PCR hot lid system with pressure detection and well plate recognition and method

Abstract

This invention relates to the field of image recognition technology and discloses a PCR hot cap system and method with pressure detection and well plate recognition. The system includes acquiring images of the reaction well positions on the sample stage and locating the center pixel coordinates; generating well plate recognition results based on the center pixel coordinates; generating a set of hot cap pressing parameters based on the well plate recognition results and outputting a hot cap pressing control command to enter the pressure sampling stage; acquiring pressure sampling values ​​output by an array-type thin-film pressure sensor and generating an initial well pressure set; stopping pressing and generating a pressure distribution map when the initial well pressure set meets the target pressure; performing pressure uniformity judgment based on the pressure distribution map; alarming and re-identifying when the pressure uniformity judgment is not valid; heating and continuously acquiring the target well pressure set when the pressure uniformity judgment is valid; and fine-tuning the pressing or lifting position when the average pressure value deviates from the target pressure by a preset deviation value, thereby achieving consumable self-adaptation and dynamic pressure compensation.

Description

Technical Field

[0001] This invention relates to the field of image recognition technology, and more specifically, to a PCR hot cap system and method with pressure detection and well plate recognition. Background Technology

[0002] In polymerase chain reaction (PCR) processes, heated caps are used to apply pressure to reaction consumables and, in conjunction with temperature control, to achieve reliable sealing, thereby reducing adverse effects such as evaporation and condensation and ensuring the stability of the reaction system during heating and cooling cycles. With the diversification of PCR application scenarios and consumable specifications, the adaptability of heated caps to different plate types and different placement states, as well as their pressure stability, have gradually become key factors affecting experimental consistency and automation levels.

[0003] However, existing heated lid systems still have two core problems that have not been solved simultaneously. First, heated lid systems lack the ability to detect reaction plate information and placement status. Specifically, they cannot automatically identify whether a reaction plate has been placed, the type of reaction plate, its placement location, or whether sample loading has been completed. Second, conventional PCR instruments typically use mechanical spring pre-tensioning or fixed motor stroke setting methods for heated lid pressure adjustment. This adjustment mechanism is not adaptable enough. When faced with different brands and specifications of reaction consumables, it cannot automatically adapt to the optimal pressure parameters, requiring manual intervention for adjustment, thereby reducing equipment production efficiency. Moreover, it is an open-loop control system, lacking real-time feedback and compensation mechanisms. When the heated lid is in a high-temperature working environment for a long time, its internal mechanical structure may undergo slight deformation due to thermal expansion, causing the initial set pressure to deviate. Existing structures cannot monitor and dynamically compensate for such deviations in real time, ultimately affecting the accuracy of experimental results.

[0004] In view of this, the present invention proposes a PCR hot cap system and method with pressure detection and well plate recognition to solve the above problems. Summary of the Invention

[0005] To overcome the above-mentioned deficiencies of the prior art, the present invention provides a PCR hot cap system and method with pressure detection and well plate recognition.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In one aspect, a PCR hot capping method with pressure detection and well plate recognition is provided, including: S10: Acquire images of the reaction well positions on the sample stage, perform well position localization processing on the reaction well position images to obtain the center pixel coordinates of each reaction well, and generate well plate recognition results based on the center pixel coordinates. S20: Generate a set of hot cover pressing parameters based on the orifice plate recognition results, and output a hot cover pressing control command after receiving the user confirmation command and enter the pressure sampling stage; S30: Collects pressure sampling values ​​output by array-type thin-film pressure sensor, generates an initial orifice pressure set based on plate type, outputs a stop pressing command when the initial orifice pressure set meets the target pressure, and generates a pressure distribution map based on the initial orifice pressure set; S40: Calculate the pressure uniformity value set based on the pressure distribution map, perform pressure uniformity judgment based on the pressure uniformity value set and the judgment threshold set, and output alarm prompt information and re-identification command when the pressure uniformity judgment is not valid, and return to execute S10 after a preset number of times. S50: When the pressure uniformity judgment is valid, output the heating control command and continuously collect the target hole pressure set during the heating stage. Calculate the average pressure value based on the target hole pressure set and compare it with the target pressure. When the pressure deviation exceeds the preset deviation value, output the downward position fine adjustment command or the upward position fine adjustment command.

[0007] In some embodiments, the aperture plate recognition result is generated based on the center pixel coordinates, including: The candidate region detection process is performed on the reaction well image, and the center pixel coordinates of the well are calculated in each candidate region. The center coordinates are then collected to obtain a set. The number of rows and columns is counted within the central coordinate set, and placement orientation information is generated based on the number of rows and columns; Based on the number of rows and columns, matching entries are retrieved from the preset plate type template set to generate candidate plate type information. The hole spacing measurement value is calculated in the center coordinate set to complete the verification of the candidate plate type information and generate plate type information. Fluorescence signals corresponding to each well location are collected, and information on the state of liquid presence in the well is generated based on the fluorescence signals. The plate type information, placement information, and information on the state of liquid presence in the well are then combined to obtain the well plate identification result.

[0008] In some embodiments, matching entries are retrieved from a preset template set based on the number of rows and columns to generate candidate template information, including: A row and column index table is constructed based on the set of center coordinates. The row and column index table contains row index, column index and corresponding center pixel coordinate entries. Calculate the hole spacing measurement value in the row and column index table and write the hole spacing measurement value into the plate type verification table. The plate type verification table contains the associated entries of candidate plate type information and hole spacing measurement value. Output board type information based on the board type verification table.

