A digital PCR analysis device and analysis method thereof

Abstract

The present invention relates to the technical field of digital PCR analysis, and in particular to a digital PCR analysis device and an analysis method thereof. The present invention comprises a droplet generation module, a PCR amplification module, a feature extraction module, a feature determination module, a ventilation module, and a fluorescence detection module. The droplet generation module is used to obtain a plurality of droplet units. The PCR amplification module is used to denature, anneal, and extend each droplet unit. The feature extraction module is used to obtain a bubble floating tendency characterization value. The feature determination module is used to screen droplet feature areas, select an annealing adjustment method, and determine whether to continue PCR amplification. The ventilation module is used to adjust the wind speed and temperature of the inlet air. The adjustment and verification unit is used to adjust and verify the droplet units. The output unit outputs the analysis results. Thus, during the denaturation process of PCR amplification, annealing can be adaptively adjusted according to the actual state of the droplet unit, thereby improving the accuracy of the digital PCR analysis results.

Description

Technical Field

[0001] The present invention relates to the technical field of digital PCR analysis, and in particular to a digital PCR analysis device and an analysis method thereof. Background Art

[0002] In areas such as tumor marker detection, pathogen detection, and genotyping, more precise quantitative analysis of nucleic acid molecules is required to achieve early diagnosis of diseases, formulate treatment plans, and monitor efficacy. Traditional PCR technology typically uses standard curves or reference genes for relative quantification, and its results are affected by multiple factors. Digital PCR, on the other hand, divides the reaction system into tiny droplet units and combines the Poisson distribution principle to calculate the absolute content of the target nucleic acid in the sample, greatly reducing the possibility of nonspecific amplification. It provides absolute quantification of the starting sample, unrestricted by standard curves and reference genes, and can more accurately determine the target nucleic acid content. However, in actual operation, the reliability of each droplet unit often faces many challenges. Temperature changes during PCR amplification can cause abnormal bubbles in the droplet unit, affecting the reliability of the micro-reaction unit and, in turn, the accuracy of the digital PCR analysis results. Therefore, real-time monitoring of the status of each micro-reaction unit during PCR amplification and improving the accuracy of digital PCR analysis results are urgent technical issues that need to be addressed.

[0003] For example, Chinese patent application publication number: CN118995402A, the invention discloses a digital PCR analysis device and a PCR analysis method, including a box assembly, a rotating assembly is provided inside the box assembly, a clamping assembly is provided on the rotating assembly, a PCR tube is placed on the clamping assembly, a PCR plate is sleeved on the PCR tube, a ventilation assembly is provided at the bottom of the rotating assembly, a filter assembly, a temperature control assembly and a blowing assembly are symmetrically provided on both sides of the box assembly, a control center is also provided on the box assembly, and a water-absorbing sponge is provided at the bottom of the box assembly; in this invention, by providing a temperature control assembly, the temperature inside the box assembly is raised and lowered, thereby fine-tuning the temperature of the PCR tube; by providing a ventilation assembly, the temperature of the PCR tube and the ambient temperature around it are cooled, thereby accelerating the reduction of the temperature of the PCR tube; by providing a blowing assembly, the air inside the box assembly and the air outside are exchanged.

[0004] The following problems also exist in the prior art:

[0005] The existing technology does not consider that changes in the thermal field distribution during PCR amplification will cause abnormal bubbles in the droplet unit, thereby affecting the accuracy of fluorescence signal detection. The existing technology cannot adaptively adjust annealing according to the actual state of the droplet unit during the denaturation process of PCR amplification, affecting the accuracy of digital PCR analysis results. Summary of the Invention

[0006] To this end, the present invention provides a digital PCR analysis device and analysis method thereof to overcome the problem that the prior art cannot adaptively adjust annealing according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby affecting the accuracy of the digital PCR analysis results.

