A method and system for detecting multiple gene loci using flow-through hybridization with sequential temperature control

Abstract

This invention relates to the technical field of gene hybridization, and more specifically, to a time-controlled temperature-controlled flow hybridization method and system for detecting multiple gene loci. The method includes: chip placement; preheating and initial hybridization; temperature fluctuation assessment; low-Tm probe hybridization; temperature fluctuation assessment; high-Tm probe hybridization; and washing and color development of the gene chip after the hybridization module discharges the hybridization solution. The solution of this invention can design a stepped temperature ramping program based on the Tm value of each probe, ensuring that each probe completes efficient binding within its optimal temperature window. Simultaneously, it can monitor and assess temperature fluctuations in real time and dynamically adjust temperature control parameters, thereby ensuring the stability and consistency of the hybridization process.

Description

Technical Field

[0001] This invention relates to the technical field of gene hybridization, and more specifically, to a time-controlled temperature-controlled method and system for detecting multiple gene loci in transduction hybridization. Background Technology

[0002] The melting temperature (Tm) is the temperature at which 50% of the DNA double helix dissociates into single strands during heating. This temperature is primarily related to the content of G (guanine) and C (cytosine) in the nucleic acid sequence; the higher the GC content, the higher the Tm value. The Tm value is an important parameter for measuring the stability of nucleic acid double helixes and plays a crucial role in molecular biology experiments. In flow-through hybridization techniques, the Tm value is essential for ensuring efficient and specific hybridization reactions.

[0003] In terms of temperature control for multi-gene locus detection during flow-through hybridization, current flow-through hybridization platforms generally employ a uniform hybridization temperature strategy, setting a constant hybridization temperature based on the average Tm value of all probes. However, this uniform hybridization temperature strategy has significant limitations when dealing with probe combinations with significantly different Tm values. On the one hand, low-Tm probes struggle to form stable double strands at higher temperatures, leading to decreased sensitivity; on the other hand, high-Tm probes may tolerate more mismatch bindings at lower temperatures, thus reducing detection specificity. Therefore, this temperature control method lacks effective control over dynamic temperature changes and cannot provide differentiated hybridization conditions based on the thermodynamic characteristics of different probes, limiting the application of probes with different Tm values ​​in complex samples or multiple mutation detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing temperature control methods that use a uniform hybridization temperature, which cannot provide differentiated hybridization conditions based on the thermodynamic characteristics of different probes. This invention provides a time-controlled, flow-guided hybridization method and system for detecting multiple gene loci. The solution of this invention can design a stepped heating program based on the Tm value of each probe, ensuring that each probe completes efficient binding within its optimal temperature window. Furthermore, it can monitor and evaluate temperature fluctuations in real time and dynamically adjust temperature control parameters, thereby ensuring the stability and consistency of the hybridization process.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A time-controlled temperature-controlled method for detecting multiple gene loci through induction hybridization is provided, comprising the following steps:

[0007] Step 1: Chip Placement: After placing the gene chip in the hybridization module, add the hybridization solution and the sample to be tested;

[0008] Step 2: Preheating and initial hybridization: The hybridization solution is heated to the initial target temperature T1 and then maintained at that temperature for a duration of M1. During the heating and heat preservation of the hybridization solution, the surface temperature of the gene chip is predicted in real time by the prediction module.

[0009] Step 3: Temperature fluctuation assessment: The predicted temperature of the gene chip obtained in Step 2 is assessed for fluctuation, and an adjustment strategy for the heating rate and holding time for the next heating is made based on the assessment results.

[0010] Step 4: Low Tm probe hybridization: According to the adjustment strategy in Step 3, the hybridization solution is heated to the low Tm probe hybridization temperature T2 and then maintained at this temperature for a duration of M2. During the heating and heat preservation of the hybridization solution, the surface temperature of the gene chip is predicted in real time by the prediction module.

[0011] Step 5: Temperature fluctuation assessment: The predicted temperature of the gene chip obtained in Step 4 is assessed for fluctuation, and an adjustment strategy for the heating rate and holding time for the next heating is made based on the assessment results.

