A pathogenic bacteria detection system and method

Abstract

The application discloses a pathogenic bacteria detection system and method. The system comprises a pretreatment module, a qPCR detection module and a data processing module. The pretreatment module comprises a sealed reaction cavity, and the sealed reaction cavity is connected with a sample inlet, a magnetic bead supply port and a fluid outlet. The outer side of the sealed reaction cavity is provided with a magnetic field generating assembly and a laser irradiation assembly. The outer periphery of the sealed reaction cavity is further provided with a lysis temperature control assembly, which is used for controlling the temperature of a lysis zone of the sealed reaction cavity at 50-70 DEG C during laser irradiation. The qPCR detection module is connected with the pretreatment module and comprises a light transmission detection cavity, a light-heat conversion heating assembly which is attached to the outer wall of the light transmission detection cavity and a light source array which emits light energy to the light-heat conversion heating assembly. The qPCR detection module further comprises a temperature sensor and a temperature control unit. The application aims to improve the three processing modules to improve the detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology, specifically to a pathogen detection system and method, which is particularly suitable for rapid on-site detection of pathogens in seafood products. Background Technology

[0002] Currently, the detection of pathogens in seafood products mainly relies on qPCR (real-time quantitative polymerase chain reaction) technology. Its basic process includes sample pretreatment (pathogen enrichment and nucleic acid extraction), qPCR amplification and detection, and fluorescence data analysis and result determination.

[0003] In the pretreatment stage, immunomagnetic bead technology has been used for the specific enrichment of pathogenic bacteria. For example, Chinese patent CN1818054A discloses a method for rapidly breaking down cells or viruses using micromagnetic beads and lasers. After mixing the magnetic beads with cells / viruses, the beads are irradiated with a laser to induce thermal expansion and vibration, thereby breaking down the cells and releasing nucleic acids, allowing for direct DNA purification. European patent EP1870449A1 also discloses a similar scheme: introducing magnetic beads into a solution containing cells / viruses to enrich the cells / viruses on the beads, and then irradiating the beads with a laser beam to break down the cells / viruses. Furthermore, the application of the photothermal effect of nanomaterials to PCR heating has been publicly reported. For instance, Chinese patent CN119464046A discloses an integrated fiber optic system for photothermal amplification and real-time PCR detection, utilizing a photothermal photosensitizer to receive heating light and generate heat, achieving photothermal cyclic amplification and real-time quantitative fluorescence detection. There are also reports in academic research on the application of the photothermal effect of nanomaterials to rapid PCR detection. In the area of ​​automated detection equipment, some attempts have been made. For example, Chinese patent CN111007050A discloses a bacterial detection system integrating magnetohydrodynamic separation and signal processing, including a separation and enrichment device, an automatic sample loading device (peristaltic pump + controller), a fluorescence reaction device, and a signal processing device, which can automate part of the process from immunomagnetic bead separation to automatic sample loading to fluorescence detection. In addition, Chinese patent CN116814414B discloses a "laser-induced graphene PCR detection device," which uses a graphene heating device to achieve heating / cooling cycles in the PCR reaction chamber.

[0004] However, the aforementioned existing technologies still have the following shortcomings: (1) During the laser lysis process after enrichment of immunomagnetic beads, the instantaneous high temperature generated by laser irradiation may cause nucleic acid degradation. There is no effective solution to this problem in the existing technology, and it is difficult to maintain the integrity of nucleic acid structure while ensuring lysis efficiency.

[0005] (2) Insufficient temperature control precision in photothermal PCR technology. Existing photothermal PCR protocols mostly focus on improving the heating and cooling rate, but pay insufficient attention to temperature uniformity and steady-state precision. Problems such as local overheating and obvious hot spots still exist. The temperature difference on the reaction surface usually reaches 3-5℃, which affects amplification efficiency and quantitative accuracy.

[0006] (3) After qPCR, the fluorescence signal analysis mainly relies on the operator's experience for baseline correction and curve identification. The interpretation results of different operators may differ, which is highly subjective and has low data processing efficiency. Summary of the Invention

[0007] To address the problems of easy degradation of nucleic acids by laser lysis, insufficient temperature control precision in photothermal PCR, and reliance on manual interpretation in the existing technologies, this invention provides a pathogen detection device and method. It achieves nucleic acid protection through temperature control of a lysis temperature control component, high-precision steady-state temperature control through a three-layer composite functional membrane and a three-stage temperature control strategy, and automatic interpretation of fluorescence signals through a built-in pathogen standard database and preprocessing algorithm, aiming to improve the accuracy, efficiency, and reliability of detection.

[0008] According to one aspect of the present invention, a pathogen detection system is provided, the method comprising: The pretreatment module includes a sealed reaction chamber, which is connected to an inlet, a magnetic bead supply port, and a fluid inlet / outlet. A magnetic field generating component and a laser irradiation component are provided on the outside of the sealed reaction chamber. A pyrolysis temperature control component is also provided on the periphery of the sealed reaction chamber. The pyrolysis temperature control component is used to control the temperature of the pyrolysis zone of the sealed reaction chamber at 50℃~70℃ during laser irradiation. The qPCR detection module, connected to the preprocessing module, includes a light-transmitting detection cavity, a photothermal conversion heating component attached to the outer wall of the light-transmitting detection cavity, and a light source array that emits light energy to the photothermal conversion heating component. The qPCR detection module further includes a temperature sensor and a temperature control unit. The temperature sensor is used to detect the temperature of the light-transmitting detection cavity in real time. The temperature control unit is signal-connected to the temperature sensor and the light source array, and is used to control the output power of the light source array according to the following strategy: When the difference between the measured temperature and the target temperature is greater than the first threshold, it is driven at full power. When the difference is less than or equal to the first threshold and greater than the second threshold, the power is driven with a linearly decreasing power. When the difference is less than or equal to the second threshold, fine-tuning is performed with a power step size of ≤2%. The temperature control unit samples the temperature sensor at a frequency ≥50Hz; The system also includes a central controller, which is connected to the preprocessing module and the qPCR detection module respectively, and is used to control the above modules to execute the detection process according to a preset timing sequence.

[0009] The above technical solution provides a pathogen detection system that, through the integrated architecture of the pretreatment module and the qPCR detection module, and the specific structural design of the lysis temperature control component, the photothermal conversion heating component and the temperature control unit, combined with a three-stage approximation control strategy and a closed-loop temperature control mechanism with a sampling frequency of ≥50Hz, achieves fully automated and high-precision detection from sample lysis to qPCR amplification.

[0010] Compared with existing technologies (such as the magnetic bead + laser pyrolysis scheme disclosed by Samsung Electronics in CN1818054A and EP1870449A1, and the photothermal PCR scheme in CN119464046A), the essential features of this scheme are threefold: Firstly, a lysis temperature control component is incorporated into the laser lysis process to maintain the lysis zone temperature between 50℃ and 70℃. The instantaneous high temperatures generated by pulsed laser irradiation can reach several hundred degrees Celsius; without control, this will lead to rapid degradation of the released nucleic acids. Existing laser lysis methods lack effective solutions to this problem, typically focusing only on lysis efficiency while neglecting nucleic acid protection. This method limits the temperature to the 50℃–70℃ range through the lysis temperature control component. This range balances the dual requirements of lysis efficiency and nucleic acid integrity—below 50℃, lysis efficiency is insufficient, while above 70℃, the risk of nucleic acid degradation increases significantly.

[0011] Secondly, in the qPCR detection module, a photothermal conversion heating component attached to the outer wall of the transparent detection chamber replaces the traditional metal heating block. This is combined with a three-stage approximation control strategy (full-power drive when the difference is greater than the first threshold, linearly decreasing drive when the difference is between the two thresholds, and fine-tuning with a step size ≤2% when the difference is less than or equal to the second threshold) and a sampling frequency ≥50Hz, forming a high-precision closed-loop temperature control mechanism. This scheme achieves a steady-state temperature control accuracy of ±0.1℃ and a heating / cooling rate ≥12℃ / s. The reaction surface temperature difference of traditional metal heating blocks is typically 3–5℃. While photothermal PCR can improve heating speed, it generally suffers from problems such as localized overheating and obvious hot spots. This scheme, through the synergy of the photothermal conversion heating component and the three-stage strategy, achieves high steady-state accuracy while rapidly heating, resolving the technical contradiction of "fast but not precise" in the field of photothermal PCR. The fine-tuning accuracy of ≤2% step size and the sampling frequency ≥50Hz in the three-stage strategy work together to control temperature overshoot below 0.1℃ and oscillation amplitude below 0.03℃. This control logic is specifically designed for the millisecond-level response characteristics of photothermal heating, rather than simply applying a conventional PID algorithm. The selection of its parameters (first threshold, second threshold, upper limit of step size, lower limit of sampling frequency) together constitutes a complete control scheme. The above accuracy cannot be achieved if any parameter is missing, and there is a synergistic relationship between the parameters.

[0012] In summary, this solution, through the synergistic cooperation of the lysis temperature control component and the three-stage closed-loop temperature control strategy, effectively solves the problem of laser lysis nucleic acid degradation while achieving high-precision steady-state temperature control for photothermal PCR. Furthermore, the timing scheduling of the central controller automates the connection between pretreatment and detection, effectively improving the accuracy, efficiency, and safety of pathogen detection.