[0007] To achieve the above objectives, the present invention provides a digital PCR analysis device, comprising:

[0008] A droplet generation module, which is used to prepare a nucleic acid sample into a PCR reaction system solution in proportion and divide the PCR reaction system solution into a plurality of droplet units;

[0009] a PCR amplification module connected to the droplet generation module, comprising a PCR plate for placing a plurality of droplet units, and a temperature control component for denaturing, annealing, and extending each droplet unit;

[0010] a feature extraction module connected to the PCR amplification module, configured to divide the PCR plate into a plurality of droplet regions and obtain a bubble floating tendency characterization value of each droplet region during the denaturation process;

[0011] a feature determination module, connected to the feature extraction module, configured to screen droplet feature regions based on a bubble rising tendency characteristic value of each droplet region, select an adjustment method for annealing each droplet unit based on the number of droplet feature regions, and determine whether to continue PCR amplification for each droplet unit;

[0012] a ventilation module connected to the PCR amplification module and the feature determination module, for adjusting the air inlet velocity to the droplet feature area and adjusting the air inlet temperature of each droplet unit in a step-by-step temperature control manner;

[0013] a fluorescence detection module connected to the PCR amplification module and the feature determination module, comprising an adjustment and verification unit for adjusting and verifying a droplet unit that has completed a single PCR amplification, and an output unit for performing fluorescence signal statistics on droplet units that have completed multiple PCR amplifications and outputting analysis results;

[0014] Wherein, the adjustment and verification unit also includes a control component for adjusting the tilt angle of the PCR plate.

[0015] Furthermore, the ventilation module further includes a plurality of ventilation holes provided below the PCR plate for ventilating the droplet area;

[0016] Each droplet area corresponds to at least one vent.

[0017] The present invention also provides a digital PCR analysis method, comprising:

[0018] Prepare the nucleic acid sample into a PCR reaction system solution according to the proportion;

[0019] Dividing the PCR reaction system solution into a plurality of droplet units, placing the units on a PCR plate, and dividing the PCR plate into a plurality of droplet areas, each droplet area comprising a plurality of droplet units;

[0020] Performing multiple PCR amplifications on the droplet units of each droplet region, wherein a single PCR amplification includes sequential denaturation, annealing, and extension;

[0021] Obtaining a bubble floating tendency characterization value of each droplet region during the denaturation process, and screening droplet characteristic regions according to the bubble floating tendency characterization value of each droplet region;

[0022] Wherein, the bubble floating tendency characterization value is determined according to the bubble characterization values corresponding to different temperature values of the droplet area;

[0023] The adjustment method for annealing each droplet unit is selected based on the number of the droplet characteristic regions, including:

[0024] Adjusting the inlet air temperature in a step-by-step temperature control manner, or adjusting the inlet air velocity for the droplet characteristic area;

[0025] performing adjustment and verification on the droplet units that have completed a single PCR amplification, and determining whether to continue PCR amplification on each droplet unit based on the result of the adjustment and verification;

[0026] The adjustment verification includes performing fluorescence signal statistics on the characteristic area of the droplet under different detection light paths and different PCR plate tilt states;

[0027] The fluorescence signal statistics of the droplet units that have completed several PCR amplifications are performed and the PCR analysis results are output.

[0028] Furthermore, obtaining the bubble characterization values corresponding to the droplet region at different temperature values includes:

[0029] Acquire liquid surface images corresponding to different temperature values of each droplet unit in the droplet region, and calculate the bubble contour area based on the liquid surface images;

[0030] The average value of the bubble contour area corresponding to each droplet unit is calculated, and the average value is determined as the bubble characterization value of the droplet area.

[0031] Furthermore, the process of determining the bubble floating tendency characterization value of each droplet region during the denaturation process includes:

[0032] Obtaining a bubble characterization value corresponding to a first denaturation temperature value of the droplet region within a preset temperature range, and determining the bubble characterization value as a first bubble characterization value;

[0033] Obtaining a bubble characterization value corresponding to a second denaturation temperature value of the droplet region within a preset temperature range, and determining the bubble characterization value as a second bubble characterization value;

[0034] Calculating a difference between the first bubble characterization value and the second bubble characterization value, and determining the difference as a bubble floating tendency characterization value corresponding to the droplet area;

[0035] The first denaturation temperature value is the upper limit of the preset temperature range, and the second denaturation temperature value is the lower limit of the preset temperature range.