[0012] Step 6: High Tm probe hybridization: Following the adjustment strategy in Step 5, heat the hybridization solution to the high Tm probe hybridization temperature T3 and maintain this temperature for a duration of M3;

[0013] Step 7: After the hybridization module drains the hybridization solution, the gene chip is washed and developed.

[0014] The time-controlled temperature-controlled hybridization multi-gene locus detection method of the present invention can design a stepped temperature rise program according to the Tm value of each probe to ensure that each probe completes efficient binding within its optimal temperature window. At the same time, it can monitor and evaluate temperature fluctuations in real time and dynamically adjust temperature control parameters to ensure the stability and consistency of the hybridization process.

[0015] Preferably, in steps two and four, the prediction module predicts the surface temperature of the gene chip, including the following steps:

[0016] S1: Data Acquisition and Preprocessing: After acquiring data from all sensors within the hybridization module, the data is processed into a feature vector X based on the data acquisition time. i =[X i1 X i2 , ...X in ], where X ij The reading of the j-th sensor at the i-th time point, 1≤j≤n; simultaneously, the actual temperature of the gene chip at each time point is acquired as the target value vector Y. i X is normalized. i and Y i Scale to the same range;

[0017] S2: Feature Engineering and Data Fusion: Combine the vector after normalization in S1 with the influencing factors to form a new feature vector;

[0018] S3: Model Training and Data Processing: Use the training set data to train the SVR model to obtain the trained SVR model. Input the feature vector obtained in S2 into the trained SVR model to obtain the predicted temperature of the chip inside the module.

[0019] In S1, all the sensors within the hybridization module include a temperature sensor, a humidity sensor, and a pressure sensor.

[0020] In S2, the influencing factors include target temperature, temperature gradient, temperature change rate, module lifecycle count, sensor drift factor, and time series characteristics.

[0021] When heating a gene chip using a hybridization solution, the heating rate of the gene chip is slower than that of the hybridization solution due to the time required for heat transfer. Furthermore, the heating of the gene chip is affected by other factors, so the temperature of the hybridization solution does not directly reflect the temperature of the gene chip. The prediction module integrates the characteristic changes of humidity and barometric pressure sensors, the rate of temperature change before reaching the target, module usage frequency, sensor drift, and other influencing factors to predict the real-time temperature of the gene chip. The predicted temperature accurately reflects the real-time temperature of the gene chip, making the method of obtaining the chip surface temperature more accurate and complete.

[0022] Preferably, in steps three and five, the fluctuation assessment includes an overall assessment and a micro-assessment, and the overall assessment includes the following steps:

[0023] Set temperature T set Then, the average value T of the measured temperature was calculated. avg Temperature standard deviation σT and temperature offset ΔT, ΔT=|T set -T avg |, set temperature T set The target temperature that the gene chip needs to be heated to is set to a temperature that is generally slightly lower than the heating temperature of the hybridization module.

[0024] The microscopic assessment includes the following steps:

[0025] Histogram statistics were performed on all temperature data, and histogram data were analyzed. Histogram data included peak position, width, number of peaks, and degree of deviation. The number of peaks included single peaks and multiple peaks.

[0026] The strategy is adjusted based on the result levels of the overall assessment and the micro-assessment.

[0027] Preferably, the result levels include normal, slight fluctuation, moderate fluctuation, and severe abnormality;

[0028] When ΔT < ±0.2℃, σT < 0.3℃, and the histogram shows a single peak, the fluctuation assessment result is normal, and the adjustment strategy is not to intervene in the original strategy.

[0029] When ΔT≤±0.5℃, σT≤0.5℃, and the histogram shows a slight shift, the fluctuation assessment result is a slight fluctuation. In this case, the adjustment strategy is to slow down the heating rate or extend the holding time.

[0030] When ΔT > ±0.5℃ or σT > 0.5℃ or the histogram has multiple peaks, the fluctuation assessment result is moderate fluctuation. At this time, the adjustment strategy is PID self-tuning or pausing the heating.