[0013] In some embodiments, the photothermal conversion heating component is a composite functional film adhered to the outer wall of the light-transmitting detection cavity, and the composite functional film comprises, from the outer wall of the light-transmitting detection cavity outwards, the following components: The graphene layer, made of graphene material, is used to adhere to the outer wall of the light-transmitting detection cavity to absorb light energy and convert it into heat energy. A carbon nanotube layer, stacked on top of the graphene layer, is made of carbon nanotube material and is used to conduct the heat generated by the graphene layer in the in-plane direction. A silicon nitride layer, stacked on top of the carbon nanotube layer, is made of silicon nitride material and is used to cover the surface of the carbon nanotube layer.

[0014] In the above technical solution, the photothermal conversion heating component is further defined as a composite functional film attached to the outer wall of the light-transmitting detection cavity. From the outer wall outward, it consists of a graphene layer, a carbon nanotube layer, and a silicon nitride layer, which respectively undertake the functions of photothermal conversion, in-plane heat equalization, and structural protection, forming a three-layer composite structure integrating "photothermal conversion-lateral heat equalization-structural protection".

[0015] Compared with existing photothermal PCR technologies (such as the scheme disclosed in CN119464046A that uses a photothermal photosensitizer to receive heating light and generate heat, or the laser-induced graphene heating device scheme disclosed in CN116814414B), existing schemes usually use a single photothermal material layer or a simple bilayer structure, which has insufficient temperature distribution uniformity, and the temperature difference on the reaction surface usually reaches 3-5℃, resulting in problems such as local overheating and obvious hot spots.

[0016] The essential feature of the three-layer composite functional membrane in this solution lies in the functional division and synergistic cooperation of the three layers: Firstly, the graphene layer serves as a photothermal conversion layer. Graphene has an absorption rate greater than 90% in the near-infrared band, converting light energy into heat energy in milliseconds through electron-phonon coupling. Plasma etching is performed on the outer wall of the cavity before thin film deposition to form a submicron-level rough structure, increasing the actual contact area between the graphene layer and the outer wall of the cavity by approximately 40%, and reducing the interfacial thermal resistance to less than 1 / 10 of that of traditional thermal grease. This interface treatment, combined with the ultra-high in-plane thermal conductivity of graphene itself (approximately 2000–4000 W / (m·K)), enables efficient heat transfer from the photothermal conversion layer to the outer wall of the cavity. This effect depends on the specific positional relationship between the graphene layer and the outer wall of the cavity; if the graphene layer is not attached to the outer wall (e.g., suspended), direct conduction with low interfacial thermal resistance cannot be achieved. Existing technologies rarely address the interface treatment between the photothermal material layer and the outer wall. This solution, by increasing the contact area through plasma etching, represents a specific optimization of the photothermal conduction path and is not a conventional choice for those skilled in the art.

[0017] Secondly, the carbon nanotube layer serves as a lateral heat equalization layer. Stacked atop the graphene layer, the carbon nanotube layer boasts an in-plane thermal conductivity ≥3800 W / (m·K), rapidly conducting and diffusing the point-like heat generated by the graphene layer along its in-plane direction. The photothermal conversion of the graphene layer exhibits point-source heat characteristics (the heating positions corresponding to the light-emitting points of the LED array differ), and without heat equalization treatment, a significant temperature gradient will still exist on the reaction surface. The ultra-high in-plane thermal conductivity of the carbon nanotube layer instantly spreads the heat laterally, eliminating hot spots in the LED array. This three-layer composite functional film can reduce the temperature difference on a 100mm×100mm reaction surface from 2.5℃ to below 0.3℃—this temperature difference compression effect is the result of the synergistic effect of the two layers. The requirement of an in-plane thermal conductivity ≥3800 W / (m·K) for the carbon nanotube layer ensures that the rate of heat diffusion laterally is much faster than the rate of heat dissipation longitudinally, thus achieving effective temperature equalization. If this value is below this range, the lateral diffusion rate is insufficient to eliminate hot spots, and the heat equalization effect will significantly deteriorate.

[0018] Thirdly, the silicon nitride layer serves as the top protective layer. The silicon nitride layer is stacked on top of the carbon nanotube layer, covering its surface. Both graphene and carbon nanotubes are carbon-based materials, and during long-term thermal cycling, they may experience structural fatigue or delamination due to thermomechanical stress, leading to a decline in heat homogenization performance. The silicon nitride layer, as a dense and hard protective coating, protects the underlying structure from repeated impacts of thermomechanical stress, ensuring long-term reliability. This feature addresses the potential problem of accuracy degradation in photothermal PCR equipment during long-term thermal cycling—a long-term stability issue often overlooked by those skilled in the art when pursuing rapid heating and cooling. The silicon nitride layer exhibits high transmittance (≥90%) in the near-infrared band, without significantly affecting the efficiency of light reaching the graphene layer, achieving a balance between protective function and optical transmittance.

[0019] It should be noted that while the in-plane thermal conductivity of the graphene layer is consistent with that of the carbon nanotube layer (both are in-plane), their functions are hierarchically differentiated: the graphene layer, located closer to the heat source (the incident surface of light energy), is responsible for efficiently converting light energy into heat energy; the carbon nanotube layer, located further away from the heat source, is responsible for spreading the converted heat laterally. This sequential functional division of "conversion first, then heat evenness" allows the graphene layer to prioritize maximizing photothermal conversion efficiency, while the carbon nanotube layer focuses on eliminating spatial non-uniformity of the converted heat. If the order of the two layers is reversed (carbon nanotube layer on the outside, graphene layer attached to the cavity), light energy must first pass through the carbon nanotube layer before reaching the graphene layer. The carbon nanotube layer will then partially absorb and scatter the light energy, leading to a decrease in photothermal conversion efficiency. Therefore, the specific stacking order of the graphene layer at the bottom and the carbon nanotube layer in the middle is irreplaceable. Overall, the three-layer structure together achieves continuous thermal management of "absorption is conversion - conversion is homogenization - homogenization is maintenance", eliminating efficiency loss caused by improper material selection and stacking order throughout the entire path from light energy incident to cavity heating.

[0020] In some embodiments, the graphene layer has a thickness of 15–30 nm, the carbon nanotube layer has an in-plane thermal conductivity ≥3800 W / (m·K) and a thickness of 0.4–0.6 mm, and the total thickness of the composite functional film is ≤1 mm.

[0021] In the above technical solution, the thickness of the graphene layer is further limited to 15-30 nm, the in-plane thermal conductivity of the carbon nanotube layer is ≥3800 W / (m·K) and the thickness is 0.4-0.6 mm, and the total thickness of the composite functional film is ≤1 mm. The three-layer composite functional film is quantitatively limited from the perspective of material parameters.

[0022] The graphene layer thickness is limited to 15–30 nm. According to the specification, the graphene layer simultaneously performs the dual functions of light energy absorption and conversion, and reducing interfacial thermal resistance. The absorption rate of graphene for near-infrared light is related to its layer count—the absorption rate of a single layer of graphene is approximately 2.3%, while when the thickness reaches 15–30 nm, the absorption rate increases to over 90%. If the thickness is less than 15 nm, light absorption is insufficient, reducing photothermal conversion efficiency and failing to provide sufficient heating power for the PCR reaction; if the thickness is greater than 30 nm, although light absorption is close to saturation, the excessively thick graphene layer increases the thermal resistance of the heat conduction path, reducing the response speed. Determining this thickness range requires balancing light absorption efficiency and heat conduction speed, as there is a constraint between the two.

[0023] Secondly, the in-plane thermal conductivity of the carbon nanotube layer is ≥3800W / (m·K). This parameter is a necessary condition for achieving "rapid lateral diffusion of point-like heat and elimination of LED spot hotspots"—when the thermal conductivity of the carbon nanotube layer is lower than this value, the heat diffusion rate is insufficient to eliminate the temperature difference in a short time, the temperature difference on the reaction surface will increase significantly, and the overall heat homogenization effect of the three-layer composite film will deteriorate.

[0024] Third, the thickness of the carbon nanotube layer is limited to 0.4–0.6 mm. Determining this thickness range requires balancing in-plane thermal conductivity and overall heat capacity. When the thickness is less than 0.4 mm, the carbon nanotube material is insufficient to form a continuous and efficient lateral thermal conductivity network; when the thickness is greater than 0.6 mm, although it facilitates in-plane heat diffusion, it increases the overall heat capacity of the composite functional film and reduces the heating and cooling rates. A carbon nanotube layer of approximately 0.5 mm thickness, combined with a graphene layer, can reduce the temperature difference across a 100 mm × 100 mm reaction surface from 2.5 °C to below 0.3 °C.

[0025] Fourth, the total thickness of the composite functional film is ≤1mm. This feature ensures that the overall thickness of the three-layer composite film is controlled below the millimeter level, preventing an increase in thermal resistance and heat capacity due to excessive film thickness. The selection of this value is intrinsically related to the thickness parameters of each layer mentioned above—while ensuring the light absorption efficiency of the graphene layer (≥15nm) and the heat uniformity within the carbon nanotube layer (≥0.4mm), the total thickness is compressed to ≤1mm, allowing the composite functional film to simultaneously meet the requirements of high light absorption, high heat uniformity, and fast response.