[0009] In some embodiments, a set of hot cap pressing parameters is generated based on the orifice plate identification results, including: The plate type is read from the orifice plate identification results, and the target pressure item and judgment threshold set item corresponding to the plate type are retrieved from the consumable parameter table to generate the basic hot cover pressure parameter set; The target pressure entries within the basic hot cap pressure parameter set are subject to factory safety value limits, and the hot cap pressure parameter set with safety value limits is output after receiving a user confirmation command.

[0010] In some embodiments, pressure sampling values ​​output by an array-type thin-film pressure sensor are acquired, and an initial set of orifice pressures is generated based on the plate shape, including: The pressure sampling values ​​output by the independent sensing units of the array-type thin-film pressure sensor are collected and aggregated according to the sampling time and sensing unit identifier to form a pressure sampling array. The plate shape is read from the plate identification results and a hole position mapping table is generated based on the plate shape. The hole position mapping table contains associated entries of hole position index and independent sensing unit identifier. Based on the pore location mapping table, the corresponding pressure sampling values ​​are retrieved in the pressure sampling array and collected according to the pore location index to form an initial pore location pressure set.

[0011] In some embodiments, reading the plate type from the plate identification result and generating a hole location mapping table based on the plate type includes: When the plate type is an 8-tube or 96-hole plate, the hole position index is associated with the independent sensing unit identifier and written into the hole position mapping table. When the plate type is a 384-well plate, the independent sensing unit identifier is associated with the 2×2 well area and written into the well mapping table.

[0012] In some embodiments, calculating a set of uniform pressure values ​​based on a pressure distribution map includes: After generating a pressure distribution map based on the initial borehole pressure set, a pressure sampling segment is selected and multiple frames of the initial borehole pressure set are obtained within the pressure sampling segment; Perform intra-frame summation on the initial pore pressure set of multiple frames to generate intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame range; Inter-frame aggregation is performed on the intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame polarity of multiple frames to obtain a set of pressure uniformity values.

[0013] In some embodiments, when the pressure deviation exceeds a preset deviation value, a fine-tuning command for the downward pressure position or a fine-tuning command for the upward pressure position is output, including: The average pressure value is calculated based on the target orifice pressure set and compared with the target pressure to generate a pressure deviation value, which is written into the deviation record table. The deviation record table includes the sampling time, average pressure value, target pressure and pressure deviation value. Based on the deviation record table, the pressure deviation value is compared with the target pressure minus the preset deviation value and the target pressure plus the preset deviation value. When the pressure deviation value falls into the target pressure minus the preset deviation value, a downward position fine adjustment command is output. When the pressure deviation value falls into the target pressure plus the preset deviation value, an upward position fine adjustment command is output.

[0014] Secondly, a PCR hot capping system with pressure detection and well plate recognition is provided, which is used to implement the above-mentioned PCR hot capping method with pressure detection and well plate recognition, including: Well plate recognition module: used to acquire reaction well position images of the sample stage, perform well position localization processing on the reaction well position images to obtain the center pixel coordinates of each reaction well, and generate well plate recognition results based on the center pixel coordinates; Interaction module: Used to generate a set of hot cap pressing parameters based on the orifice plate recognition results, and outputs hot cap pressing control command and enters the pressure sampling stage after receiving user confirmation command; Pressure sampling module: used to collect pressure sampling values ​​output by array-type thin film pressure sensor, generate an initial orifice pressure set based on plate type, output a stop pressing command when the initial orifice pressure set meets the target pressure, and generate a pressure distribution map based on the initial orifice pressure set; Uniformity judgment module: It is used to calculate the pressure uniformity value set based on the pressure distribution map, perform pressure uniformity judgment based on the pressure uniformity value set and the judgment threshold set, and output alarm prompt information and re-identification command when the pressure uniformity judgment is not valid, and return to the execution orifice plate recognition module after a preset number of times. Heating adjustment module: When the pressure uniformity judgment is met, it outputs heating control command and continuously collects the target hole pressure set during the heating stage. It calculates the average pressure value based on the target hole pressure set and compares it with the target pressure. When the pressure deviation exceeds the preset deviation value, it outputs a downward position fine adjustment command or an upward position fine adjustment command.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, images of the reaction orifices on the sample stage are acquired and the orifice location is processed to obtain the center pixel coordinates of each reaction orifice. Based on these center pixel coordinates, orifice plate identification results are generated, ensuring that the presence, type, and placement of the reaction orifice plate are structurally confirmed before pressing. A set of hot cap pressing parameters is generated based on the orifice plate identification results. Upon receiving a user confirmation command, a hot cap pressing control command is output, and the pressure sampling stage begins. The pressing stroke follows the plate shape to an adjustable diameter. Pressure sampling values ​​from an array of thin-film pressure sensors are acquired. An initial orifice pressure set is generated based on the plate shape. When the initial orifice pressure set meets the target pressure, a stop pressing command is output, and a pressure distribution map is generated. This transforms the hot cap pressure from a fixed stroke to a closed-loop convergence of "sampling—judgment—stop." A pressure uniformity set is calculated based on the pressure distribution map and combined with the judgment... The system performs pressure uniformity judgment based on a set threshold. When the pressure uniformity judgment fails, it outputs an alarm message and a re-identification command, returning to execution S10 a preset number of times. This prevents placement misalignment, plate shape mismatch, or abnormal local stress from occurring before heating and returns the system to the identification link. When the pressure uniformity judgment is successful, it outputs a heating control command and continuously collects the target orifice pressure set during the heating phase. Based on the target orifice pressure set, it calculates the average pressure value and compares it with the target pressure. When the pressure deviation exceeds a preset deviation value, it outputs a downward pressure position fine-tuning command or an upward pressure position fine-tuning command. This allows pressure drift caused by factors such as thermal expansion to be monitored and dynamically compensated in real time during the heating process. Thus, under the constraint of the same link between orifice plate identification and pressure feedback, it achieves automatic adaptation of different consumables and stable pressure maintenance, improving sealing consistency and reducing manual intervention. Attached Figure Description