[0031] When ΔT > ±1.0℃ or σT > 1.0℃, the fluctuation assessment result is severely abnormal. At this time, the adjustment strategy is to stop heating, cool down, and issue an alarm signal.

[0032] The present invention also provides a time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system, including a reagent tank containing a number of reagents, a pipette holder holding a number of pipette tips, a hybridization reaction module, a drainage module, a pipetting module, a drive module, a camera module, and a control module;

[0033] The hybridization reaction module is used to place the gene chip. The hybridization reaction module is provided with a number of reaction wells. The hybridization reaction module can heat the reaction wells.

[0034] The pipetting module can pick up the pipette tip on the pipette tip holder, draw reagent from the reagent tank, and place the reagent into the reaction well of the hybridization reaction module;

[0035] The camera module is used to capture images of chip hybridization;

[0036] The driving module is used to drive the camera module and the pipetting module to move, and both the camera module and the pipetting module are mounted on the driving module;

[0037] The drainage module is connected to the hybridization reaction module to drain the reagents from the reaction orifice of the hybridization reaction module;

[0038] The control module is connected to the hybridization reaction module, the drainage module, the drive module, the pipetting module, and the camera module. The control module is used to control the system to operate according to the above-described time-controlled temperature-controlled flow-guided hybridization multi-gene locus detection method.

[0039] In operation, the time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system of the present invention first places the gene chip to be detected in the hybridization reaction module. Then, the control module controls the pipetting module and the drive module to start working. The drive module first moves the pipetting module towards the pipette tip holder until it approaches and picks up the pipette tip from the holder. Then, the drive module moves the pipetting module towards the reagent tank until the pipetting module controls the pipette tip to approach the reagent tank and draw the reagent from it. After the pipette tip draws the reagent, the drive module moves the pipetting model towards the hybridization reaction model until the pipette tip approaches the reaction well of the hybridization reaction module and injects liquid into the well. After the liquid is injected into the reaction well, the control module controls the hybridization reaction module and the camera module to operate. The hybridization reaction module operates according to steps two to seven of the time-controlled temperature-controlled flow-through hybridization multi-gene locus detection method described above, while the camera module captures images of the chip hybridization.

[0040] The time-controlled temperature-controlled hybridization multi-gene locus detection system of the present invention can design a stepped temperature rise program according to the Tm value of each probe to ensure that each probe completes efficient binding within its optimal temperature window. At the same time, it can monitor and evaluate temperature fluctuations in real time and dynamically adjust temperature control parameters to ensure the stability and consistency of the hybridization process.

[0041] Furthermore, the hybridization reaction module includes a temperature control component and a partition chamber component, with a gene chip placed between the partition chamber component and the temperature control component. The reaction wells are arranged in an array on the partition chamber component. The temperature control component is used to heat the reagents within the reaction wells.

[0042] Furthermore, a temperature sensor, a humidity sensor, and a pressure sensor are fixedly installed on the hybridization reaction module. Several of each of these sensors are provided, with one corresponding to each reaction well. The temperature control component also has one temperature sensor. The temperature sensor corresponding to the reaction well monitors the reagent temperature within the well, the humidity sensor monitors the ambient humidity within the well, and the pressure sensor monitors the ambient air pressure within the well. The temperature sensor on the temperature control component monitors the temperature of the heat sink of the temperature control component.