[0026] In some embodiments, the temperature control unit controls the output power of the light source array according to the following strategy: When the difference between the measured temperature and the target temperature is greater than 2°C, the light source array is driven at full power. When the difference is ≤2℃ and >0.2℃, the power The temperature is linearly reduced, allowing it to smoothly approach the target value with an overshoot of <0.1℃; When the difference is ≤0.2℃, fine-tuning is performed with power increments of ≤2% to ensure that the temperature oscillation amplitude near the target value is ≤0.03℃.

[0027] In the above technical solution, the first threshold is further limited to 2℃ and the second threshold is limited to 0.2℃, and a specific calculation formula for the linear reduction of power is defined. And the quantitative control effect of steady-state oscillation amplitude ≤0.03℃.

[0028] Compared with existing temperature control technologies (such as conventional PID control, on / off control, etc.), the three-stage approximation strategy of this solution has the following essential characteristics: First, the power control logic in the three-segment approximation strategy differs fundamentally from PID control. A conventional PID controller's output is based on a continuous linear combination of proportional, integral, and derivative terms, resulting in a continuous and stepless control process. This solution, however, employs discrete three-segment logic—executing three distinct control modes—"full power," "linear decrease," and "step fine-tuning"—depending on the error's range (>2℃, ≤2℃ and >0.2℃, ≤0.2℃). This discrete segmented control strategy can proactively switch control modes according to different stages of temperature approaching the target value. While segmented control is not uncommon in industrial control, this solution sets the segment boundaries at two values ​​separated by an order of magnitude: 2℃ and 0.2℃. The intermediate segment uses a linear decrease formula to directly and linearly correlate the error with the output power, and the final segment uses a 2% step fine-tuning step—this combination is not a conventional variation of the PID controller, nor is it a solution that those skilled in the art would readily conceive of when faced with the common need to "improve temperature control accuracy."

[0029] Secondly, the selection of the boundary values ​​(2℃ and 0.2℃) and step size (≤2%) for the three temperature ranges is technically critical. In photothermal heating systems, the response time of the light source array is in the millisecond range, and the temperature change rate is extremely fast (≥12℃ / s). If the first threshold is greater than 2℃ (e.g., 5℃), the system will heat at full power until the error drops below 5℃. Due to thermal inertia, the overshoot will increase significantly (potentially exceeding 0.5℃). If the first threshold is less than 2℃ (e.g., 1℃), the full-power heating range is too short, and the system's advantage of rapid temperature rise cannot be fully utilized, prolonging the total time to reach the target temperature. Similarly, if the second threshold is greater than 0.2℃ (e.g., 0.5℃), the linear decreasing control will enter the fine-tuning stage too early, resulting in a slow approximation speed. If it is less than 0.2℃ (e.g., 0.1℃), the linear decreasing range is too narrow, and when switching to fine-tuning, the temperature is still far from the target value, requiring a long fine-tuning time. A fine-tuning step size of ≤2% is matched with a switching threshold of 0.2℃. If the step size is greater than 2% (e.g., 5%), the oscillation amplitude near the target value will exceed 0.03℃; if the step size is too small (e.g., 0.5%), unnecessary adjustment time will be added. The above parameters are interrelated and mutually constrained, and are not adjustable in isolation, so their combination has synergy.

[0030] Thirdly, the precise quantification of the control effect (overshoot < 0.1℃ and oscillation amplitude ≤ 0.03℃) provides a basis for verifying the rationality of the above parameter selection. Overshoot and oscillation amplitude are two independent indicators for measuring stability in a temperature control system—overshoot focuses on the maximum overshoot during the process of approaching the target value, while oscillation amplitude focuses on the temperature fluctuation range after entering steady state. In a photothermal heating system, since the heating power is applied through the light source array with a millisecond-level response speed, the temperature disturbance caused by power adjustment is more severe than that of a traditional metal heating block. Achieving overshoot < 0.1℃ and oscillation amplitude ≤ 0.03℃ simultaneously is significantly more difficult than in a traditional temperature control system. This scheme uses a three-stage approximation strategy to actively avoid overheating in the "rapid heating stage" (with early intervention in the linear decrease stage) and to suppress oscillations with a very small step size (step size ≤ 2%) in the "steady-state holding stage," so that both can be satisfied simultaneously in the same control scheme—avoiding a trade-off between speed and stability.

[0031] In some embodiments, the pyrolysis temperature control assembly includes a sandwiched ice-water bath circulation structure and a pulsed intermittent laser output control module. The sandwiched ice-water bath circulation structure is used to cool the pyrolysis zone of the sealed reaction chamber, and the pulsed intermittent laser output control module is used to start and stop the laser irradiation assembly in stages. The two work together to maintain the pyrolysis zone of the sealed reaction chamber at 50°C to 70°C.

[0032] In the above technical solution, the specific implementation of the pyrolysis temperature control component is further defined: it includes a sandwich ice-water bath circulation structure and a pulsed intermittent laser output control module. The sandwich ice-water bath circulation structure is used to cool the pyrolysis zone of the sealed reaction chamber, and the pulsed intermittent laser output control module is used to start and stop the laser irradiation component in segments. The two work together to maintain the temperature of the pyrolysis zone at 50℃~70℃.

[0033] Compared to existing technologies (such as the magnetic bead + laser lysis schemes disclosed in CN1818054A and EP1870449A1), existing laser lysis technologies only focus on the cell disruption effect of laser irradiation, neglecting the destructive effect of the instantaneous high temperature of the laser on released nucleic acids, and failing to propose any temperature control measures. The essential feature of this scheme is that it addresses the unrecognized technical problem of "nucleic acid degradation due to instantaneous high temperature" during laser lysis. Through the synergistic combination of active temperature control (a sandwiched ice-water bath circulation structure) and heat source control (pulsed intermittent laser output), it achieves active temperature management during the lysis process.

[0034] On one hand, the sandwiched ice-water bath circulation structure provides continuous basic cooling capacity. A sandwich layer is set around the lysis zone, and ice water circulates within it, continuously carrying away the heat accumulated in the lysis zone. The temperature control target for the lysis zone is 50℃~70℃—this temperature range is technically critical: below 50℃, the heat generated by the laser is excessively carried away, and the intracellular vapor pressure is insufficient to effectively break down the cell wall, resulting in decreased lysis efficiency; above 70℃, the nucleic acid degradation rate accelerates significantly. The temperature control range of 50℃~70℃ ensures the heat accumulation required for cell lysis while avoiding the risk of nucleic acid degradation.

[0035] On the other hand, the pulsed intermittent laser output control module actively controls the input intensity and duration of the heat source by segmenting the laser's start and stop. Unlike relying solely on external cooling, the pulsed intermittent output provides the instantaneous high temperature required for decomposition during laser operation, and then uses an ice-water bath to cool the laser down during laser shutdown. The intermittent output of the laser (rather than continuous output) ensures that each pulse generates sufficient thermal shock to achieve decomposition, while preventing the temperature from accumulating above 70°C.

[0036] More importantly, the interlayered ice-water bath provides continuous basic cooling capacity, suppressing the rate of temperature rise and peak value; the pulsed intermittent laser control module determines the timing and duration of heat input; the continuous cooling rate of the ice-water bath and the heat generation rate of the intermittent laser output are matched to each other, jointly determining the temperature profile of the pyrolysis zone. Without the ice-water bath, even with segmented start and stop of the pulsed intermittent laser, the temperature will still gradually increase over multiple pulse cycles; without pulsed intermittent laser control, the ice-water bath can only passively cool down and cannot actively regulate the heat source input.

[0037] In some embodiments, the preprocessing module further includes a magnetic bead storage and allocation unit, the magnetic bead storage and allocation unit comprising: The annular rotary storage bin has multiple independent and sealed storage compartments, each of which stores immunomagnetic beads specific to different pathogens. Each of the aforementioned storage compartments is equipped with an RFID electronic tag, which stores the type code of the corresponding magnetic bead and the cumulative number of times it has been used; An RFID reader / writer is used to read and update the information in the RFID electronic tag; The management controller is configured to: read the cumulative number of times the target magnetic bead has been used each time a detection is initiated; allow material discharge if the number of times has not reached 40; and increment the cumulative number of times used by 1 after the magnetic bead has been detected and returned to the corresponding storage compartment; issue an alert when the cumulative number of times used reaches 38; and lock the material discharge permission of the storage compartment when the number of times used reaches 40.

[0038] In the above technical solution, the preprocessing module is further defined to include a magnetic bead storage and distribution unit, which adopts a ring-shaped rotary storage bin with RFID electronic tags and management controllers to realize the classification, storage, identification, usage tracking and life expiration warning of specific immunomagnetic beads of different pathogens, thus constructing a complete intelligent management system for the entire life cycle of magnetic beads.

[0039] Compared to existing technologies (such as CN1818054A, EP1870449A1, etc.), the magnetic beads in existing magnetic bead + laser pyrolysis solutions are all single-use and discarded after use. Even if some magnetic bead recycling solutions exist, they are only simple physical recycling and lack means to track the usage status of the magnetic beads and manage their lifespan.