[0016] Figure 1 This is a schematic flowchart of a PCR hot cap method with pressure detection and well plate recognition according to the present invention. Figure 2 This is a schematic diagram of the structure of a PCR hot cap system with pressure detection and well plate recognition according to the present invention; Figure 3 This is a schematic diagram of the perforated plate identification and alarm in this invention; Figure 4 This is a schematic diagram of the hot cap being pressed down and heated in this invention; Figure 5 This is a schematic diagram of real-time pressure control in this invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. In the following detailed description, many specific details are set forth to provide a thorough understanding of the exemplary embodiments described. However, it will be apparent to those skilled in the art that the described embodiments may be practiced without some or all of these specific details. In other exemplary embodiments, well-known structures have not been described in detail to avoid unnecessarily obscuring the concepts of this disclosure. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention. Furthermore, the various aspects described in the embodiments may be combined arbitrarily without conflict.

[0018] The user information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for analysis, data stored, data displayed, etc.) involved in this application are all information and data authorized by the user or fully authorized by all parties, and the collection, use and processing of the relevant data must comply with relevant regulations.

[0019] Example 1 Figure 1 This disclosure illustrates a PCR hot cap method with pressure detection and well plate recognition according to at least one embodiment, comprising: S10: Acquire images of the reaction well positions on the sample stage, perform well position localization processing on the reaction well position images to obtain the center pixel coordinates of each reaction well, and generate well plate recognition results based on the center pixel coordinates. The aperture plate recognition result is generated based on the center pixel coordinates, including: The candidate region detection process is performed on the reaction well image, and the center pixel coordinates of the well are calculated in each candidate region. The center coordinates are then collected to obtain a set. The number of rows and columns is counted within the central coordinate set, and placement orientation information is generated based on the number of rows and columns; Based on the number of rows and columns, matching entries are retrieved from the preset plate type template set to generate candidate plate type information. The hole spacing measurement value is calculated in the center coordinate set to complete the verification of the candidate plate type information and generate plate type information. Fluorescence signals corresponding to each well location are collected, and information on the state of liquid presence in the well is generated based on the fluorescence signals. The plate type information, placement information, and information on the state of liquid presence in the well are then combined to obtain the well plate identification result.

[0020] It should be noted that the core of the well plate identification process, which revolves around the generation of well plate identification results, lies in converting the reaction well plate image of the sample stage into a set of central coordinates that can be used for counting and ranging. This allows the subsequent determination of plate type information and placement orientation information to be independent of manual selection. The central coordinate set provides a unified spatial index for each well plate and supports the statistics of row and column numbers. The row and column numbers are used to characterize the scale features of the well plate in two-dimensional arrangement and serve as the retrieval key for the preset plate type template set. The verification of candidate plate type information and well spacing measurement values ​​is used to eliminate misjudgments that may be caused by counting alone and to fix the plate type information. The fluorescence signal is used to distinguish the reflection difference between the sample stage surface and the well plate material and to further distinguish the background fluorescence difference caused by the presence of reaction liquid in the well. This generates information on the state of liquid presence in the well and combines it with the plate type information and placement orientation information to form the well plate identification result.

[0021] For example, in an 8-tube array scenario, the set of center coordinates can be represented as: (120,80),(160,80),(200,80),(240,80),(280,80),(320,80),(360,80),(400,80), where the row and column numbers correspond to 1 and 8 respectively, and the hole spacing measurement value is 40 pixels. The preset plate type template sets the entries that match 1×8 to output candidate plate type information, and the plate type information is verified by the hole spacing measurement value to be an 8-tube array. At the same time, the fluorescence signals of each hole position can be collected as 12,11,13,28,30,29,12,11, where 12,11,13 The wells 28, 30, and 29 correspond to the low fluorescence signal of the plate, while the wells 28, 30, and 29 correspond to the background fluorescence signal of the reaction liquid inside the well. This information is then combined with the plate type information and placement orientation information to obtain the well plate identification result. Compared with the existing method that relies on manual selection of plate type and manual confirmation of sample addition, this method can incorporate the plate type information, placement orientation information, and liquid presence information inside the well into the same well plate identification result before the hot cap is pressed down. This information can then be used as the input basis for the generation of subsequent hot cap pressing parameter sets, reducing the risk of process interruption and whole plate experiment failure due to incorrect plate type selection or failure to add sample.

[0022] It should be noted that, as Figure 3 As shown, the abnormal alarm path for orifice plate recognition is described. After the orifice plate is loaded, the reaction well position image acquisition and well position positioning processing are triggered to obtain the center coordinate set and form the orifice plate recognition result. Then, the plate type information, placement orientation information and liquid presence status information in the orifice plate recognition result are compared with preset information. When the comparison does not meet the preset requirements, the stop process is entered and an alarm prompt is output. This is to avoid triggering the hot cap pressure control command when the orifice plate is not placed, the placement orientation is deviated, or the sample addition is not completed, which would cause the pressure sampling value to be distorted and subsequent heating to be started incorrectly.