[0043] Furthermore, the drainage module includes a drive pump, a pressure buffer chamber, a pressure sensor, a first solenoid valve, and a second solenoid valve. The first solenoid valve, the second solenoid valve, the pressure sensor, and the drive pump are all connected to the pressure buffer chamber. The other end of the first solenoid valve is connected to the reaction port, and the other end of the second solenoid valve is connected to a waste bottle. During operation, the drainage module first closes the first and second solenoid valves to seal the pressure buffer chamber. The drive pump is then turned on in the forward direction to create negative pressure in the pressure buffer chamber. Once the pressure sensor detects that the pressure in the pressure buffer chamber has reached the set value, the drive pump is turned off. Then, the second solenoid valve is opened to connect the pressure buffer chamber to the reaction port, drawing the reagent from the reaction port into the pressure buffer chamber. The first solenoid valve is then closed. After the first solenoid valve closes, the drive pump is turned on in the reverse direction to create positive pressure in the pressure buffer chamber. Once the pressure sensor detects that the pressure in the pressure buffer chamber has reached the set value, the drive pump continues to operate and the second solenoid valve is opened. At this point, the waste liquid in the pressure buffer chamber is discharged into the waste bottle by air pressure. The pressure buffer chamber design avoids the problem of the mechanical pump directly contacting the reagent, extending the equipment's lifespan and improving the system's reliability.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The time-controlled temperature-controlled flow hybridization multi-gene locus detection method and system of the present invention can design a stepped temperature rise program according to the Tm value of each probe to ensure that each probe completes efficient binding at its optimal temperature window. At the same time, it can monitor and evaluate temperature fluctuations in real time and dynamically adjust temperature control parameters to ensure the stability and consistency of the hybridization process. Attached Figure Description

[0046] Figure 1 A flowchart of a time-controlled temperature-controlled flow-through hybridization method for detecting multiple gene loci;

[0047] Figure 2 This is a schematic diagram of a time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system;

[0048] Figure 3 This is a schematic diagram of the hybridization reaction module of a time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system;

[0049] Figure 4 This is a schematic diagram of the drainage module of a time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system, excluding the connecting tube.

[0050] In the attached diagram: 1. Drive unit module; 2. Pipette module; 3. Pipette tip holder; 4. Reagent tank; 5. Hybridization reaction module; 6. Drainage module; 7. Camera module; 8. Gene chip; 501. Temperature control component; 502. Separation chamber component; 511. Heat sink; 601. First solenoid valve; 602. Second solenoid valve; 603. Drive pump; 604. Pressure buffer chamber. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments. The accompanying drawings are for illustrative purposes only, representing schematic diagrams rather than actual physical objects, and should not be construed as limiting the scope of this patent. To better illustrate the embodiments of the present invention, some components in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0052] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0053] Example 1

[0054] This embodiment is a first embodiment of a time-controlled temperature-controlled flow hybridization method for detecting multiple gene loci, including the following steps:

[0055] Step 1: Chip Placement: After placing the gene chip in the hybridization module, add the hybridization solution and the sample to be tested;

[0056] Step 2: Preheating and initial hybridization: The hybridization solution is heated to the initial target temperature T1 and then maintained at that temperature for a duration of M1. During the heating and heat preservation of the hybridization solution, the surface temperature of the gene chip is predicted in real time by the prediction module.

[0057] Step 3: Temperature fluctuation assessment: The predicted temperature of the gene chip obtained in Step 2 is assessed for fluctuation, and an adjustment strategy for the heating rate and holding time for the next heating is made based on the assessment results.

[0058] Step 4: Low Tm probe hybridization: According to the adjustment strategy in Step 3, the hybridization solution is heated to the low Tm probe hybridization temperature T2 and then maintained at this temperature for a duration of M2. During the heating and heat preservation of the hybridization solution, the surface temperature of the gene chip is predicted in real time by the prediction module.

[0059] Step 5: Temperature fluctuation assessment: The predicted temperature of the gene chip obtained in Step 4 is assessed for fluctuation, and an adjustment strategy for the heating rate and holding time for the next heating is made based on the assessment results.

[0060] Step 6: High Tm probe hybridization: Following the adjustment strategy in Step 5, heat the hybridization solution to the high Tm probe hybridization temperature T3 and maintain this temperature for a duration of M3;

[0061] Step 7: After the hybridization module drains the hybridization solution, the gene chip is washed and developed.

[0062] Specifically, in steps two and four, the prediction module predicts the surface temperature of the gene chip, including the following steps:

[0063] S1: Data Acquisition and Preprocessing: After acquiring data from all sensors within the hybridization module, the data is processed into a feature vector X based on the data acquisition time. i =[X i1 X i2 , ...X in ], where X ij The reading of the j-th sensor at the i-th time point, 1≤j≤n; simultaneously, the actual temperature of the gene chip at each time point is acquired as the target value vector Y. i X is normalized. i and Y i Scale to the same range;

[0064] S2: Feature Engineering and Data Fusion: Combine the vector after normalization in S1 with the influencing factors to form a new feature vector;

[0065] S3: Model Training and Data Processing: Use the training set data to train the SVR model to obtain the trained SVR model. Input the feature vector obtained in S2 into the trained SVR model to obtain the predicted temperature of the chip inside the module.