[0040] The key feature of this solution lies in the construction of a complete intelligent management system for the entire lifecycle of magnetic beads: Firstly, a circular rotary storage bin combined with RFID tags enables the identification and traceability of magnetic beads. Each storage compartment is equipped with an RFID tag, storing information such as the type code of the magnetic beads in that compartment and the cumulative number of times they have been used. The RFID reader reads the tag information each time a test is initiated, ensuring that the type of magnetic bead used is consistent with the detection target. This mechanism solves the problem of magnetic bead type confusion or misuse in scenarios involving parallel detection of multiple bacterial species.

[0041] Secondly, the management controller monitors the lifespan of magnetic beads in real time by accumulating the number of uses. During repeated use, the antibody activity on the surface of the magnetic beads gradually decreases, leading to a decline in capture efficiency. A warning threshold of 38 uses is used to alert operators in advance to prepare for replacement, preventing inaccurate test results due to insufficient capture efficiency at the 39-40 use stage. A 40-use lockout ensures that the magnetic beads will not be used further when antibody activity is insufficient, guaranteeing the reliability of test results. The management controller is configured to automatically increment the usage count by 1 after each test, enabling real-time updates of the usage count.

[0042] In some embodiments, the magnetic bead storage and distribution unit further includes a weighing sensor disposed at the bottom of each of the storage compartments, for acquiring the weight data of the corresponding magnetic beads in real time.

[0043] In the above technical solution, each storage compartment is further equipped with a weighing sensor at the bottom to obtain the weight data of the corresponding magnetic beads in real time, so as to realize the quantitative control of magnetic bead discharge and real-time verification of inventory.

[0044] In magnetic bead reuse scenarios, with increasing usage, the actual usable quantity of magnetic beads may deviate from the initial value due to wear, aggregation, or adhesion to the bead walls. If discharge control relies solely on the initial loading amount, insufficient discharge may lead to decreased capture efficiency, while excessive discharge may result in waste.

[0045] In this solution, load cells are installed at the bottom of each storage compartment to acquire the weight data of the corresponding magnetic beads in real time. This weight data can be used for control at the following three levels: Firstly, closed-loop control of the discharge volume. During the magnetic bead discharge process, the management controller determines whether the amount of discharged magnetic beads has reached the target value based on the real-time weight change feedback from the weighing sensor, and automatically stops discharging when the target value is reached. This eliminates the discharge volume deviation caused by factors such as magnetic bead wear, aggregation, or wall adhesion, ensuring the accuracy and consistency of the amount of immunomagnetic beads used in each test.

[0046] Secondly, real-time verification of magnetic bead inventory. The weighing sensor reads the total weight of the magnetic beads each time a test is initiated and compares it with the expected remaining quantity recorded in the RFID tag. If the deviation exceeds a threshold, an alarm is triggered, prompting operators to check for magnetic bead leakage, clumping, or abnormal wear. This avoids the risk of test failure due to insufficient actual usable quantity caused by unexpected magnetic bead wear.

[0047] Third, the weighing data provides a verification basis for confirming the reset of the magnetic beads after reflux. After the test is completed, the magnetic beads are refluxed to the storage compartment. The weighing sensor measures the weight after reflux and compares it with the weight before discharge to confirm whether all the magnetic beads have been recovered. If the reflux flow rate is found to be significantly lower than the discharge rate, an abnormal alarm will be issued to avoid magnetic beads remaining in the pipeline or reaction chamber, which may cause cross-contamination or blockage.

[0048] In some embodiments, the light source array is a near-infrared LED array, which is arranged in a honeycomb pattern below the light-transmitting detection cavity, and a microlens array light-uniforming structure is provided between the near-infrared LED array and the light-transmitting detection cavity.

[0049] In the above technical solution, the light source array is further defined as a near-infrared LED array, which is arranged in a honeycomb pattern below the light-transmitting detection cavity, and a microlens array light-uniforming structure is provided between the LED array and the cavity. The uniformity of light energy incident during photothermal conversion heating is optimized from two dimensions: the light source arrangement and the light path uniformity.

[0050] Compared to existing photothermal PCR methods, current technologies typically use lasers or ordinary LEDs as heating sources, but rarely pay attention to the spatial arrangement of the light source or the homogenization of light energy. Even when some methods mention "array arrangement," they are mostly simple rectangular matrix arrangements that do not consider the issues of light spot overlap and uniformity, and do not set up a dedicated light homogenizing structure.

[0051] Firstly, the honeycomb arrangement offers superior optical uniformity compared to the traditional rectangular matrix arrangement. In a rectangular matrix arrangement, the LED light-emitting points are aligned in rows and columns, resulting in a significant periodic variation in light intensity across the reaction surface—the light intensity is enhanced in row-aligned areas, while the light intensity is relatively weaker in inter-row areas, forming regular stripes or grid-like light spots. In a honeycomb arrangement, each light-emitting point maintains an equal distance from its adjacent points (in a regular hexagonal arrangement, the spacing between adjacent points is consistent), leading to a more uniform overlapping distribution of light spots on the reaction surface compared to a rectangular matrix. This eliminates the stripe-like uneven areas caused by row and column alignment in rectangular arrangements. Furthermore, a honeycomb arrangement can accommodate more LEDs within the same area (the fill rate of a hexagonal close-packed LED is higher than that of a rectangular arrangement), resulting in a higher light power density per unit area, which is beneficial for rapid heating. This arrangement addresses the issue of uniform spatial distribution of light energy in "area heating" scenarios, and is not a conventional choice for point or line heating scenarios.

[0052] Secondly, the microlens array homogenizing structure further eliminates the light spot hotspots of the LED array. The emitted light from a single LED exhibits a Lambertian distribution, with high intensity at the center and weak intensity at the edges. Even with a honeycomb arrangement, spatial non-uniformity of the central hotspot still exists in the illumination area corresponding to each LED. The microlens array redistributes and diffuses the emitted light from each LED, making the light energy distribution on the outer wall of the light-transmitting detection cavity more uniform. This structure allows light energy to be uniformly incident on the outer wall of the cavity after homogenization. Combined with the characteristic of quartz material having a transmittance of >90% in the near-infrared band, it ensures that light energy efficiently reaches the graphene light-absorbing layer while eliminating local hotspots caused by individual LED differences (brightness inconsistencies between LEDs).

[0053] Third, the orientation of the LED array "located below the light-transmitting detection cavity" ensures that light energy is incident vertically from bottom to top. Combined with the layout of the light-transmitting detection cavity located above the LED array, this ensures that light energy evenly covers the entire bottom area of ​​the light-transmitting detection cavity.

[0054] In some embodiments, the system further includes a fluorescence acquisition and analysis module, which includes a fluorescence acquisition component and a data processing unit. The fluorescence acquisition component is used to acquire fluorescence signals in the light-transmitting detection cavity, and the data processing unit is used to perform noise reduction filtering and baseline correction preprocessing on the fluorescence signals, and compare the preprocessed fluorescence signals with a built-in pathogen database to output pathogen detection results.

[0055] In the above technical solution, the system is further defined to include a fluorescence acquisition and analysis module, which acquires fluorescence signals in real time during the qPCR amplification process through a fluorescence acquisition component, and performs preprocessing such as noise reduction filtering and baseline correction through a data processing unit before comparing with a built-in pathogen database, and automatically outputs pathogen detection results, thus constructing a fully automated analysis system from fluorescence signal acquisition to result interpretation.

[0056] Compared with existing technologies (such as manual interpretation methods or general data analysis software that come with conventional qPCR instruments), the fluorescence signal analysis after qPCR in existing technologies mainly relies on the operator's experience for baseline correction and curve identification. Even if some software provides automated analysis functions, they are mostly general algorithms that are not specifically optimized for the complex matrix of seafood samples, nor do they have built-in dedicated databases for specific detection scenarios.

[0057] Firstly, the multi-stage signal preprocessing (noise reduction filtering and baseline correction) is designed to meet the specific needs of seafood sample testing. Even after preprocessing, seafood samples (especially fish, shrimp, shellfish, and crabs) may still contain residual matrix impurities, oils, pigments, and other interfering substances. These substances may generate non-specific fluorescence signals or cause baseline drift. Simultaneously, the instrument's own noise level and changes in ambient light can also affect the accuracy of the fluorescence signal. This solution uses noise reduction filtering to remove high-frequency noise, random noise, and transient abnormal peaks, and baseline correction to correct baseline drift and offset issues, enabling the effective amplified signal to be extracted from complex background noise. In traditional manual interpretation methods, baseline correction relies on the operator's experience, and different personnel may set significantly different baselines for the same set of data. This solution automatically completes the uniform baseline correction through algorithms, eliminating the subjectivity of human judgment and making data preprocessing repeatable and consistent.

[0058] Secondly, the data processing unit compares the preprocessed signal with the built-in pathogen database to automate precise strain typing, positive / negative identification, and nucleic acid quantification. Unlike general public databases, this database is a dedicated database adapted to the complex substrates of seafood and the qPCR detection system of this device. Its threshold system and amplification judgment rules are set according to SN / T 1870-2016 and ISO 22119 standards, and have been iteratively optimized through thousands of real seafood sample tests, resulting in precise Ct threshold ranges and amplification efficiency correction parameters suitable for complex substrate scenarios. When faced with interference from PCR inhibitors in seafood substrates, the standard curves of general databases may deviate from reality, leading to inaccurate quantification. This solution, through a database specifically optimized for seafood substrates, makes the judgment results superior to those relying on general databases in terms of compliance, accuracy, and comparability.