[0023] Furthermore, each well location is irradiated by an excitation light source, and the fluorescence signal returned from each well location is collected. The fluorescence signal intensity corresponding to each well location is compared with a preset fluorescence reference range. When the fluorescence signal intensity of the corresponding well location is higher than the preset empty hole reference intensity, it is determined that there is liquid in the well location. When the fluorescence signal intensity of the corresponding well location is not higher than the preset empty hole reference intensity, it is determined that there is no liquid in the well location. The determination results of each well location are collected to form information on the presence of liquid in the well.

[0024] Based on the number of rows and columns, matching entries are retrieved from a preset template set to generate candidate template information, including: A row and column index table is constructed based on the set of center coordinates. The row and column index table contains row index, column index and corresponding center pixel coordinate entries. Calculate the hole spacing measurement value in the row and column index table and write the hole spacing measurement value into the plate type verification table. The plate type verification table contains the associated entries of candidate plate type information and hole spacing measurement value. Output board type information based on the board type verification table.

[0025] Furthermore, the generation and output of candidate plate type information are discussed. The row and column index table is used to organize the set of center coordinates according to the row and column indices, so that each hole position has a definite position number in the row and column directions and retains the corresponding center pixel coordinate entries. This provides a stable data entry for subsequent calculation of hole spacing measurement values. The hole spacing measurement value represents the pixel distance between the center pixel coordinates of adjacent rows or adjacent columns of holes. It is used to constrain the geometric scale of hole position arrangement and reduce plate type confusion caused by matching only the number of rows and columns.

[0026] Optionally, the plate type verification table is used to establish an association between candidate plate type information and hole spacing measurement values. This allows the output of plate type information to be constrained by both candidate plate type information and hole spacing measurement values ​​simultaneously, rather than by a single counting condition. For example, in a 96-hole plate scenario, the row and column index table can contain entries such as (row 1, column 1, (120, 80)), (row 1, column 2, (140, 80)), and (row 2, column 1, (120, 100)). The hole spacing measurement value can be obtained by the difference of the center pixel coordinates of (row 1, column 1) and (row 1, column 2) to obtain 20 pixels and write it into the plate type verification table to form an association entry (candidate plate type information is 96-hole plate, hole spacing measurement value is 20 pixels). Subsequently, the plate type information is output as 96-hole plate based on the plate type verification table. This method, compared to judging solely by the number of rows and columns, can maintain a clear output diameter of plate type information and reduce the risk of incorrect selection of pressing parameters due to misjudgment when hole counts are similar or there are missing holes.

[0027] S20: Generate a set of hot cover pressing parameters based on the orifice plate recognition results, and output a hot cover pressing control command after receiving the user confirmation command and enter the pressure sampling stage; A set of hot cap pressing parameters is generated based on the orifice plate recognition results, including: The plate type is read from the orifice plate identification results, and the target pressure item and judgment threshold set item corresponding to the plate type are retrieved from the consumable parameter table to generate the basic hot cover pressure parameter set; The target pressure entries within the basic hot cap pressure parameter set are subject to factory safety value limits, and the hot cap pressure parameter set with safety value limits is output after receiving a user confirmation command.

[0028] It should be noted that the formation of the hot cap pressing parameter set and the triggering of the pressing control command revolve around the process. The purpose of using the orifice plate identification result as input is to bind the plate type information and the judgment threshold set entries required for subsequent pressure judgment to the same process entry point. This ensures that the parameter calling path remains clear even when different consumable specifications correspond to different pressure orifices. The consumable parameter table is used to store the correspondence between the plate type and the target pressure entries and the judgment threshold set entries. The basic hot cap pressing parameter set is used to carry the target pressure entries and judgment threshold set entries retrieved from the consumable parameter table and serves as the parameter entry point for the subsequent control stage.

[0029] S30: Collects pressure sampling values ​​output by array-type thin-film pressure sensor, generates an initial orifice pressure set based on plate type, outputs a stop pressing command when the initial orifice pressure set meets the target pressure, and generates a pressure distribution map based on the initial orifice pressure set; The pressure sampling values ​​output by the array-type thin-film pressure sensor are collected, and an initial set of orifice pressures is generated based on the plate shape, including: The pressure sampling values ​​output by the independent sensing units of the array-type thin-film pressure sensor are collected and aggregated according to the sampling time and sensing unit identifier to form a pressure sampling array. The plate shape is read from the plate identification results and a hole position mapping table is generated based on the plate shape. The hole position mapping table contains associated entries of hole position index and independent sensing unit identifier. Based on the pore location mapping table, the corresponding pressure sampling values ​​are retrieved in the pressure sampling array and collected according to the pore location index to form an initial pore location pressure set.

[0030] It should be noted that the collection path from the pressure sampling value to the initial orifice pressure set is discussed. The array-type thin-film pressure sensor is composed of multiple independent sensing units. The pressure sampling values ​​output by the independent sensing units are discrete multi-point measurement results. The purpose of collecting them into a pressure sampling array according to the sampling time and sensing unit identifier is to organize the multi-point pressure values ​​at the same sampling time into a searchable data structure, thereby avoiding the problem of mixed sampling times or unclear sensing unit sources in the subsequent collection process.