[0036] Further, screening the droplet characteristic region includes,

[0037] If the bubble floating tendency characterization value corresponding to the droplet region meets the droplet bubble characterization condition, the droplet region is selected as a droplet characteristic region;

[0038] The droplet bubble characterization condition is that the bubble floating tendency characterization value corresponding to the droplet region exceeds the average value of the bubble floating tendency characterization values corresponding to each droplet region.

[0039] Furthermore, the adjustment method for annealing each droplet unit includes:

[0040] If the number of the droplet characteristic areas meets the overall adjustment conditions, the inlet air temperature is adjusted in a step-by-step temperature control manner;

[0041] If the number of the droplet characteristic areas does not meet the overall adjustment condition, adjusting the air inlet velocity of the droplet characteristic areas;

[0042] The overall adjustment condition is that the proportion of the number of the droplet characteristic regions to the total number of droplet regions exceeds a preset proportion threshold.

[0043] Furthermore, the temperature adjustment step amount of the step temperature control method is negatively correlated with the bubble floating tendency characterization value, and the air inlet rate is negatively correlated with the bubble floating tendency characterization value.

[0044] Furthermore, the adjustment and verification of the droplet unit that completes the single PCR amplification includes:

[0045] Performing fluorescence signal statistics of the droplet unit in the characteristic area for several times under different light paths, and obtaining fluorescence signal statistics corresponding to the different light paths;

[0046] The tilt angle of the PCR plate is adjusted, and fluorescence signal statistics of the droplet units in the characteristic area at different tilt angles are performed using the same optical path to obtain fluorescence signal statistics corresponding to different tilt angles.

[0047] Further, determining whether to continue PCR amplification for each droplet unit includes:

[0048] Calculate the difference between each fluorescence signal statistic and any other fluorescence signal statistic;

[0049] If the difference does not exceed the preset difference threshold, it is determined to continue PCR amplification for each droplet unit.

[0050] Compared with the prior art, the present invention has the following beneficial effects: the present invention is provided with a droplet generation module, a PCR amplification module, a feature extraction module, a feature determination module, a ventilation module, and a fluorescence detection module; the nucleic acid sample is prepared into a PCR reaction system solution in proportion by the droplet generation module, and the PCR reaction system solution is divided into a plurality of droplet units; the PCR amplification module denatures, anneals, and extends each droplet unit; the feature extraction module divides the PCR plate into a plurality of droplet areas; a bubble floating tendency characterization value of each droplet area during the denaturation process is obtained; and the feature determination module selects the droplet characteristic area according to the bubble floating tendency characterization value of each droplet area. Based on the number of droplet characteristic regions, an adjustment method for annealing each droplet unit is selected, and whether to continue PCR amplification for each droplet unit is determined. The ventilation module adjusts the air inlet rate to the droplet characteristic region, and the air inlet temperature of each droplet unit is adjusted using a stepped temperature control method. The adjustment and verification unit adjusts and verifies the droplet units that have completed a single PCR amplification. The output unit collects fluorescence signal statistics for droplet units that have completed multiple PCR amplifications and outputs the analysis results. Consequently, annealing can be adaptively adjusted according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby improving the accuracy of digital PCR analysis results.

[0051] In particular, the present invention determines the bubble floating tendency characterization value by the difference between the bubble characterization value corresponding to the first denaturation temperature value of the droplet area within the preset temperature range and the bubble characterization value corresponding to the second denaturation temperature value within the preset temperature range. It can be understood that in the PCR denaturation process, temperature is a key factor affecting the bubble state. According to thermodynamics theory, for bubbles in the PCR reaction system, temperature increase will intensify the thermal motion of gas molecules, making bubbles easier to float. By calculating the difference in bubble contour area on the liquid surface of the droplet unit corresponding to the two temperature values, the bubble change state of each droplet unit in the droplet area during the denaturation process can be intuitively reflected. The present invention determines the bubble floating tendency characterization value by the difference between the bubble characterization value corresponding to the first denaturation temperature value of the droplet area within the preset temperature range and the bubble characterization value corresponding to the second denaturation temperature value within the preset temperature range. Furthermore, the trend of bubble floating in each droplet area is quantified, thereby improving the accuracy of the digital PCR analysis results.

