling frequency of 1kHz. The stability of the pressure waveform is judged by a standard that the difference between the maximum and minimum pressure values ​​within 500ms is ≤5Pa. If the difference is >5Pa, the motion parameters are adjusted, such as decreasing the acceleration value, extending the acceleration time, and the test is repeated until the pressure waveform meets the stability standard. When the uniform speed is >11mm / s, the deceleration value during the deceleration phase is adjusted to 150-200mm / s², and the deceleration time is adjusted to 30-50ms to ensure the final velocity is ≤1mm / s. For example: Setting the uniform speed to 15 mm / s, adjusting the deceleration phase parameters to a deceleration value of 200 mm / s² and a deceleration time of 40 ms, the calculated final velocity is 15 - 200 × 0.04 = 7 mm / s. This still does not meet the requirement. Further extending the deceleration time to 75 ms, the final velocity becomes 15 - 200 × 0.075 = 0 mm / s, meeting the requirement of a final velocity ≤ 1 mm / s. During the test, the air pressure sensor 10 transmits data showing a maximum air pressure of 99172 Pa and a minimum of 99168 Pa within a continuous 500 ms. The difference is 4 Pa ​​≤ 5 Pa, indicating a stable air pressure waveform. This motion parameter is the fixed motion parameter for liquid surface detection.

[0041] Step 4: The processor controls the gripper mechanism of the pipette body 2 to load the pipette tip and moves it to a preset position above the reagent liquid surface via the Z-axis drive unit 3, 10-20mm away from the initial calibration position of the liquid surface. For example, for high-viscosity PCR buffer, the preset position is set to 20mm; for low-viscosity alcohol, the preset position is set to 15mm. The air pressure calibration process in Step 2 is executed again to ensure that the air pressure benchmark is consistent in this acquisition environment. Then, the Z-axis drive unit 3 is driven to lower the pipette body 2 according to the fixed motion parameters determined in Step 3, so that the pipette tip contacts the reagent liquid surface. During this process, the processor controls the air pressure sensor 10 to collect air pressure waveform data throughout the liquid contact process at a sampling frequency of 1kHz. After the acquisition is completed, the processor controls the pipette body 2 to unload the pipette tip into the waste pipette tip box, replace it with a new pipette tip, and repeat the above acquisition process 3-5 times to obtain multiple acquisitions of air pressure waveform data to ensure data reliability. When the pipette tip contacts the reagent liquid surface, the pressure waveform collected by the pressure sensor 10 will show a significant step phenomenon. This step phenomenon meets the requirements of a step increment ≥ 10 Pa and a step duration of 5-20 ms. For example, when collecting alcohol reagent, the step increment is 12 Pa and the duration is 10 ms. This step waveform data is the core basis for subsequent feature information extraction.

[0042] Step 5: Filter the collected pressure waveform data using the host computer software. After noise reduction using the Kalman filter algorithm, extract feature information such as the pressure step increment range, step waveform acquisition time range, maximum step value range, minimum step value range, and step threshold band. Assign a unique identifier to the current reagent and upload the unique identifier and corresponding feature information to the cloud server via TCP / IP protocol to establish a reagent pressure waveform database. This database supports access and sharing from multiple intelligent PCR and sample processing systems described in this invention: devices register and authenticate on the cloud server using their unique hardware identifier, such as a MAC address, and gain data access permissions after successful authentication; integrated devices that do not store target reagent information initiate a data acquisition request by inputting the target reagent name or unique identifier through the host computer software. After verifying permissions, the cloud server transmits the unique identifier and feature information corresponding to the reagent to the requesting device in AES encrypted format; after receiving the data, the requesting device automatically stores it in the local SD card database, which is directly used for the subsequent liquid level detection feature matching process, without repeating the data acquisition and feature extraction process of steps 4-5.