[0059] Third, the data processing unit can further employ machine learning curve similarity matching algorithms to perform point fitting and feature extraction (such as starting point, upward slope, inflection point, peak interval, plateau characteristics, etc.) on the amplification curve of the test sample. This is then compared dimension-by-dimensionally with the features of the standard curves of 13 pathogenic bacteria in the database, calculating feature overlap and parameter matching. The trained model then achieves precise matching of bacterial characteristics. Regarding the differentiation of similar strains, traditional manual interpretation or single-parameter comparison (such as relying solely on Ct values) is insufficient to effectively distinguish between different pathogenic bacterial strains with high homology and similar signal characteristics. Multi-dimensional curve feature comparison utilizes the full information of the amplification curve (not just the single parameter Ct value) for comprehensive judgment, achieving higher-precision typing and identification. The machine learning model continuously optimizes matching parameters through big data training, ensuring that the system's interpretation accuracy continuously improves with the accumulation of data. This fully automated analysis process replaces traditional manual interpretation, solving the problem of inconsistent interpretation results among different operators.

[0060] Fourth, the data processing unit has a built-in dedicated ETL automated parsing script that can directly identify and parse the raw CSV data files exported by the device. It automatically completes the classification, extraction, cleaning, and storage of fluorescence data, Ct values, curve quality scores, amplification status, and judgment results, and automatically generates a standardized visual test report within 5 minutes. This end-to-end automated processing significantly improves data analysis efficiency and ensures the objectivity and traceability of test results.

[0061] According to another aspect of the present invention, a method for detecting pathogenic bacteria is provided, based on the above-described system, comprising: S1. The sample to be tested is introduced into the closed reaction chamber. At the same time, the corresponding immunomagnetic beads are called according to the type of target pathogen and delivered to the closed reaction chamber so that the immunomagnetic beads can bind to the target pathogen in the sample. S2. Apply a magnetic field to the outside of the sealed reaction chamber to enrich the immunomagnetic beads bound to the target pathogens and discharge the unbound waste liquid. S3. Introduce cleaning solution into the sealed reaction chamber to wash the enriched immunomagnetic beads. S4. The target pathogenic bacteria in the sealed reaction chamber are irradiated with a pulsed laser to cause them to lyse and release nucleic acid, and the temperature of the lysis zone in the sealed reaction chamber is controlled at 50℃~70℃ during the laser irradiation process. S5. Transfer the liquid containing the lysed nucleic acid to the qPCR detection chamber; S6. Heat is generated by irradiating the photothermal conversion heating component attached to the outer wall of the qPCR detection chamber to heat the qPCR detection chamber for amplification reaction; during the heating process, the temperature of the qPCR detection chamber is collected in real time at a sampling frequency of ≥50Hz, and the heating power is controlled according to the following strategy: When the difference between the measured temperature and the target temperature is greater than the first threshold, it is driven at full power. When the difference is less than or equal to the first threshold and greater than the second threshold, the power is driven with a linearly decreasing power. When the difference is less than or equal to the second threshold, fine-tuning is performed with a power step size of ≤2%. Simultaneously, fluorescence signals during the amplification process are acquired in real time; S7. Perform preprocessing on the collected fluorescence signal, compare the preprocessed fluorescence signal with the pathogenic bacteria standard amplification feature database, and output the pathogenic bacteria detection result; The above steps S1 to S7 are all automatically controlled and executed by the central controller according to a preset timing sequence.

[0062] In the above technical solution, to better utilize the system, this application proposes a method for detecting pathogens. Each step corresponds to a module of the system, and its specific principles have been described above and will not be repeated here. Attached Figure Description

[0063] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0064] Figure 1 This is a schematic diagram of the system framework of an embodiment of the pathogen detection system of the present invention; Figure 2 This is a schematic diagram of the preprocessing module of an embodiment of the pathogen detection system of the present invention; Figure 3 This is a schematic diagram of a magnetic bead storage and distribution unit according to an embodiment of the pathogen detection system of the present invention; Figure 4 This is a schematic diagram of superparamagnetic beads according to an embodiment of the pathogen detection system of the present invention; Figure 5 This is a schematic diagram of a 2×3 array of neodymium iron boron permanent magnets in an embodiment of the pathogen detection system of the present invention; Figure 6 This is a schematic diagram of the qPCR detection module of an embodiment of the pathogen detection system of the present invention; Figure 7 This is a schematic diagram of the data processing of the fluorescence acquisition and analysis module in an embodiment of the pathogen detection system of the present invention; Figure 8 This is a schematic flowchart of an embodiment of the pathogen detection method of the present invention. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] Example 1 Please see Figure 1 This embodiment provides a pathogen detection system, which includes a preprocessing module, a qPCR detection module, a fluorescence acquisition and analysis module, and a central controller.

[0067] Please see Figure 2 The pretreatment module includes a sealed reaction chamber 1, which is connected to an inlet 11, a magnetic bead supply port 12, and a fluid inlet / outlet 13. A magnetic field generating assembly 14 and a laser irradiation assembly 15 are located on the outer side of the sealed reaction chamber 1. A pyrolysis temperature control assembly 2 is also located around the sealed reaction chamber 1 to control the temperature of the sealed reaction chamber 1 between 50°C and 70°C during laser irradiation.

[0068] The pyrolysis temperature control component 2 includes a sandwiched ice-water bath circulation structure 21 and a pulsed intermittent laser output control module 22. The sandwiched ice-water bath circulation structure 21 is used to cool the pyrolysis zone of the sealed reaction chamber 1, and the pulsed intermittent laser output control module 22 is used to start and stop the laser irradiation component 15 in stages. Together, they maintain the pyrolysis zone of the sealed reaction chamber 1 at 50℃~70℃ during laser irradiation. The sandwiched ice-water bath circulation structure 21 is provided with an ice water inlet 211 and an ice water outlet 212 (e.g., ...). Figure 2 As shown, the pyrolysis zone is continuously cooled by circulating ice water, and the segmented start-stop control of the pulse intermittent laser output control module 22 enables precise adjustment of the temperature of the pyrolysis zone.

[0069] Please see Figure 2 and Figure 5 The magnetic field generating component 14 includes a 2×3 array of NdFeB permanent magnets 141, capable of constructing a high gradient magnetic field of ≥150T / m. In this embodiment, high-voltage electrical energy can be stored in a capacitor bank and instantaneously discharged to a pulse coil through switching devices such as thyristors, generating a strong pulsed magnetic field with an amplitude of several Tesla and a duration of milliseconds. This pulsed magnetic field directly acts on the NdFeB permanent magnets 141, changing their magnetic domain orientation to complete magnetization, or applying a reverse decaying magnetic field to achieve demagnetization. This controls the output of a 450–600 mT pulsed magnetic field, used for magnetic separation and enrichment of immunomagnetic beads bound with target pathogens. After magnetic separation, the central controller controls the magnetic field generating component 14 to maintain the magnetic field (or applies a reverse decaying magnetic field followed by an enrichment magnetic field), keeping the magnetic beads bound with target pathogens in an enriched state. Subsequently, the unbound supernatant waste liquid is pumped from the fluid inlet / outlet 13 to a waste liquid collection container via a waste liquid pump. The magnetic field is maintained during waste discharge to ensure that the magnetic beads are not discharged with the waste liquid. The waste liquid pump is a diaphragm pump that is independent of the fluid transport peristaltic pump, and the waste liquid collection container is a closed waste liquid tank (not shown in the figure).

[0070] The laser irradiation component 15 includes a pulsed laser generator, whose output light path is guided to the sealed reaction chamber 1. In this embodiment, laser lysis relies on the triple action of photothermal effect, photomechanical shock wave, and photochemical oxidation to achieve endogenous cell lysis: the pulsed laser irradiates the buffer solution inside the chamber, causing polar molecules such as water and inorganic salts to oscillate and rub at high speed, rapidly generating heat and pressure inside the cell. The intracellular vapor pressure breaks down the cell wall of pathogenic bacteria, increasing the cell disruption efficiency by 3 to 5 times compared to traditional external heating in a water bath; at the same time, the laser can break the hydrogen bonds and van der Waals forces between nucleic acids and proteins, accelerating the release of intracellular nucleic acids. This application does not specifically limit the laser's optical path design; those skilled in the art can choose a suitable optical path layout based on the actual equipment layout.

[0071] The preprocessing module also includes a magnetic bead storage and allocation unit. See also... Figure 3 The magnetic bead storage and distribution unit includes a ring-shaped rotary storage bin 4, with multiple independent, sealed storage compartments 41 (13 sets in this embodiment). Each storage compartment 41 stores immunomagnetic beads specific to different pathogenic bacteria. Each storage compartment 41 is equipped with an RFID tag 42, which stores the type code and cumulative number of uses of the corresponding magnetic bead. An RFID reader 43 is used to read and update the information in the RFID tag 42. Each storage compartment 41 is individually equipped with a weighing sensor 45 at its bottom to acquire the weight data of the corresponding magnetic bead in real time, so as to realize quantitative dispensing and inventory verification.

[0072] The magnetic bead storage and distribution unit is also equipped with a management controller 44, which is configured to: read the cumulative number of times the target magnetic bead has been used each time the detection is started; allow material discharge if the number of times has not reached 40; and increment the cumulative number of times of use by 1 after the magnetic bead has been detected and returned to the corresponding storage compartment; issue an alarm when the cumulative number of times of use reaches 38; and lock the material discharge permission of the storage compartment when the number of times of use reaches 40.