[0066] In S1, all the sensors within the hybridization module include a temperature sensor, a humidity sensor, and a barometric pressure sensor.

[0067] In S2, influencing factors include target temperature, temperature gradient, temperature change rate, module lifetime count, sensor drift factor, and time series characteristics.

[0068] Specifically, in steps three and five, the fluctuation assessment includes an overall assessment and a micro-assessment. The overall assessment includes the following steps:

[0069] Set temperature T set Then, the average value T of the measured temperature was calculated. avg Temperature standard deviation σT and temperature offset ΔT, ΔT=|T set -T avg |;

[0070] Micro-assessment includes the following steps:

[0071] Histogram statistics were performed on all temperature data, and histogram data were analyzed. Histogram data included peak position, width, number of peaks, and degree of deviation. The number of peaks included single peaks and multiple peaks.

[0072] The strategy is adjusted based on the output levels of the overall assessment and the micro-assessment results.

[0073] Preferably, the result grades include normal, slight fluctuation, moderate fluctuation, and severe abnormality;

[0074] When ΔT < ±0.2℃, σT < 0.3℃, and the histogram shows a single peak, the fluctuation assessment result is normal, and the adjustment strategy is not to intervene in the original strategy.

[0075] When ΔT≤±0.5℃, σT≤0.5℃, and the histogram shows a slight shift, the fluctuation assessment result is a slight fluctuation. In this case, the adjustment strategy is to slow down the heating rate or extend the holding time.

[0076] When ΔT > ±0.5℃ or σT > 0.5℃ or the histogram has multiple peaks, the fluctuation assessment result is moderate fluctuation. At this time, the adjustment strategy is PID self-tuning or pausing the heating.

[0077] When ΔT > ±1.0℃ or σT > 1.0℃, the fluctuation assessment result is severely abnormal. At this time, the adjustment strategy is to stop heating, cool down, and issue an alarm signal.

[0078] Specifically, taking actual data as an example, after the gene chip is placed in the hybridization module, the hybridization solution and the sample to be tested are heated. At this time, the temperature of the hybridization solution is room temperature. Then the temperature control component of the hybridization module starts to work, that is, the temperature is raised to 42°C and maintained for 10 minutes. 42°C is T1; 10 minutes is M1. During these 10 minutes, the temperature of the gene chip surface is predicted by the temperature sensor at a frequency of once per second.

[0079] After 10 minutes, 600 temperature data points were predicted. At this point, the temperature was set at 42℃, which is T. set Then, calculate the average value T of the 600 temperature data points. avgCalculate the temperature standard deviation σT and the temperature offset ΔT. Simultaneously, create a histogram of 600 temperature data points with temperature as the vertical axis and time as the horizontal axis, and analyze the peak position, width, number of peaks (single-peak / multi-peak), and degree of deviation of the histogram.

[0080] Finally, based on the histogram data and the average value T of 600 temperature data points... avg The output adjustment strategy includes temperature standard deviation σT and temperature offset ΔT.

[0081] The beneficial effects of this embodiment are as follows:

[0082] The time-controlled temperature-controlled hybridization multi-gene locus detection method of this embodiment can design a stepped temperature rise program according to the Tm value of each probe to ensure that each probe completes efficient binding within its optimal temperature window. At the same time, it can monitor and evaluate temperature fluctuations in real time and dynamically adjust temperature control parameters to ensure the stability and consistency of the hybridization process.

[0083] Example 2

[0084] This embodiment is an example of a time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system, such as... Figure 2 As shown, it includes a reagent tank 4 containing a number of reagents, a pipette holder 3 holding a number of pipette tips, a hybridization reaction module 5, a drainage module 6, a pipetting module 2, a drive module, a camera module 7, and a control module.