[0043] Step 6: During liquid level detection, the operator selects the target reagent through the visual interface of the host computer software. After receiving the instruction, the processor calls the corresponding feature information of the target reagent from the reagent pressure waveform database via the network; controls the pipette body 2 to load the corresponding specification tip and complete the pressure calibration in step 2, and drives the Z-axis drive unit 3 to drive the pipette body 2 to descend according to the fixed motion parameters determined in step 3; during this process, the pressure waveform features of the tip touching the liquid are collected in real time, and the real-time features are matched item by item with the target reagent feature information stored in the database. The matching algorithm adopts the Euclidean distance matching method; if the matching degree of all feature information is ≥95%, it is determined that the tip of the pipette body 2 has detected the liquid surface, and the processor immediately sends a stop signal to the Z-axis drive unit 3 to control it to stop moving, and at the same time sends a signal of successful detection to the host computer software, completing the liquid level detection process; if the matching degree is <95%, it continues to descend 0.5mm and collects matching again, until three consecutive matching failures are reported and a detection failure signal is sent.

[0044] Pressure-temperature proportionality coefficient calculation In a closed system, when the pipette is not aspirating or dispensing liquid, the air volume V and the amount of substance n inside the pipette can be considered constant. According to the ideal gas law PV=nRT, where P is pressure, V is volume, n is amount of substance, R is the ideal gas constant with a value of 8.314 J / (mol・K), and T is absolute temperature, it can be deduced that P is proportional to T, i.e., P=K×T, where K=nR / V is the temperature-pressure proportionality coefficient.

[0045] At a standard ambient temperature of 23℃ (296.15K), 1000 sets of air pressure data were continuously collected within the sealed body of the pipette using air pressure sensor 10. The average value was then substituted into the calculation formula of the pressure sensor chip to obtain the current average air pressure P = 0.001297 PSI, which is equivalent to 8.9426 Pa in SI units. Substituting P = 8.9426 Pa and T = 296.15 K into P = K × T, we calculated K = 8.9426 / 296.15 ≈ 0.0302 Pa / K. This parameter is specific to the sealed space of the self-developed pipette on this platform and provides core physical parameter support for air pressure calibration.

[0046] Workflow Before the experiment begins, the operator uses the host computer software to complete the initial calibration of reagent liquid levels and prepare related materials. The processor initializes each functional module and completes the gas pressure calibration and motion parameter planning for the Z-axis drive unit 3. After the experiment starts, the processor controls the material transfer module to take out the pipette tip box, PCR plate, and reagents from the material stack in the refrigeration module 9 and transfer them to the operating area. After the pipette body 2 is loaded with pipette tips, it accurately performs liquid spot spraying, aspiration, and discharge operations with the support of the liquid level detection method to complete the PCR system construction. A cleaning action is performed after each liquid operation. Then, the PCR plate is transferred to the sealing module 6 for sealing via the material transfer module, and then transferred to the PCR reaction module 8 to perform the PCR reaction. After the reaction is completed, the PCR plate and related materials are returned to the material stack via the material transfer module. The processor records the entire process data to form a traceable archive. The entire process realizes full automation of PCR system construction and sample processing without manual intervention, effectively solving the problems of low throughput, large error, and fragmented process in traditional operations.

[0047] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A smart PCR and sample processing system for agricultural breeding, characterized in that, Includes: an operating table, and a pipette body (2), a pressure sensor (10), a Z-axis drive unit (3), a material storage module (1), a refrigeration module (9), a material transfer module, a sealing module (6), a PCR reaction module (8), a pneumatic adjustment unit, and a processor, all mounted on the operating table. The pressure sensor (10) is placed inside the pipette body (2) and has a temperature detection function, used to synchronously collect the pressure data and temperature data inside the sealed pipette body (2); the pipette body (2) is located on the output end of the Z-axis drive unit (3) for lifting and lowering; the pneumatic adjustment unit is connected to the pipette body (2) for adjusting the internal pressure of the pipette body (2); The processor is communicatively connected to the pipette body (2), the pressure sensor (10), the Z-axis drive unit (3), the material transfer module, the sealing module (6), the PCR reaction module (8), and the pneumatic adjustment unit; The processor is configured to: The control material transfer module takes out the pipette tip box, PCR plate and reagents from the material storage module (1) in the refrigeration module (9) and transfers them to the operating table work area; Control the pipette body (2) to load the pipette tip, and based on the detection data of the pressure sensor (10), complete the pressure calibration through the pneumatic adjustment unit, drive the Z-axis drive unit (3) to drive the pipette body (2) to perform liquid dotting, aspiration and discharge operations, and complete the construction of the PCR system; The material transfer module controls the transfer of the PCR plate carrying the PCR system to the sealing module (6) for sealing, and then to the PCR reaction module (8) to perform the PCR reaction; after the reaction is completed, the material transfer module controls the return of the PCR plate and related materials to the material storage module (1). At the same time, the processing procedures, reagent consumption, and equipment operating parameters of each breeding sample are recorded to form a traceable data archive.