[0031] Furthermore, the orifice mapping table is used to establish the correspondence between orifice indexes and independent sensor unit identifiers, enabling the pressure sampling values ​​in the pressure sampling array to be aggregated into an initial orifice pressure set according to the orifice index. The plate type serves as the basis for generating the orifice mapping table to adapt to the differences in the coverage relationship of sensor units under different orifice arrangements, ensuring that the same sensor array can still form a fixed-diameter orifice index system under different plate types. For example, the orifice mapping table may contain related entries such as (orifice index 1, sensor unit identifier A01), (orifice index 2, sensor unit identifier A02), (orifice index 3, sensor unit identifier A03), etc., for the pressure sampling array. At a certain sampling moment, entries such as (A01, 158), (A02, 160), and (A03, 161) can be included. After searching based on the pore location mapping table and aggregating by pore location index, the initial pore location pressure set is obtained as (pore location index 1, 158), (pore location index 2, 160), and (pore location index 3, 161). This is different from the method of using only a single point pressure value as the basis for stopping pressure. The determination of stopping pressure and the generation of pressure distribution map are both based on the initial pore location pressure set after the pore location index is aggregating. This makes it easier to express the overall pressure level and the pressure difference of each pore location in the subsequent stages and reduce the risk of local overpressure or local underpressure going undetected.

[0032] Read the plate type from the orifice plate identification results and generate an orifice location mapping table based on the plate type, including: When the plate type is an 8-tube or 96-hole plate, the hole position index is associated with the independent sensing unit identifier and written into the hole position mapping table. When the plate type is a 384-well plate, the independent sensing unit identifier is associated with the 2×2 well area and written into the well mapping table.

[0033] Furthermore, the correlation between the orifice location mapping table and the orifice location under different plate types is discussed. The function of the orifice location mapping table is to convert the pressure sampling value from the independent sensing unit identifier field to the orifice location index field, so that when the initial orifice location pressure set is collected according to the orifice location index, there is a definite retrieval entry. In plate types with a small number of orifices or a large spacing between orifices, the effective coverage of an independent sensing unit can usually correspond to a single orifice location. Therefore, the orifice location index is written into the orifice location mapping table in a one-to-one association with the independent sensing unit identifier, so that each orifice location index has a unique sensing unit identifier source in the pressure sampling array.

[0034] It should be noted that in plate designs with a large number of holes and small hole spacing, the pressure-sensing area of ​​an independent sensing unit may cover multiple adjacent hole areas. In this case, the independent sensing unit identifier is associated with the coverage of the 2×2 hole area and written into the hole location mapping table. This ensures that the pressure sampling values ​​of the same independent sensing unit can be grouped into the corresponding hole location index range, thereby guaranteeing that the hole location mapping table remains usable even with high-density hole arrangement and maintaining a clear hole location index system. For example, the hole location mapping table may include (independent sensing unit identifier B07, hole location index 21 to...). The coverage association entry of the orifice index 24 indicates that the four orifice indices corresponding to the independent sensing unit identifier B07 cover a 2×2 orifice area. When the pressure sampling array contains (B07, 152) at a certain sampling time, the initial orifice pressure set can be classified into the same pressure sampling value source within the range of orifice index 21 to orifice index 24. This reduces the risk of pressure distribution map distortion caused by mismatch between orifice index and pressure sampling value source, and maintains the stability of the input aperture for subsequent uniformity judgment, compared to the forced one-to-one association method under high-density plate type.

[0035] S40: Calculate the pressure uniformity value set based on the pressure distribution map, perform pressure uniformity judgment based on the pressure uniformity value set and the judgment threshold set, and output alarm prompt information and re-identification command when the pressure uniformity judgment is not valid, and return to execute S10 after a preset number of times. The set of uniform pressure values ​​is calculated based on the pressure distribution map, including: After generating a pressure distribution map based on the initial borehole pressure set, a pressure sampling segment is selected and multiple frames of the initial borehole pressure set are obtained within the pressure sampling segment; Perform intra-frame summation on the initial pore pressure set of multiple frames to generate intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame range; Inter-frame aggregation is performed on the intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame polarity of multiple frames to obtain a set of pressure uniformity values.

[0036] It should be noted that the pressure distribution map is used to represent the value distribution of the initial orifice pressure set in the orifice index dimension; the pressure sampling segment is used to limit the pressure signal to enter a stable sampling interval after the hot cap stops pressing down; the multi-frame initial orifice pressure set is used to reflect the orifice pressure state at different times within the same pressure sampling segment; the intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame range are used to extract the overall level and dispersion from the single-frame orifice pressure set; and the inter-frame aggregation is used to aggregate the calculation results of multiple frames into a pressure uniformity set to support subsequent pressure uniformity judgment.

[0037] Furthermore, the purpose of selecting pressure sampling segments after the pressure distribution map is generated is to avoid writing pressure fluctuations caused by the pressure transient into the pressure uniformity set. When obtaining multiple frames of initial orifice pressure sets within the pressure sampling segment, multiple frames of orifice pressure sets can be extracted sequentially according to the sampling time sequence while keeping the orifice index unchanged, so that each frame corresponds to the orifice index system under the same plate type. Thus, in the intra-frame aggregation stage, the pressure of each orifice can be statistically analyzed with a unified caliber and the inter-frame aggregation can be compared.

[0038] Intra-frame calculations are performed based on the orifice pressure data F1, F2, ..., Fn to calculate the mean pressure value, range, variance, standard deviation, and coefficient of variation: It should be noted that the average pressure value formula is used to group the orifice pressure sets F1, F2, ..., Fn into a single pressure characterization quantity, which facilitates comparison with the target pressure F. Its expression is: ; In the formula, This indicates the number of holes involved in the settlement. Indicates the hole position number. Indicates the sequence number is The orifice pressure sampling value, This represents the average pressure value of the pressure set at the orifice.

[0039] Furthermore, the extreme value and range formulas are used to characterize the fluctuation boundary of the orifice pressure within the orifice plate range and form a definite dispersion index. Their expressions are as follows: , , ; In the formula, This represents the maximum pressure value in the set of orifice pressures. R represents the minimum pressure value in the pore pressure set, and R represents the range of the pore pressure set.