[0085] The hybridization reaction module 5 is used to place the gene chip 8. The hybridization reaction module 5 is provided with several reaction wells, and the hybridization reaction module 5 can heat the reaction wells.

[0086] The pipetting module 2 can pick up the pipette tip on the pipette tip holder 3, draw reagent from the reagent tank 4, and place the reagent into the reaction well of the hybridization reaction module 5;

[0087] Camera module 7 is used to capture images of the chip hybridization process;

[0088] The drive module is used to drive the movement of the camera module 7 and the pipetting module 2. Both the camera module 7 and the pipetting module 2 are mounted on the drive module.

[0089] The drainage module 6 is connected to the hybridization reaction module 5 to drain the reagents from the reaction wells of the hybridization reaction module 5;

[0090] The control module is connected to the hybridization reaction module 5, the drainage module 6, the drive module, the pipetting module 2, and the camera module 7. The control module is used to control the system to work according to the above-mentioned time-controlled temperature-controlled flow-guided hybridization multi-gene locus detection method.

[0091] The working principle or process of this embodiment is as follows:

[0092] In this embodiment, the time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system first places the gene chip 8 to be detected into the hybridization reaction module 5. Then, the control module controls the pipetting module 2 and the drive module to start working. The drive module first moves the pipetting module 2 towards the pipette tip holder 3 until it approaches and picks up the pipette tip on the holder 3. Then, the drive module moves the pipetting module 2 towards the reagent tank 4 until the pipetting module 2 controls the pipette tip to approach the reagent tank 4 and draw the reagent from it. After the pipette tip draws the reagent, the drive module moves the pipetting model towards the hybridization reaction model until the pipette tip approaches the reaction well of the hybridization reaction module 5 and injects liquid into the reaction well. After the liquid is injected into the reaction well, the control module controls the hybridization reaction module 5 and the camera module 7 to work. The hybridization reaction module 5 works according to steps two to seven of the time-controlled temperature-controlled flow-through hybridization multi-gene locus detection method described above, while the camera module 7 captures images of the chip hybridization.

[0093] The beneficial effects of this embodiment are as follows:

[0094] The time-controlled temperature-controlled hybridization multi-gene locus detection system of this embodiment can design a stepped temperature ramping program based on the Tm value of each probe to ensure that each probe completes efficient binding within its optimal temperature window. At the same time, it can monitor and evaluate temperature fluctuations in real time and dynamically adjust temperature control parameters to ensure the stability and consistency of the hybridization process.

[0095] Example 3

[0096] This embodiment is the third embodiment of a time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system. This embodiment is based on Embodiment 2, as follows: Figures 2-4 As shown, the structure of the system is further defined.

[0097] Specifically, the hybridization reaction module 5 includes a temperature control component 501 and a partition chamber component. The space between the partition chamber component and the temperature control component 501 is used to place the gene chip 8, and the reaction wells are arranged in an array on the partition chamber component.

[0098] Specifically, temperature sensors, humidity sensors, and pressure sensors are fixedly installed on the hybridization reaction module 5. Several of each type of sensor are provided, with one corresponding to each reaction well. The temperature control component 501 also has one temperature sensor. The temperature sensor corresponding to each reaction well monitors the reagent temperature within the well, the humidity sensor monitors the ambient humidity, and the pressure sensor monitors the ambient air pressure. The temperature sensor on the temperature control component 501 monitors the temperature of the heat sink 511 of the temperature control component 501.

[0099] Specifically, the drainage module 6 includes a drive pump 603, a pressure buffer chamber 604, a pressure sensor, a first solenoid valve 601, and a second solenoid valve 602. The first solenoid valve 601, the second solenoid valve 602, the pressure sensor, and the drive pump 603 are all connected to the pressure buffer chamber 604. The other end of the first solenoid valve 601 is connected to a reaction port, and the other end of the second solenoid valve 602 is used to connect to a waste liquid bottle. The drive pump 603 is a peristaltic pump.