[0073] In this embodiment, the annular rotary storage bin 4 is made of 6061-T6 aluminum alloy anodized material, which is corrosion-resistant and does not easily generate impurities that contaminate the magnetic beads. The bin body can be driven by a single motor or dual motors working together. The drive unit is required to achieve high-precision positioning of the rotary table within ±0.05mm, and can accurately rotate to the corresponding magnetic bead bin position according to the detection items. The motor drive method is not the core innovation of this application. Those skilled in the art can choose a suitable drive method according to the actual equipment layout, which will not be elaborated here.

[0074] In this embodiment, please refer to Figure 4The immunomagnetic beads were all based on Fe3O4@SiO2 core-shell superparamagnetic beads with a particle size of 500 nm. Antibody-directed immobilization was achieved using the EDC / NHS carboxyl activation method. Each type of magnetic bead was conjugated with a specific antibody against the corresponding pathogen, with the conjugation amount consistently ranging from 45 to 60 μg / mg. After being discharged from the bead storage chamber, the magnetic beads were pumped into the sealed reaction chamber 1 along with the buffer solution, where they underwent an immunobinding reaction with the target pathogen in the sample.

[0075] In this embodiment, the magnetic bead storage and distribution unit is connected to the sealed reaction chamber 1 via an independent discharge pipeline. Each storage compartment 41 is connected to an independent discharge branch pipeline, and each discharge branch pipeline is selectively connected to the main manifold pipeline via an electrically controlled valve group. The end of the main manifold pipeline is connected to the magnetic bead supply port 12 of the sealed reaction chamber 1. Both the discharge branch pipeline and the main manifold pipeline are made of PEEK material. A peristaltic pump is installed on the main manifold pipeline to provide fluid transport power. When the magnetic beads are discharged, the management controller 44 controls the annular rotary storage bin 4 to rotate to the target magnetic bead bin position, aligning the target storage compartment 41 with the discharge position, and opens the electrically controlled valve and peristaltic pump of the corresponding compartment. The buffer solution carries the magnetic beads into the sealed reaction chamber 1 through the discharge branch pipeline, the main manifold pipeline, and the magnetic bead supply port 12. The discharge weight is monitored in real time by a weighing sensor 45 to achieve quantitative discharge. During the magnetic bead recirculation, under the traction of a directional magnetic field, the magnetic beads enter the recirculation pipeline from the sealed reaction chamber 1 through the fluid inlet / outlet 13. The recirculation pipeline is also made of PEEK material and is independent of the discharge pipeline. The recirculation pipeline is selectively connected to the recovery inlet of each storage compartment 41 through another electrically controlled valve group, allowing the magnetic beads to return to their original corresponding storage compartment 41 according to their type. After the magnetic bead recirculation is completed, the management controller 44 controls the cleaning fluid to flush the discharge pipeline and the recirculation pipeline to remove residues.

[0076] The pretreatment module also includes a fluid delivery component (not shown in the figure), connected between the sealed reaction chamber 1 and the light-transmitting detection chamber 31, for automatically delivering the lysed nucleic acid solution from the sealed reaction chamber 1 to the light-transmitting detection chamber 31. In this embodiment, the fluid delivery pipeline uses corrosion-resistant independent PEEK pipelines and electrically controlled solenoid valves, and different types of magnetic beads are equipped with dedicated delivery pipelines to prevent cross-contamination between different types. The specific implementation method of fluid delivery is not the core innovation of this application; those skilled in the art can design suitable delivery methods based on the actual equipment layout, which will not be elaborated here.

[0077] In this embodiment, the fluid delivery assembly includes a transfer pipeline and a peristaltic pump. One end of the transfer pipeline is connected to the fluid inlet / outlet 13 of the sealed reaction chamber 1, and the other end is connected to the liquid inlet of the light-transmitting detection chamber 31. After lysis, the central controller controls the opening of the electrically controlled valve at the fluid inlet / outlet 13 and the peristaltic pump to pump the lysed nucleic acid solution into the light-transmitting detection chamber 31 via the transfer pipeline. The transfer pipeline is made of PEEK material and is independent of the magnetic bead discharge pipeline. The transfer volume of the nucleic acid solution is monitored in real time by a weighing sensor 45, and the transfer automatically stops when a preset volume is reached.

[0078] Please see Figure 6 The qPCR detection module 3 includes a light-transmitting detection cavity 31, a photothermal conversion heating component 32 attached to the outer wall of the light-transmitting detection cavity 31, and a light source array 33 that emits light energy to the photothermal conversion heating component 32.

[0079] In this embodiment, the transmissive detection chamber 31 is pre-filled with qPCR amplification reaction solution (lyophilized or liquid form) before detection begins. The nucleic acid solution is delivered to the transmissive detection chamber 31 via a fluid delivery assembly and mixed with the pre-filled amplification reaction solution. A vent hole (not shown in the figure) is provided at the top of the transmissive detection chamber 31 to balance the internal and external air pressure. Alternatively, the qPCR reaction solution can be pre-mixed with the nucleic acid solution in a separate sample delivery line before entering the transmissive detection chamber 31, and then injected together. The mixing method can be passive mixing (achieved through line length and flow rate) or active mixing (achieved through a small static mixer).

[0080] The photothermal conversion heating component 32 is a composite functional film attached to the outer wall of the light-transmitting detection cavity 31. The composite functional film includes a graphene layer 321, a carbon nanotube layer 322, and a silicon nitride layer 323. The three-layer composite film is deposited sequentially on the outer wall of the light-transmitting detection cavity 31 using a combination of magnetron sputtering and chemical vapor deposition.

[0081] The composite functional membrane comprises, from its inner surface to its outer surface during use, the following: The graphene layer 321 (thickness 15-30nm) is made of graphene material and is closely attached to the outer wall of the light-transmitting detection cavity 31. On the one hand, it absorbs near-infrared light and converts it into heat energy, and on the other hand, it fills the microscopic unevenness of the outer wall of the cavity and reduces the contact thermal resistance. A carbon nanotube layer 322 (thickness 0.4-0.6 mm) is stacked on top of the graphene layer 321. It is made of carbon nanotube material and has an in-plane thermal conductivity ≥3800 W / (m·K). It is used to quickly conduct and diffuse the heat generated by the graphene layer 321 along the in-plane direction to eliminate the temperature unevenness caused by point heat sources. A silicon nitride layer 323, stacked on top of the carbon nanotube layer 322, is made of silicon nitride material and covers the surface of the carbon nanotube layer 322 to protect the underlying structure from thermomechanical stress and ensure long-term reliability.

[0082] The total thickness of the composite functional membrane 32 is ≤1mm.

[0083] In this embodiment, the composite functional film is deposited on the outer side of the light-transmitting detection cavity 31 by deposition. Before the film is deposited, the outer wall of the cavity is plasma etched to form a submicron-level rough structure, which increases the actual contact area between the subsequently deposited graphene layer 321 and the outer wall of the cavity by about 40%, and reduces the interface thermal resistance to less than 1 / 10 of that of traditional thermal grease.

[0084] Through the synergistic design of near-infrared LEDs and a three-layer composite functional film, millisecond-level photothermal conversion and uniform heating of the reaction surface can be achieved, reducing the temperature difference of the 100mm×100mm reaction surface from the traditional 3-5℃ to below 0.3℃.

[0085] The light source array 33 is a near-infrared LED array with a peak wavelength of 850nm, arranged in a honeycomb pattern below the light-transmitting detection cavity 31. A microlens array homogenizing structure 34 is provided between the near-infrared LED array and the light-transmitting detection cavity 31. The LED light source, combined with a 0.1% precision programmable constant current drive power supply, achieves stepless dimming, with the drive current continuously adjustable within the range of 0-100% and a response time ≤50μs. After being homogenized by the microlens array, the near-infrared light is perpendicularly incident on the outer wall of the light-transmitting detection cavity 31. The quartz material has a transmittance of >90% in the near-infrared band, ensuring that the light energy efficiently reaches the graphene light-absorbing layer.

[0086] In this embodiment, the deposition of the composite functional membrane is performed after the light-transmitting detection cavity 31 is manufactured and before the entire device is assembled. Specifically, the light-transmitting detection cavity 31 is placed in a vacuum coating apparatus, and a graphene layer 321 (magnetron sputtering), a carbon nanotube layer 322 (chemical vapor deposition), and a silicon nitride layer 323 (chemical vapor deposition) are deposited sequentially. After deposition, the coated light-transmitting detection cavity 31 is assembled into the qPCR detection module 3, completing the integration of components such as the light source array 33, the microlens array homogenizing structure 34, and the heat dissipation structure (heat pipe 35 and heat dissipation fins 36). The composite functional membrane is deposited on the outer wall of the cavity and does not come into contact with the reaction liquid, so there is no need to consider the impact of the deposited material on biocompatibility.