[0040] In this embodiment, the deviation formula is used to establish a difference relationship between the pressure at each orifice and the average pressure value, so as to transform the dispersion of the orifice pressure into a computable deviation sequence. Its expression is: ; In the formula, This represents the pressure deviation value at orifice number i. This represents the pressure sampling value at orifice number i. This represents the average pressure value of the pressure set at the orifice.

[0041] Optionally, the variance formula is used to square the deviation sequence and eliminate the effects of positive and negative cancellation, thereby obtaining the variance corresponding to the dispersion of the orifice pressure. Its expression is: ; In the formula, This represents the variance of the pressure set at the orifice. Indicates the number of holes. This represents the pressure sampling value at orifice number i.

[0042] It should be noted that the standard deviation formula is used to restore the variance to a dispersion measure with the same dimensions as the pressure value, so as to determine the threshold with the standard deviation. For comparisons of quantities with the same dimensions, the expression is: ; In the formula, This represents the standard deviation of the pressure set at the orifice. Let n represent the variance of the pressure set at each orifice, and n represent the number of orifices. This represents the pressure sampling value at orifice number i. This represents the average pressure value of the pressure set at the orifice.

[0043] Furthermore, the coefficient of variation formula is used to normalize the standard deviation to the relative dispersion based on the average pressure value, thereby maintaining comparability even under scenarios with differences in plate shape or target pressure. Its expression is: ; In the formula, Let σ represent the coefficient of variation of the pore pressure set, and let σ represent the standard deviation of the pore pressure set. This represents the average pressure of the pressure set at the orifice.

[0044] Uniformity judgment criteria: The standard deviation (absolute dispersion) of pressure at all orifices must not exceed a preset upper limit. ,Right now ; The relative dispersion (coefficient of variation) of the pressure must not exceed a preset upper limit. ,Right now ; The maximum pressure fluctuation range must not exceed the preset upper limit. ,Right now ; Judgment process: When , , If the pressure is uniform, it is judged as uniform; if any one of the three conditions is not met, it is judged as uneven pressure.

[0045] Furthermore, such as Figure 4As shown, the linkage process between hot cap pressing and heating is as follows: After the hot cap is pressed, the pressure detection module collects the pressure sampling values ​​of each independent sensing unit according to the sampling time and aggregates them into a hole pressure set. The average pressure value of the hole pressure set is compared with the target pressure F. If the comparison does not meet the target pressure F, the pressing continues and the sampling is repeated at the next sampling time. When the comparison meets the target pressure F, the pressing stops and a pressure distribution map is generated based on the hole pressure set. Then, a pressure uniformity value set is formed based on the pressure distribution map and a pressure uniformity judgment is performed. If the pressure uniformity judgment is not valid, an alarm prompt is output and the process is terminated. If the pressure uniformity judgment is valid, a heating control command is output to enter the heating stage.

[0046] S50: When the pressure uniformity judgment is valid, output the heating control command and continuously collect the target hole pressure set during the heating stage. Calculate the average pressure value based on the target hole pressure set and compare it with the target pressure. When the pressure deviation exceeds the preset deviation value, output the downward position fine adjustment command or the upward position fine adjustment command.

[0047] When the pressure deviation exceeds the preset deviation value, a fine-tuning command for the downward pressure position or the upward pressure position is output, including: The average pressure value is calculated based on the target orifice pressure set and compared with the target pressure to generate a pressure deviation value, which is written into the deviation record table. The deviation record table includes the sampling time, average pressure value, target pressure and pressure deviation value. Based on the deviation record table, the pressure deviation value is compared with the target pressure minus the preset deviation value and the target pressure plus the preset deviation value. When the pressure deviation value falls into the target pressure minus the preset deviation value, a downward position fine adjustment command is output. When the pressure deviation value falls into the target pressure plus the preset deviation value, an upward position fine adjustment command is output.

[0048] In this embodiment, the target orifice pressure set is continuously collected during the heating stage because when the hot cap is in heating condition, the reaction consumables and the hot cap assembly will experience stress changes due to thermal expansion and contraction. Determining the pressure uniformity only during the pressing stage is insufficient to ensure that the pressure is maintained near the target pressure during the subsequent heating stage. Therefore, after the heating control command is output, the orifice pressure is collected according to the sampling time, and the average pressure value is calculated for the target orifice pressure set. The average pressure value is then compared with the target pressure to obtain the pressure deviation value, thereby providing verifiable data for position fine-tuning.

[0049] In this embodiment, as Figure 5As shown, the real-time pressure control path during the heating stage continuously collects the target orifice pressure set and calculates the average pressure value after the heating control command is output. The average pressure value is compared with the target pressure F to obtain the pressure deviation value, and the pressure deviation value is compared with the allowable deviation ΔF. When the pressure deviation value is greater than the allowable deviation ΔF, the position of the hot cover is adjusted to change the stress level. After the adjustment is completed, the system returns to the next sampling time to continue collecting and comparing. When the pressure deviation value is not greater than the allowable deviation ΔF, the current position of the hot cover is maintained and heating sampling continues, so that the stress drift caused by thermal expansion and contraction can be recorded and corrected by the position fine-tuning closed loop.