2. The intelligent PCR and sample processing system for agricultural breeding according to claim 1, characterized in that, The pipette body (2) includes a single-channel spraying unit for precise spot spraying of liquid, a multi-channel pipetting unit for batch liquid transfer, and a transport gripper for gripping and transporting PCR plates.

3. The intelligent PCR and sample processing system for agricultural breeding according to claim 1, characterized in that, The material transfer module includes a lifting transfer shaft (4) for transporting materials from the material storage module (1) to the operating table, a material transport shaft (5) for transporting sealed PCR plates to the PCR reaction area, and a six-axis robotic arm (7) for transporting PCR plates to the PCR reaction module (8) and returning them after the reaction; the refrigeration module (9) is a refrigerator, and the material storage module (1) is a material stack and is located inside the refrigerator.

4. The intelligent PCR and sample processing system for agricultural breeding according to claim 1, characterized in that, The processor is also configured to control the pipette body (2) to perform a cleaning action after each liquid dosing, aspiration and discharge operation. The cleaning method is deionized water rinsing + nitrogen drying, wherein the deionized water rinsing time is 3-5s, the nitrogen drying time is 2-3s, and the nitrogen pressure is 0.1-0.3MPa.

5. A method for detecting liquid level in a pipette used in an integrated intelligent PCR device for agricultural breeding, characterized in that, The intelligent PCR and sample processing system for agricultural breeding as described in any one of claims 1-4 includes the following steps: Step 1: Build a detection system through the operating table of the integrated device, use the processor to connect the pipette body (2), the air pressure sensor (10), the Z-axis drive unit (3) and the pneumatic adjustment unit, and complete the initial calibration of the reagent liquid level through the host computer software, and prepare the reagents, pipette tips, pipette tip box and waste pipette tip box required for the experiment. Step 2: Start the air pressure calibration process through the processor and control the air pressure sensor (10) to synchronously collect the initial air pressure value inside the sealed pipette body (2). Compared with the initial temperature value Simultaneously, the air pressure value P of the external environment is collected by the air pressure sensor (10); calibration is based on the formula P = Calculate the calibration air pressure value, and adjust the internal air pressure of the pipette body (2) to the calibration value P using the pneumatic adjustment unit to ensure that |Pcalibration - Pexternal| ≤ 3Pa; where, Standard ambient temperature; Step 3: The processor plans the motion parameters of the Z-axis drive unit (3), which include an initial velocity of 5-15 mm / s, acceleration parameters of 50-200 mm / s², acceleration time of 10-50 ms, constant speed parameters, deceleration parameters, and stopping parameters; the pipette body (2) is controlled to perform lifting and lowering tests according to the motion parameters, and the air pressure waveform uploaded by the air pressure sensor (10) is acquired in real time. The difference between the maximum and minimum air pressure values ​​within 500 ms is ≤5 Pa as the standard to judge whether the air pressure waveform is stable. If the difference is >5 Pa, the motion parameters are adjusted and the test is repeated until the air pressure waveform meets the stability standard. The motion parameters are determined to be the fixed motion parameters for liquid surface detection. When the constant speed is >11 mm / s, the deceleration value of the deceleration stage is adjusted to 150-200 mm / s² and the deceleration time is 30-50 ms to ensure that the final speed is ≤1 mm / s. Step 4: The processor controls the pipette body (2) to load the pipette tip, move it to the preset position above the reagent liquid surface and execute the air pressure calibration process of Step 2 again. Then, the Z-axis drive unit (3) drives the pipette body (2) to descend according to the motion parameters determined in Step 3, so that the pipette tip contacts the reagent liquid surface. At the same time, the air pressure sensor (10) is controlled to collect the air pressure waveform data during the contact process. After the collection is completed, the pipette body (2) is controlled to unload the pipette tip into the waste pipette tip box, replace it with a new pipette tip and repeat the above collection process 3-5 times to obtain the air pressure waveform data collected multiple times. Step 5: Analyze the collected air pressure waveform data through the host computer software, extract feature information such as air pressure step increment range, step waveform acquisition time range, maximum step value range, minimum step value range and step threshold band, assign a unique identifier to the current reagent, associate the unique identifier with the corresponding feature information and store it to establish a reagent air pressure waveform database. Step 6: When performing liquid level detection, the target reagent is selected through the host computer software. The processor calls the feature information corresponding to the reagent in the database, controls the pipette body (2) to load the pipette tip and complete the air pressure calibration in step 2, and drives the Z-axis drive unit (3) to descend according to the motion parameters determined in step 3. The air pressure waveform characteristics during the pipette tip contact with the liquid are collected in real time and matched with the feature information in the database item by item. If the matching degree of all feature information is ≥95%, it is determined that the liquid level has been detected. The processor immediately controls the Z-axis drive unit (3) to stop moving and sends a successful detection signal to the host computer software.