[0087] The qPCR detection module 3 also includes a temperature sensor and a temperature control unit (not shown in the figure). The temperature sensor is used to detect the temperature of the light-transmitting detection cavity 31 in real time. The temperature control unit is signal-connected to the temperature sensor and the light source array 33, and is used to control the output power of the light source array 33 according to a three-stage strategy: When the difference between the measured temperature and the target temperature is |e| > 2℃, the light source array 33 is driven at full power to achieve rapid heating. When the difference |e|≤2℃ and |e|>0.2℃, the power The temperature is linearly reduced, allowing it to smoothly approach the target value with an overshoot of <0.1℃; When the difference |e|≤0.2℃, fine-tuning is performed with power increments of ≤2% to ensure that the temperature oscillation amplitude near the target value is ≤0.03℃.

[0088] The temperature control unit samples the temperature sensor at a frequency ≥50Hz.

[0089] The target temperature is dynamically set by the central controller according to the current stage of qPCR amplification, including denaturation temperature, annealing temperature, and extension temperature.

[0090] In this embodiment, a temperature sensor (not shown in the figure) is embedded in the inner wall of the pyrolysis zone of the sealed reaction chamber 1. The temperature sensor's probe extends into the pyrolysis zone and is immersed in the pyrolysis liquid to detect the liquid phase temperature of the pyrolysis zone in real time. The temperature sensor is installed near the focal spot area of ​​the laser irradiation component 15 to avoid direct laser irradiation of the probe, which could lead to abnormal readings. The jacketed ice-water bath circulation structure 21 and the pulsed intermittent laser output control module 22 are both connected to the central controller. When the temperature sensor detects that the temperature of the pyrolysis zone exceeds 70°C, the central controller controls the pulsed intermittent laser output control module 22 to shut down the laser irradiation component 15 while maintaining the jacketed ice-water bath circulation; when the temperature drops below 50°C, the laser irradiation component 15 is restarted. This cycle is repeated to precisely maintain the temperature of the pyrolysis zone within the range of 50°C to 70°C. The single-time on-time of the pulsed laser is preferably 0.5 to 2 seconds, and the off-time is dynamically adjusted according to the real-time temperature.

[0091] In this embodiment, the qPCR detection module 3 also includes a heat dissipation structure, comprising heat dissipation pipes 35 (7 copper heat pipes) and heat dissipation fins 36 (interdigitated structure). The heat dissipation pipes 35 are embedded in the heat dissipation fins 36 (e.g., Figure 6 As shown, 361 is the heat pipe embedding hole, and 362 and 363 are the left and right interdigitated fins. The heat pipe utilizes phase change heat transfer to quickly remove excess heat from the hot end. The interdigitated fin structure replaces traditional flat or needle-shaped fins, significantly increasing the effective heat dissipation area within the same volume. The overall thermal resistance is as low as 0.1℃ / W, effectively preventing heat accumulation and thermal drift, providing a physical basis for high-precision temperature control of ±0.1℃.

[0092] Please see Figure 7The fluorescence acquisition and analysis module includes a fluorescence acquisition component and a data processing unit. The fluorescence acquisition component is used to acquire fluorescence signals in the light-transmitting detection cavity 31. The data processing unit has a built-in pathogenic bacteria standard amplification characteristic database, which is used to preprocess the acquired fluorescence signals, compare the preprocessed fluorescence signals with the database, and output the pathogenic bacteria detection results.

[0093] In this embodiment, the fluorescence acquisition component is disposed above or to the side of the light-transmitting detection cavity 31. The fluorescence acquisition component includes a fluorescence excitation light source, an emission filter, and a photodetector. The fluorescence excitation light source emits excitation light to illuminate the sample inside the light-transmitting detection cavity 31. After the sample is excited, it generates fluorescence. The fluorescence is filtered by the emission filter to remove stray light and then received by the photodetector and converted into an electrical signal. Those skilled in the art can select appropriate fluorescence excitation light source wavelengths and filter parameters according to the type of fluorescent dye used in the actual detection; these will not be elaborated here. The fluorescence excitation light source can be disposed on the side or bottom of the light-transmitting detection cavity 31, and placed on different sides of the light-transmitting detection cavity 31 from the heating light source array 33, to avoid mutual interference between the heating light and the excitation light.

[0094] In this embodiment, the database is stored in the local storage of the data processing unit (such as a solid-state drive or embedded flash memory). The database can be updated periodically via a USB interface or network communication interface on the device. After the device is powered on, the data processing unit automatically loads the database into memory for real-time comparison and analysis during the detection process.

[0095] In this embodiment, the data processing unit addresses issues in seafood sample detection such as matrix impurity interference, instrument noise, ambient light interference, baseline drift, and non-specific signal fluctuations. It employs a multi-level signal preprocessing algorithm to standardize and correct the original fluorescence signal, eliminating invalid interference signals and retaining valid amplified signals. The system combines machine learning curve fitting and comparison algorithms, threshold determination algorithms, and quantitative conversion mathematical models to complete multi-dimensional intelligent analysis and fully automated computation.

[0096] The pathogenic bacteria standard amplification feature database is a dedicated database constructed for multiple pathogenic bacteria in seafood samples. Its threshold system and amplification judgment rules are set according to SN / T 1870-2016 and / or ISO 22119 standards, and have been iteratively optimized using thousands of real seafood sample test data. The database is built using the PostgreSQL relational database management system, following the third normal form of database design, and consists of five core data tables: pathogens (pathogenic bacteria strain library), samples (sample information library), qpcr_runs (test batch library), wells (test well position data), and amplification_curves (amplification curve feature library).

[0097] The system has a built-in dedicated ETL automated parsing script that can directly recognize and parse the raw CSV data files exported by the device. It automatically completes the classification, extraction, cleaning, and storage of raw fluorescence data, Ct detection values, curve quality scores, amplification status, and final judgment results. Employing a machine learning curve similarity matching algorithm, it performs point fitting and feature extraction on the amplification curve of the sample to be tested, comparing it dimension-by-dimensionally with the characteristics of pathogenic bacteria standard curves in the database. This completes accurate bacterial typing, positive / negative determination, and nucleic acid quantification (CFU / g), and automatically generates a standardized visual test report within 5 minutes.

[0098] The central controller is connected to the preprocessing module, the qPCR detection module, the fluid delivery assembly, and the fluorescence acquisition and analysis module, respectively, and is used to control the above modules to execute the detection process according to a preset timing sequence.

[0099] In this embodiment, the central controller is connected via an SPI serial communication bus to the preprocessing module (including the magnetic field generating component 14, the laser irradiation component 15, and the lysis temperature control component 2), the qPCR detection module 3 (including the temperature sensor, the light source array 33, and the fluorescence acquisition component), the fluid transport component, and the fluorescence acquisition and analysis module, respectively, to achieve high-speed data interaction and synchronous control between the modules. For example, the central controller executes the detection process according to the following timing sequence: t0: Start the magnetic bead discharge; t1: Immunobinding is complete, and the magnetic field is activated; t2: Enrichment complete, waste discharge initiated; t3: Waste discharge completed, start cleaning fluid delivery; t4: Cleaning complete, activate the magnetic field to maintain operation, then activate waste discharge; t5: Waste discharge complete, magnetic field shut off, laser pyrolysis initiated; t6: Cracking complete, fluid transfer initiated; t7: Transfer complete, start qPCR amplification.

[0100] The trigger condition for each of the above steps is the feedback of the completion signal of the previous step. Some steps can be judged as completed based on the weighing signal of the weighing sensor 45 or the temperature signal of the temperature sensor.

[0101] Example 2 Please see Figure 8This embodiment provides a method for detecting pathogenic bacteria, which can be executed based on the pathogenic bacteria detection system described in Embodiment 1. In this embodiment, the sealed reaction chamber in step S1 is the sealed reaction chamber with reference numeral 1 in Embodiment 1; the temperature control in step S4 is achieved by the pyrolysis temperature control component with reference numeral 2 in Embodiment 1, specifically by using the interlayer ice-water bath circulation structure 21 and the pulsed intermittent laser output control module 22 to control the temperature of the pyrolysis zone at 50℃~70℃; the fluid transfer in step S5 is achieved by the fluid transport component described in Embodiment 1.

[0102] The method includes the following steps: S1. The sample to be tested is introduced into the closed reaction chamber 1. At the same time, the corresponding immunomagnetic beads are called according to the type of target pathogen and delivered to the closed reaction chamber 1 so that the immunomagnetic beads can bind to the target pathogen in the sample.

[0103] In this step, the immunomagnetic beads come from the magnetic bead storage and distribution unit, rotate through the annular rotary storage bin 4 to the target magnetic bead bin, and are sent into the sealed reaction chamber 1 through the magnetic bead supply port 12.

[0104] S2. Apply a magnetic field to the outside of the sealed reaction chamber 1 to enrich the immunomagnetic beads bound to the target pathogens and discharge the unbound waste liquid.

[0105] In this step, the magnetic field is applied by the magnetic field generating component 14 (2×3 array of neodymium iron boron permanent magnets 141), and the enriched waste liquid is discharged through the fluid inlet / outlet 13.

[0106] S3. A cleaning solution is introduced into the sealed reaction chamber 1 to wash the enriched immunomagnetic beads in order to remove unbound nonspecific adsorbents.

[0107] S4. The target pathogenic bacteria in the sealed reaction chamber 1 are irradiated with a pulsed laser to cause them to lyse and release nucleic acid. During the laser irradiation process, the temperature of the lysis zone in the sealed reaction chamber 1 is controlled at 50℃~70℃ by the lysis temperature control component 2.