[0100] The working principle or process of this embodiment is as follows:

[0101] When the drainage module 6 is working, it first closes the first solenoid valve 601 and the second solenoid valve 602 to seal the pressure buffer chamber 604. Then, it opens the drive pump 603 in the forward direction to create negative pressure in the pressure buffer chamber 604. Once the pressure sensor detects that the pressure in the pressure buffer chamber 604 has reached the set value, it closes the drive pump 603. Next, it opens the second solenoid valve 602 to connect the pressure buffer chamber 604 to the reaction port, drawing the reagent from the reaction port into the pressure buffer chamber 604. Then, it closes the first solenoid valve 601. After the first solenoid valve 601 closes, it opens the drive pump 603 in the reverse direction to create positive pressure in the pressure buffer chamber 604. Once the pressure sensor detects that the pressure in the pressure buffer chamber 604 has reached the set value, it continues to operate the drive pump 603 and opens the second solenoid valve 602. At this point, the waste liquid in the pressure buffer chamber 604 is discharged into the waste liquid bottle by air pressure.

[0102] The beneficial effects of this embodiment are as follows:

[0103] By using the pressure buffer chamber 604, the problem of direct contact between the mechanical pump and the reagent can be avoided, thus extending the equipment life and improving the system reliability.

[0104] In the specific implementation of the above embodiments, the technical features can be combined in any non-contradictory way. For the sake of brevity, not all possible combinations of the above technical features are described. However, as long as the combination of these technical features is not contradictory, it should be considered to be within the scope of this specification.

[0105] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A time-controlled temperature-controlled method for detecting multiple gene loci through transduction hybridization, characterized in that, The steps include: Step 1: Chip Placement: After placing the gene chip in the hybridization module, add the hybridization solution and the sample to be tested; Step 2: Preheating and initial hybridization: The hybridization solution is heated to the initial target temperature T1 and then maintained at that temperature for a duration of M1. During the heating and heat preservation of the hybridization solution, the surface temperature of the gene chip is predicted in real time by the prediction module. Step 3: Temperature fluctuation assessment: The predicted temperature of the gene chip obtained in Step 2 is assessed for fluctuation, and an adjustment strategy for the heating rate and holding time for the next heating is made based on the assessment results. Step 4: Low Tm probe hybridization: According to the adjustment strategy in Step 3, the hybridization solution is heated to the low Tm probe hybridization temperature T2 and then maintained at this temperature for a duration of M2. During the heating and heat preservation of the hybridization solution, the surface temperature of the gene chip is predicted in real time by the prediction module. Step 5: Temperature fluctuation assessment: The predicted temperature of the gene chip obtained in Step 4 is assessed for fluctuation, and an adjustment strategy for the heating rate and holding time for the next heating is made based on the assessment results. Step 6: High Tm probe hybridization: Following the adjustment strategy in Step 5, heat the hybridization solution to the high Tm probe hybridization temperature T3 and maintain this temperature for a duration of M3; Step 7: After the hybridization module discharges the hybridization solution, the gene chip is washed and developed. The prediction module predicts the surface temperature of the gene chip using the following steps: S1: Data Acquisition and Preprocessing: After acquiring the data collected by all sensors in the hybridization module, the data is formed into a feature vector Xi=[Xi1, Xi2, ...Xin] according to the data acquisition time, where Xij is the reading of the j-th sensor at the i-th time point, 1≤j≤n; at the same time, the actual temperature of the gene chip at each time point is acquired as the target value vector Yi, and normalization processing is used to scale Xi and Yi to the same range; S2: Feature Engineering and Data Fusion: Combine the vector after normalization in S1 with the influencing factors to form a new feature vector; S3: Model Training and Data Processing: Use the training set data to train the SVR model to obtain the trained SVR model. Input the feature vector obtained in S2 into the trained SVR model to obtain the predicted temperature of the chip inside the module. In steps three and five, the fluctuation assessment includes an overall assessment and a micro-assessment. The overall assessment includes the following steps: After setting the temperature Tset, calculate the average value of the measured temperature Tavg, the standard deviation of the temperature σT, and the temperature offset ΔT, where ΔT = |Tset - Tavg|. The microscopic assessment includes the following steps: Histogram statistics were performed on all temperature data, and histogram data were analyzed. Histogram data included peak position, width, number of peaks, and degree of deviation. The number of peaks included single peaks and multiple peaks. The strategy is adjusted based on the results of the overall assessment and the micro-assessment. The result levels include normal, slight fluctuation, moderate fluctuation, and severe abnormality; When ΔT < ±0.2℃, σT < 0.3℃, and the histogram shows a single peak, the fluctuation assessment result is normal, and the adjustment strategy is not to intervene in the original strategy. When ΔT≤±0.5℃, σT≤0.5℃, and the histogram shifts, the fluctuation assessment result is a slight fluctuation. In this case, the adjustment strategy is to slow down the heating rate or extend the holding time. When ΔT > ±0.5℃ or σT > 0.5℃ or the histogram has multiple peaks, the fluctuation assessment result is moderate fluctuation. At this time, the adjustment strategy is PID self-tuning or pausing the heating. When ΔT > ±1.0℃ or σT > 1.0℃, the fluctuation assessment result is severely abnormal. At this time, the adjustment strategy is to stop heating, cool down, and issue an alarm signal.