[0052] In particular, the present invention screens the droplet characteristic areas through the bubble floating tendency characterization value of each droplet area. It can be understood that, the larger the bubble floating tendency characterization value of the droplet area, the greater the difference in bubble contour area on the surface of the droplet unit liquid at different denaturation stages in the area, which characterizes that the bubble floating tendency in the area is stronger, the more bubbles are on the droplet unit liquid surface, and the more seriously the area is affected by bubbles. The present invention screens the droplet characteristic areas through the bubble floating tendency characterization value of each droplet area, thereby achieving an intuitive reflection of the bubble status in different droplet areas and improving the accuracy of the digital PCR analysis results.

[0053] In particular, the present invention selects an adjustment method for annealing each droplet unit by the number of droplet characteristic regions. It can be understood that by adopting an adjustment method that is compatible with the proportion of the number of droplet characteristic regions in the total number of droplet regions, over-adjustment or under-adjustment can be avoided. When overall adjustment is required, step temperature control can simultaneously have a positive impact on a large number of droplet units, quickly improving reaction conditions. When only local adjustment is required, adjusting the air inlet rate according to the droplet characteristic region can concentrate resources to solve key problems and improve the efficiency of the entire PCR experiment. The present invention selects an adjustment method for annealing each droplet unit by the number of droplet characteristic regions, thereby achieving adaptive adjustment of annealing according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0054] In particular, when the number of droplet characteristic areas meets the overall adjustment conditions, the present invention selects a stepped temperature control method to adjust the inlet air temperature. It can be understood that the number of droplet characteristic areas meets the overall adjustment conditions, which means that there are more droplet areas affected by bubbles as a whole, and a suitable temperature adjustment method is needed to alleviate the impact of bubbles on the accuracy of PCR analysis results. During the PCR amplification process, condensation water is easily generated due to rapid temperature changes. The condensation water will take away part of the heat, resulting in uneven temperature distribution of the droplet units, affecting the accuracy and reliability of the analysis. The stepped temperature control method can regularly adjust the inlet air temperature by setting different temperature steps, making the temperature change more gentle and orderly, avoiding the generation of condensation water caused by rapid temperature changes, and helping to balance the temperature differences between the droplet units, thereby improving the uniformity and stability of the overall reaction. When the number of droplet characteristic areas meets the overall adjustment conditions, the present invention selects a stepped temperature control method to adjust the inlet air temperature, thereby achieving adaptive adjustment of annealing and improving the accuracy of digital PCR analysis results.

[0055] In particular, when the number of droplet characteristic areas does not meet the overall adjustment conditions, the present invention selects to adjust the air inlet rate of the droplet characteristic areas. It can be understood that the number of droplet characteristic areas does not meet the overall adjustment conditions, which means that there are fewer droplet areas affected by bubbles as a whole. Targeted adjustment of the air inlet rate of these droplet characteristic areas can more accurately solve local problems and directly change the heat dissipation rate of the droplet units in these areas, thereby quickly and accurately adjusting their temperature, making the annealing process more in line with the requirements and avoiding unnecessary impacts on other normal droplet areas on the entire PCR plate. When the number of droplet characteristic areas does not meet the overall adjustment conditions, the present invention selects to adjust the air inlet rate of the droplet characteristic areas, thereby achieving adaptive adjustment of annealing and improving the accuracy of digital PCR analysis results.

[0056] In particular, the temperature adjustment step amount of the step temperature control method of the present invention is negatively correlated with the bubble floating tendency characterization value. It can be understood that the larger the bubble floating tendency characterization value, the more heat accumulation in the droplet characteristic area, and the smaller the temperature adjustment step amount is required to make the temperature environment change in the droplet characteristic area more gentle, avoiding the generation of condensed water caused by rapid temperature changes. Furthermore, during the denaturation process of PCR amplification, the annealing is adaptively adjusted according to the actual state of the droplet unit, thereby improving the accuracy of the digital PCR analysis results.