[0108] In this step, the pulsed laser is generated by the laser irradiation component 15, and the pyrolysis temperature control component 2 includes a sandwich ice-water bath circulation structure 21 and a pulsed intermittent laser output control module 22. The two work together to achieve precise temperature control of 50℃~70℃.

[0109] S5. The liquid after lysis and release of nucleic acid is automatically transferred to the qPCR detection chamber (i.e., the light-transmitting detection chamber 31 described in Example 1) through the fluid delivery component.

[0110] S6. Heat is generated by irradiating the photothermal conversion heating component 32 attached to the outer wall of the qPCR detection chamber to heat the qPCR detection chamber for amplification reaction; during the heating process, the temperature of the qPCR detection chamber is collected in real time at a sampling frequency of ≥50Hz, and the heating power is controlled according to the following strategy: When the difference between the measured temperature and the target temperature is greater than the first threshold, it is driven at full power. When the difference is less than or equal to the first threshold and greater than the second threshold, the power is driven with a linearly decreasing power. When the difference is less than or equal to the second threshold, fine-tuning is performed with a power step size of ≤2%. Simultaneously, fluorescence signals are acquired in real time during the amplification process.

[0111] In this step, the photothermal conversion heating component 32 is the three-layer composite functional film (graphene layer 321, carbon nanotube layer 322, silicon nitride layer 323) described in Example 1. The heating power is controlled by the temperature control unit, and the target temperature can be set according to different amplification stages (such as 95°C denaturation, 60°C annealing extension, etc.).

[0112] S7. Perform preprocessing on the collected fluorescence signal, and compare the preprocessed fluorescence signal with the pathogenic bacteria standard amplification feature database described in Example 1 to output the pathogenic bacteria detection result.

[0113] In this step, the preprocessing includes noise reduction filtering and baseline correction. The database is the seafood pathogen database described in Example 1. The output results include pathogen typing, positive and negative determination, and / or nucleic acid quantification data.

[0114] The above steps S1 to S7 are all automatically controlled and executed by the central controller according to a preset timing sequence.

[0115] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A pathogen detection system, characterized in that, The system includes: The pretreatment module includes a sealed reaction chamber, which is connected to an inlet, a magnetic bead supply port, and a fluid inlet / outlet. A magnetic field generating component and a laser irradiation component are provided on the outside of the sealed reaction chamber. A pyrolysis temperature control component is also provided on the periphery of the sealed reaction chamber. The pyrolysis temperature control component is used to control the temperature of the pyrolysis zone of the sealed reaction chamber at 50℃~70℃ during laser irradiation. The qPCR detection module, connected to the preprocessing module, includes a light-transmitting detection cavity, a photothermal conversion heating component attached to the outer wall of the light-transmitting detection cavity, and a light source array that emits light energy to the photothermal conversion heating component. The qPCR detection module further includes a temperature sensor and a temperature control unit. The temperature sensor is used to detect the temperature of the light-transmitting detection cavity in real time. The temperature control unit is signal-connected to the temperature sensor and the light source array, and is used to control the output power of the light source array according to the following strategy: When the difference between the measured temperature and the target temperature is greater than the first threshold, it is driven at full power. When the difference is less than or equal to the first threshold and greater than the second threshold, the power is driven with a linearly decreasing power. When the difference is less than or equal to the second threshold, fine-tuning is performed with a power step size of ≤2%. The temperature control unit samples the temperature sensor at a frequency ≥50Hz; The system also includes a central controller, which is connected to the preprocessing module and the qPCR detection module respectively, and is used to control the above modules to execute the detection process according to a preset timing sequence.

2. The pathogen detection system as described in claim 1, characterized in that, The photothermal conversion heating component is a composite functional film attached to the outer wall of the light-transmitting detection cavity, and the composite functional film comprises, from the outer wall of the light-transmitting detection cavity outwards, the following components: The graphene layer, made of graphene material, is used to adhere to the outer wall of the light-transmitting detection cavity to absorb light energy and convert it into heat energy. A carbon nanotube layer, stacked on top of the graphene layer, is made of carbon nanotube material and is used to conduct the heat generated by the graphene layer in the in-plane direction. A silicon nitride layer, stacked on top of the carbon nanotube layer, is made of silicon nitride material and is used to cover the surface of the carbon nanotube layer.

3. The pathogen detection system as described in claim 2, characterized in that, The graphene layer has a thickness of 15–30 nm, the carbon nanotube layer has an in-plane thermal conductivity ≥3800 W / (m·K) and a thickness of 0.4–0.6 mm, and the total thickness of the composite functional film is ≤1 mm.

4. The pathogen detection system as described in claim 1, characterized in that, The temperature control unit controls the output power of the light source array according to the following strategy: When the difference between the measured temperature and the target temperature is greater than 2°C, the light source array is driven at full power. When the difference is ≤2℃ and >0.2℃, the power The temperature is linearly reduced, allowing it to smoothly approach the target value with an overshoot of <0.1℃; When the difference is ≤0.2℃, fine-tuning is performed with power increments of ≤2% to ensure that the temperature oscillation amplitude near the target value is ≤0.03℃.

5. The pathogen detection system as described in claim 1, characterized in that, The pyrolysis temperature control component includes a sandwiched ice-water bath circulation structure and a pulsed intermittent laser output control module. The sandwiched ice-water bath circulation structure is used to cool the pyrolysis zone of the sealed reaction chamber, and the pulsed intermittent laser output control module is used to start and stop the laser irradiation component in stages. The two work together to maintain the pyrolysis zone of the sealed reaction chamber at 50℃~70℃.

6. The pathogen detection system as described in claim 1, characterized in that, The preprocessing module further includes a magnetic bead storage and allocation unit, which includes: The annular rotary storage bin has multiple independent and sealed storage compartments, each of which stores immunomagnetic beads specific to different pathogens. Each of the aforementioned storage compartments is equipped with an RFID electronic tag, which stores the type code of the corresponding magnetic bead and the cumulative number of times it has been used; An RFID reader / writer is used to read and update the information in the RFID electronic tag; The management controller is configured to: read the cumulative number of times the target magnetic bead has been used each time a detection is initiated; allow material discharge if the number of times has not reached 40; and increment the cumulative number of times used by 1 after the magnetic bead has been detected and returned to the corresponding storage compartment; issue an alert when the cumulative number of times used reaches 38; and lock the material discharge permission of the storage compartment when the number of times used reaches 40.

7. The pathogen detection system as described in claim 6, characterized in that, The magnetic bead storage and distribution unit also includes a weighing sensor installed at the bottom of each of the storage compartments, for real-time acquisition of the weight data of the corresponding magnetic beads.

8. The pathogen detection system as described in claim 1, characterized in that, The light source array is a near-infrared LED array, which is arranged in a honeycomb pattern below the light-transmitting detection cavity. A microlens array light-uniforming structure is provided between the near-infrared LED array and the light-transmitting detection cavity.

9. The pathogen detection system as described in claim 1, characterized in that, The system also includes a fluorescence acquisition and analysis module, which includes a fluorescence acquisition component and a data processing unit. The fluorescence acquisition component is used to acquire fluorescence signals in the light-transmitting detection cavity. The data processing unit is used to perform noise reduction filtering and baseline correction preprocessing on the fluorescence signals, and compare the preprocessed fluorescence signals with the built-in pathogen database to output pathogen detection results.

10. A method for detecting pathogenic bacteria, based on the system described in any one of claims 1 to 9, characterized in that, include: S1. The sample to be tested is introduced into the closed reaction chamber. At the same time, the corresponding immunomagnetic beads are called according to the type of target pathogen and delivered to the closed reaction chamber so that the immunomagnetic beads can bind to the target pathogen in the sample. S2. Apply a magnetic field to the outside of the sealed reaction chamber to enrich the immunomagnetic beads bound to the target pathogens and discharge the unbound waste liquid. S3. Introduce cleaning solution into the sealed reaction chamber to wash the enriched immunomagnetic beads. S4. The target pathogenic bacteria in the sealed reaction chamber are irradiated with a pulsed laser to cause them to lyse and release nucleic acid, and the temperature of the lysis zone in the sealed reaction chamber is controlled at 50℃~70℃ during the laser irradiation process. S5. Transfer the liquid containing the lysed nucleic acid to the qPCR detection chamber; S6. Heat is generated by irradiating the photothermal conversion heating component attached to the outer wall of the qPCR detection chamber to heat the qPCR detection chamber for amplification reaction; during the heating process, the temperature of the qPCR detection chamber is collected in real time at a sampling frequency of ≥50Hz, and the heating power is controlled according to the following strategy: When the difference between the measured temperature and the target temperature is greater than the first threshold, it is driven at full power. When the difference is less than or equal to the first threshold and greater than the second threshold, the power is driven with a linearly decreasing power. When the difference is less than or equal to the second threshold, fine-tuning is performed with a power step size of ≤2%. Simultaneously, fluorescence signals during the amplification process are acquired in real time; S7. Perform preprocessing on the collected fluorescence signal, compare the preprocessed fluorescence signal with the pathogenic bacteria standard amplification feature database, and output the pathogenic bacteria detection result; The above steps S1 to S7 are all automatically controlled and executed by the central controller according to a preset timing sequence.

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