[0050] It should be noted that, to avoid frequent alternation between downward and upward fine-tuning commands during the heating phase due to instantaneous pressure fluctuations, delayed response of the hot cover to thermal deformation, or delay in the adjustment of the actuator position, an adjustment waiting phase can be entered after each output of a downward or upward fine-tuning command. During the adjustment waiting phase, the control system continuously performs target orifice pressure set acquisition for at least two sampling cycles. After the completion of the at least two sampling cycles, the average pressure value is calculated based on the latest acquired target orifice pressure set and compared with the target pressure for a new round of judgment. If the recalculated pressure deviation still exceeds the preset deviation value, the corresponding position fine-tuning command is output. If the recalculated pressure deviation does not exceed the preset deviation value, the current hot cover position remains unchanged and pressure acquisition during the heating phase continues, thereby suppressing back-and-forth adjustments caused by short-term disturbances and improving the stability of pressure control during the heating phase.

[0051] It should be noted that the deviation record table is used to collect the key quantities of each sampling in the same table to avoid the loss of traceability of instructions due to the scattered sources of position fine-tuning. The sampling time is used to identify the heating stage time point corresponding to the pressure deviation value, the average pressure value is used to describe the overall stress level of the current orifice pressure set, the target pressure is used to keep the pressure setting diameter consistent with the plate type, and the pressure deviation value is used to express the difference between the average pressure value and the target pressure.

[0052] Furthermore, the interval discrimination compares the pressure deviation value with the pressure intervals corresponding to the target pressure minus the preset deviation value and the target pressure plus the preset deviation value. This is to convert the continuous pressure deviation into two types of discrete control commands and avoid repeated jitter. When the pressure deviation value falls to the side of the target pressure minus the preset deviation value, it indicates that the average pressure value is relatively small. The corresponding output is a downward pressure fine-tuning command to increase the force. When the pressure deviation value falls to the side of the target pressure plus the preset deviation value, it indicates that the average pressure value is relatively large. The corresponding output is an upward pressure fine-tuning command to reduce the force. In this way, the pressure deviation is corrected by feedback during the heating stage, and the impact of the set pressure drift caused by thermal expansion on the experimental process is reduced.

[0053] Example 2 Please see Figure 2 As shown, based on the same inventive concept, this embodiment discloses a system that includes, for details not covered in this embodiment, please refer to the relevant parts of Embodiment 1. Well plate recognition module: used to acquire reaction well position images of the sample stage, perform well position localization processing on the reaction well position images to obtain the center pixel coordinates of each reaction well, and generate well plate recognition results based on the center pixel coordinates; Interaction module: Used to generate a set of hot cap pressing parameters based on the orifice plate recognition results, and outputs hot cap pressing control command and enters the pressure sampling stage after receiving user confirmation command; Pressure sampling module: used to collect pressure sampling values ​​output by array-type thin film pressure sensor, generate an initial orifice pressure set based on plate type, output a stop pressing command when the initial orifice pressure set meets the target pressure, and generate a pressure distribution map based on the initial orifice pressure set; Uniformity judgment module: It is used to calculate the pressure uniformity value set based on the pressure distribution map, perform pressure uniformity judgment based on the pressure uniformity value set and the judgment threshold set, and output alarm prompt information and re-identification command when the pressure uniformity judgment is not valid, and return to execute S10 after a preset number of times. Heating adjustment module: When the pressure uniformity judgment is met, it outputs heating control command and continuously collects the target hole pressure set during the heating stage. It calculates the average pressure value based on the target hole pressure set and compares it with the target pressure. When the pressure deviation exceeds the preset deviation value, it outputs a downward position fine adjustment command or an upward position fine adjustment command.

[0054] The detailed description above, in conjunction with the accompanying drawings, describes examples but does not represent all examples that can be implemented or fall within the scope of the claims. The terms “example” and “exemplary” are used in this specification to mean “serving as an example, instance or illustration” and do not mean “superior to or better than other examples”.

[0055] Throughout this specification, the phrase "an embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the use of these phrases may refer to more than one embodiment. Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0056] It should also be noted that these embodiments may be described as processes depicted as flowcharts, structural diagrams, or block diagrams. Although a flowchart may describe the operations as sequential processes, many of these operations can be performed in parallel or concurrently, and the order of these operations may be rearranged.

Claims

1. A PCR hot capping method with pressure detection and well plate recognition, characterized in that, include: S10: Acquire images of the reaction well positions on the sample stage, perform well position localization processing on the reaction well position images to obtain the center pixel coordinates of each reaction well, and generate well plate recognition results based on the center pixel coordinates. S20: Generate a set of hot cover pressing parameters based on the orifice plate recognition results, and output a hot cover pressing control command after receiving the user confirmation command and enter the pressure sampling stage; S30: Collects pressure sampling values ​​output by array-type thin-film pressure sensor, generates an initial orifice pressure set based on plate type, outputs a stop pressing command when the initial orifice pressure set meets the target pressure, and generates a pressure distribution map based on the initial orifice pressure set; S40: Calculate the pressure uniformity value set based on the pressure distribution map, perform pressure uniformity judgment based on the pressure uniformity value set and the judgment threshold set, and output alarm prompt information and re-identification command when the pressure uniformity judgment is not valid, and return to execute S10 after a preset number of times. S50: When the pressure uniformity judgment is valid, output the heating control command and continuously collect the target hole pressure set during the heating stage. Calculate the average pressure value based on the target hole pressure set and compare it with the target pressure. When the pressure deviation exceeds the preset deviation value, output the downward position fine adjustment command or the upward position fine adjustment command.