[0057] In particular, the air inlet rate of the droplet characteristic area of the present invention is negatively correlated with the bubble floating tendency characterization value. It can be understood that the air inlet rate affects the heat exchange rate. The larger the bubble floating tendency characterization value, the more heat accumulates in the droplet characteristic area, and the smaller the air inlet rate is needed to reduce the heat exchange rate to avoid condensation caused by rapid temperature changes, which affects the accuracy of the digital PCR analysis results. Furthermore, the annealing is adaptively adjusted according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0058] In particular, the present invention performs fluorescence signal statistics of different optical paths for the droplet units within the characteristic area several times to obtain the fluorescence signal statistics corresponding to different optical paths. It can be understood that the presence of bubbles will cause the optical path to undergo refraction, scattering, and other phenomena, affecting the transmission and detection of the fluorescence signal. Different optical paths are affected by bubbles to different degrees. By performing statistics on the fluorescence signals of multiple optical paths, the differences in signals under different optical paths can be compared and analyzed, thereby verifying whether the current annealing adjustment method is effective, thereby improving the accuracy of the digital PCR analysis results.

[0059] In particular, the present invention adjusts the tilt angle of the PCR plate, and uses the same optical path to perform fluorescence signal statistics on the droplet units in the characteristic area under different tilt angles, so as to respectively obtain the fluorescence signal statistics corresponding to different tilt angles. It can be understood that adjusting the tilt angle of the PCR plate can remove the bubbles remaining in the droplet units, and different tilt angles have different degrees of influence on the bubbles. By obtaining the fluorescence signal statistics corresponding to different tilt angles, comparing and analyzing the differences in fluorescence signal statistics at different tilt angles, it is possible to verify whether the current annealing adjustment method is effective, thereby improving the accuracy of the digital PCR analysis results.

[0060] In particular, the present invention determines whether to continue PCR amplification for each droplet unit by calculating the difference between the statistics of each fluorescence signal and the statistics of any other fluorescence signal. It can be understood that the larger the difference, the more serious the impact of the bubble in the current droplet unit, and the more serious the impact on the PCR analysis, and the PCR amplification should be stopped in time; the smaller the difference, the weaker the impact of the bubble in the current droplet unit, and the higher the reliability of the PCR analysis, and each droplet unit can continue PCR amplification, thereby improving the accuracy of the digital PCR analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a functional block diagram of a digital PCR analysis device according to an embodiment of the present invention;

[0062] Figure 2 This is a diagram showing the steps of the digital PCR analysis method according to an embodiment of the present invention;

[0063] Figure 3 A logic flow chart for screening droplet feature regions according to an embodiment of the present invention;

[0064] Figure 4 A logic flow chart for selecting an adjustment method for annealing each droplet unit according to an embodiment of the present invention. DETAILED DESCRIPTION

[0065] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0066] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0067] It should be noted that, in the description of the present invention, terms such as "upper", "lower", "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0068] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted" and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; or internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0069] See also Figure 1 , which is a functional block diagram of a digital PCR analysis device according to an embodiment of the present invention. A digital PCR analysis device according to the present invention includes:

[0070] A droplet generation module, which is used to prepare a nucleic acid sample into a PCR reaction system solution in proportion and divide the PCR reaction system solution into a plurality of droplet units;

[0071] a PCR amplification module connected to the droplet generation module, comprising a PCR plate for placing a plurality of droplet units, and a temperature control component for denaturing, annealing, and extending each droplet unit;

[0072] a feature extraction module connected to the PCR amplification module, configured to divide the PCR plate into a plurality of droplet regions and obtain a bubble floating tendency characterization value of each droplet region during the denaturation process;

[0073] a feature determination module, connected to the feature extraction module, configured to screen droplet feature regions based on a bubble rising tendency characteristic value of each droplet region, select an adjustment method for annealing each droplet unit based on the number of droplet feature regions, and determine whether to continue PCR amplification for each droplet unit;

[0074] a ventilation module connected to the PCR amplification module and the feature determination module, for adjusting the air inlet velocity to the droplet feature area and adjusting the air inlet temperature of each droplet unit in a step-by-step temperature control manner;

[0075] a fluorescence detection module connected to the PCR amplification module and the feature determination module, comprising an adjustment and verification unit for adjusting and verifying a droplet unit that has completed a single PCR amplification, and an output unit for performing fluorescence signal statistics on droplet units that have completed multiple PCR amplifications and outputting analysis results;

[0076] Wherein, the adjustment and verification unit also includes a control component for adjusting the tilt angle of the PCR plate.