6. The pipette level detection method according to claim 5, characterized in that, The initial velocity in step 3 ranges from 5 to 15 mm / s. The acceleration value and acceleration time during the acceleration phase correspond to the following: when the acceleration value is 50-100 mm / s², the acceleration time is 30-50 ms; when the acceleration value is 101-200 mm / s², the acceleration time is 10-29 ms. This correspondence is fixed in the processor by a preset algorithm. This preset algorithm is used to maintain the correspondence between acceleration and acceleration time based on the real-time motion feedback of the Z-axis drive unit, ensuring that the stability of the air pressure waveform during the movement of the Z-axis drive unit meets the judgment criterion that the difference between the maximum and minimum air pressure values ​​within 500 ms is ≤ 5 Pa in step 3.

7. The pipette level detection method according to claim 5, characterized in that, The reagent pressure waveform database established in step 5 is deployed on a cloud server, supporting multiple integrated intelligent PCR devices for agricultural breeding to access and share it through the following methods: Access Authentication: Devices complete registration and authentication on the cloud server using their unique hardware identifier. Once authentication is successful, data access permissions are granted. Data Acquisition: For integrated devices that do not store target reagent information, a data acquisition request is initiated by inputting the target reagent name or unique identifier through the host computer software. After the cloud server verifies the permissions, it transmits the unique identifier and characteristic information corresponding to the reagent to the requesting device in an encrypted format. Data application: After the requesting device receives the data, it automatically stores it in the local database and directly uses it for the feature matching process of subsequent liquid level detection, without having to repeat the data acquisition and feature extraction process in steps 4-5.

8. The pipette level detection method according to claim 5, characterized in that, During the air pressure calibration process in step 2, if the difference between the internal air pressure of the pipette body (2) and the external air pressure is greater than 3Pa after the first adjustment, the collection-calculation-adjustment process is repeated until the difference is less than or equal to 3Pa. The time interval between each repeated adjustment is 100ms.

9. The pipette level detection method according to claim 5, characterized in that, The specific height of the preset position mentioned in step 4 can be adjusted by the host computer software according to the reagent type, with an adjustment accuracy of ±0.5mm.

10. The pipette level detection method according to claim 5, characterized in that, When the pipette tip contacts the reagent liquid surface in step 4, the step phenomenon of the gas pressure waveform collected by the gas pressure sensor (10) meets the following conditions: the step increment is ≥10Pa and the step duration is 5-20ms. This step waveform data is the core basis for feature information extraction.

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