2. The PCR hot cap method with pressure detection and well plate recognition according to claim 1, characterized in that, The aperture plate recognition result is generated based on the center pixel coordinates, including: The candidate region detection process is performed on the reaction well image, and the center pixel coordinates of the well are calculated in each candidate region. The center coordinates are then collected to obtain a set. The number of rows and columns is counted within the central coordinate set, and placement orientation information is generated based on the number of rows and columns; Based on the number of rows and columns, matching entries are retrieved from the preset plate type template set to generate candidate plate type information. The hole spacing measurement value is calculated in the center coordinate set to complete the verification of the candidate plate type information and generate plate type information. Fluorescence signals corresponding to each well location are collected, and information on the state of liquid presence in the well is generated based on the fluorescence signals. The plate type information, placement information, and information on the state of liquid presence in the well are then combined to obtain the well plate identification result.

3. The PCR hot cap method with pressure detection and well plate recognition according to claim 2, characterized in that, Based on the number of rows and columns, matching entries are retrieved from a preset template set to generate candidate template information, including: A row and column index table is constructed based on the set of center coordinates. The row and column index table contains row index, column index and corresponding center pixel coordinate entries. Calculate the hole spacing measurement value in the row and column index table and write the hole spacing measurement value into the plate type verification table. The plate type verification table contains the associated entries of candidate plate type information and hole spacing measurement value. Output board type information based on the board type verification table.

4. The PCR hot cap method with pressure detection and well plate recognition according to claim 3, characterized in that, A set of hot cap pressing parameters is generated based on the orifice plate recognition results, including: The plate type is read from the orifice plate identification results, and the target pressure item and judgment threshold set item corresponding to the plate type are retrieved from the consumable parameter table to generate the basic hot cover pressure parameter set; The target pressure entries within the basic hot cap pressure parameter set are subject to factory safety value limits, and the hot cap pressure parameter set with safety value limits is output after receiving a user confirmation command.

5. The PCR hot cap method with pressure detection and well plate recognition according to claim 1, characterized in that, The pressure sampling values ​​output by the array-type thin-film pressure sensor are collected, and an initial set of orifice pressures is generated based on the plate shape, including: The pressure sampling values ​​output by the independent sensing units of the array-type thin-film pressure sensor are collected and aggregated according to the sampling time and sensing unit identifier to form a pressure sampling array. The plate shape is read from the plate identification results and a hole position mapping table is generated based on the plate shape. The hole position mapping table contains associated entries of hole position index and independent sensing unit identifier. Based on the pore location mapping table, the corresponding pressure sampling values ​​are retrieved in the pressure sampling array and collected according to the pore location index to form an initial pore location pressure set.

6. The PCR hot cap method with pressure detection and well plate recognition according to claim 5, characterized in that, Read the plate type from the orifice plate identification results and generate an orifice location mapping table based on the plate type, including: When the plate type is an 8-tube or 96-hole plate, the hole position index is associated with the independent sensing unit identifier and written into the hole position mapping table. When the plate type is a 384-well plate, the independent sensing unit identifier is associated with the 2×2 well area and written into the well mapping table.

7. The PCR hot cap method with pressure detection and well plate recognition according to claim 1, characterized in that, The set of uniform pressure values ​​is calculated based on the pressure distribution map, including: After generating a pressure distribution map based on the initial borehole pressure set, a pressure sampling segment is selected and multiple frames of the initial borehole pressure set are obtained within the pressure sampling segment; Perform intra-frame summation on the initial pore pressure set of multiple frames to generate intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame range; Inter-frame aggregation is performed on the intra-frame average pressure value, intra-frame standard deviation, intra-frame coefficient of variation, and intra-frame polarity of multiple frames to obtain a set of pressure uniformity values.

8. The PCR hot cap method with pressure detection and well plate recognition according to claim 7, characterized in that, When the pressure deviation exceeds the preset deviation value, a fine-tuning command for the downward pressure position or the upward pressure position is output, including: The average pressure value is calculated based on the target orifice pressure set and compared with the target pressure to generate a pressure deviation value, which is written into the deviation record table. The deviation record table includes the sampling time, average pressure value, target pressure and pressure deviation value. Based on the deviation record table, the pressure deviation value is compared with the target pressure minus the preset deviation value and the target pressure plus the preset deviation value. When the pressure deviation value falls into the target pressure minus the preset deviation value, a downward position fine adjustment command is output. When the pressure deviation value falls into the target pressure plus the preset deviation value, an upward position fine adjustment command is output.

9. A PCR hot cap system with pressure detection and well plate recognition, used to implement the PCR hot cap method with pressure detection and well plate recognition as described in any one of claims 1-8, characterized in that, include: Well plate recognition module: used to acquire reaction well position images of the sample stage, perform well position localization processing on the reaction well position images to obtain the center pixel coordinates of each reaction well, and generate well plate recognition results based on the center pixel coordinates; Interaction module: Used to generate a set of hot cap pressing parameters based on the orifice plate recognition results, and outputs hot cap pressing control command and enters the pressure sampling stage after receiving user confirmation command; Pressure sampling module: used to collect pressure sampling values ​​output by array-type thin film pressure sensor, generate an initial orifice pressure set based on plate type, output a stop pressing command when the initial orifice pressure set meets the target pressure, and generate a pressure distribution map based on the initial orifice pressure set; Uniformity judgment module: It is used to calculate the pressure uniformity value set based on the pressure distribution map, perform pressure uniformity judgment based on the pressure uniformity value set and the judgment threshold set, and output alarm prompt information and re-identification command when the pressure uniformity judgment is not valid, and return to the execution orifice plate recognition module after a preset number of times. Heating adjustment module: When the pressure uniformity judgment is met, it outputs heating control command and continuously collects the target hole pressure set during the heating stage. It calculates the average pressure value based on the target hole pressure set and compares it with the target pressure. When the pressure deviation exceeds the preset deviation value, it outputs a downward position fine adjustment command or an upward position fine adjustment command.

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