2. The time-controlled temperature-controlled flow-through hybridization method for detecting multiple gene loci according to claim 1, characterized in that, In S2, the influencing factors include target temperature, temperature gradient, temperature change rate, module lifecycle count, sensor drift factor, and time series characteristics.

3. The time-controlled temperature-controlled flow-through hybridization method for detecting multiple gene loci according to claim 1, characterized in that, In S1, all the sensors within the hybridization module include a temperature sensor, a humidity sensor, and a pressure sensor.

4. A time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system, characterized in that, It includes a reagent tank (4) containing a number of reagents, a pipette holder (3) holding a number of pipette tips, a hybridization reaction module (5), a drainage module (6), a pipetting module (2), a drive module, a camera module (7), and a control module; The hybridization reaction module (5) is used to place the gene chip (8). The hybridization reaction module (5) is provided with a plurality of reaction wells. The hybridization reaction module (5) can heat the reaction wells. The pipetting module (2) can pick up the pipette tip on the pipette tip holder (3) and draw the reagent from the reagent tank (4) and place the reagent into the reaction well of the hybridization reaction module (5); The camera module (7) is used to capture images of chip hybridization; The driving module is used to drive the camera module (7) and the pipetting module (2) to move. The camera module (7) and the pipetting module (2) are both mounted on the driving module. The drainage module (6) is connected to the hybridization reaction module (5) to drain the reagents in the reaction orifice of the hybridization reaction module (5); The control module is connected to the hybridization reaction module (5), the drainage module (6), the drive module, the pipetting module (2), and the camera module (7). The control module is used to control the system to operate according to the time-controlled temperature-controlled flow-guided hybridization multi-gene locus detection method as described in any one of claims 1-3.

5. The time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system according to claim 4, characterized in that, The hybridization reaction module (5) includes a temperature control component (501) and a partition chamber component. The partition chamber component and the temperature control component (501) are used to place the gene chip (8). The reaction wells are arranged in an array on the partition chamber component.

6. The time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system according to claim 5, characterized in that, A temperature sensor, a humidity sensor, and a pressure sensor are fixedly installed on the hybridization reaction module (5). Several of each of the temperature sensor, humidity sensor, and pressure sensor are provided. Each reaction hole has a corresponding temperature sensor, humidity sensor, and pressure sensor. The temperature control component (501) also has a temperature sensor.

7. The time-controlled temperature-controlled flow-through hybridization multi-gene locus detection system according to claim 4, characterized in that, The drainage module (6) includes a drive pump (603), a pressure buffer chamber (604), a pressure sensor, a first solenoid valve (601), and a second solenoid valve (602). The first solenoid valve (601), the second solenoid valve (602), the pressure sensor, and the drive pump (603) are all connected to the pressure buffer chamber (604). The other end of the first solenoid valve (601) is connected to the reaction port, and the other end of the second solenoid valve (602) is used to connect to the waste liquid bottle.

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