[0077] Specifically, the present invention does not limit the specific structure of the droplet generation module. Preferably, it can be a droplet generator that prepares the nucleic acid sample into a PCR reaction system solution in proportion and divides the PCR reaction system solution into several droplet units, which will not be repeated here.

[0078] Specifically, the present invention does not limit the specific structure of the PCR plate. Preferably, it can be a multi-well plate structure for placing a plurality of droplet units, which will not be described in detail here.

[0079] Specifically, the present invention does not limit the specific structure of the temperature control component. Preferably, it can be implemented by cooperating with a microfluidic channel and a temperature controller. A microfluidic channel is designed inside or around the PCR plate, and a heat exchange fluid circulates in the channel. The temperature of the heat exchange fluid is controlled by a temperature controller. No further details are given here.

[0080] Specifically, the present invention does not limit the specific structure of the feature extraction module. Preferably, it can be implemented by an industrial camera in conjunction with a processor. The liquid surface image of the droplet unit in each droplet area is obtained by the industrial camera, the bubble area is obtained based on edge detection, and the data received from the industrial camera is received by the processor to obtain the bubble floating trend characterization value. No further details are given here.

[0081] Specifically, the present invention does not limit the specific structure of the feature determination module. Preferably, it can be a microprocessor used in a computer to screen the droplet feature area, select the adjustment method for annealing each droplet unit, and determine whether to continue PCR amplification for each droplet unit. The details will not be repeated here.

[0082] Specifically, the present invention does not limit the specific structure of the adjustment and verification unit. Preferably, it can be a multi-light path fluorescence detector to perform fluorescence signal statistics on the droplet unit, which will not be described in detail here.

[0083] Specifically, the present invention does not limit the specific structure of the output unit. Preferably, it can be a microprocessor used in a computer to output the PCR analysis results, which will not be described in detail here.

[0084] Specifically, the present invention does not limit the specific structure of the control component. Preferably, it can be an electric rotating table, which is driven by a motor to accurately control the inclination angle of the PCR plate relative to the horizontal ground. It will not be repeated here.

[0085] Specifically, the ventilation module further includes a plurality of ventilation holes arranged below the PCR plate for ventilating the droplet area;

[0086] Each droplet area corresponds to at least one vent.

[0087] Specifically, the present invention does not limit the specific structure of the ventilation module. It can control the inlet air temperature by controlling the heater on the air inlet duct, and control the inlet air rate by controlling the blowing unit on the air inlet duct. Preferably, it can be a programmable logic controller. The control of functional devices by a programmable logic controller is an existing technology and will not be repeated here.

[0088] See also Figure 2 As shown, it is a step diagram of the digital PCR analysis method of an embodiment of the present invention. The present invention also provides a digital PCR analysis method, including:

[0089] Step S100, preparing a PCR reaction system solution from a nucleic acid sample according to a certain proportion;

[0090] Step S200, dividing the PCR reaction system solution into a plurality of droplet units, placing the units on a PCR plate, and dividing the PCR plate into a plurality of droplet areas, each droplet area containing a plurality of droplet units;

[0091] Step S300 , performing several PCR amplifications on the droplet units of each droplet region, wherein a single PCR amplification includes denaturation, annealing, and extension performed sequentially;

[0092] Step S400, obtaining a bubble floating tendency characterization value of each droplet region during the denaturation process, and screening droplet characteristic regions according to the bubble floating tendency characterization value of each droplet region;

[0093] Wherein, the bubble floating tendency characterization value is determined according to the bubble characterization values corresponding to different temperature values of the droplet area;

[0094] Step S500, selecting an adjustment method for annealing each droplet unit based on the number of the droplet characteristic regions, including:

[0095] Adjusting the inlet air temperature in a step-by-step temperature control manner, or adjusting the inlet air velocity for the droplet characteristic area;

[0096] Step S600, performing adjustment verification on the droplet units that have completed a single PCR amplification, and determining whether to continue PCR amplification on each droplet unit based on the result of the adjustment verification;

[0097] The adjustment verification includes performing fluorescence signal statistics on the characteristic area of the droplet under different detection light paths and different PCR plate tilt states;

[0098] Step S700 , performing fluorescence signal statistics on the droplet units that have completed several rounds of PCR amplification, and outputting PCR analysis results.

[0099] Specifically, obtaining the bubble characterization values corresponding to the droplet region at different temperature values includes:

[0100] Acquire liquid surface images corresponding to different temperature values of each droplet unit in the droplet region, and calculate the bubble contour area based on the liquid surface images;

[0101] The average value of the bubble contour area corresponding to each droplet unit is calculated, and the average value is determined as the bubble characterization value of the droplet area.

[0102] Specifically, the process of determining the bubble floating tendency characterization value of each droplet region during the denaturation process includes:

[0103] Obtaining a bubble characterization value corresponding to a first denaturation temperature value of the droplet region within a preset temperature range, and determining the bubble characterization value as a first bubble characterization value;

[0104] Obtaining a bubble characterization value corresponding to a second denaturation temperature value of the droplet region within a preset temperature range, and determining the bubble characterization value as a second bubble characterization value;

[0105] Calculating a difference between the first bubble characterization value and the second bubble characterization value, and determining the difference as a bubble floating tendency characterization value corresponding to the droplet area;

[0106] The first denaturation temperature value is the upper limit of the preset temperature range, and the second denaturation temperature value is the lower limit of the preset temperature range.

[0107] Specifically, the preset temperature range can be set by those skilled in the art according to the accuracy requirements of PCR analysis. The higher the accuracy requirements, the larger the temperature range is set. Preferably, the temperature range can be [94, 98], with the interval unit being °C.

[0108] Specifically, the present invention determines the bubble floating tendency characterization value by the difference between the bubble characterization value corresponding to the first denaturation temperature value of the droplet area within the preset temperature range and the bubble characterization value corresponding to the second denaturation temperature value within the preset temperature range. It can be understood that in the PCR denaturation process, temperature is a key factor affecting the bubble state. According to thermodynamics theory, for bubbles in the PCR reaction system, the increase in temperature will intensify the thermal motion of gas molecules, making it easier for bubbles to float. By calculating the difference in bubble contour area on the liquid surface of the droplet unit corresponding to the two temperature values, the bubble change state of each droplet unit in the droplet area during the denaturation process can be intuitively reflected. The present invention determines the bubble floating tendency characterization value by the difference between the bubble characterization value corresponding to the first denaturation temperature value of the droplet area within the preset temperature range and the bubble characterization value corresponding to the second denaturation temperature value within the preset temperature range. Furthermore, the trend of bubble floating in each droplet area is quantified, thereby improving the accuracy of the digital PCR analysis results.

[0109] See also Figure 3 As shown, it is a logic flow chart of screening the droplet characteristic area according to an embodiment of the present invention. Screening the droplet characteristic area includes:

[0110] If the bubble floating tendency characterization value corresponding to the droplet region meets the droplet bubble characterization condition, the droplet region is selected as a droplet characteristic region;

[0111] If the bubble floating tendency characterization value corresponding to the droplet area does not meet the droplet bubble characterization condition, the droplet area is not screened;

[0112] The droplet bubble characterization condition is that the bubble floating tendency characterization value corresponding to the droplet region exceeds the average value of the bubble floating tendency characterization values corresponding to each droplet region.

[0113] Specifically, the present invention screens the droplet characteristic area through the bubble floating tendency characterization value of each droplet area. It can be understood that, the larger the bubble floating tendency characterization value of the droplet area, the greater the difference in bubble contour area on the surface of the droplet unit liquid surface at different denaturation stages in the area, which characterizes that the bubble floating tendency in the area is stronger, the more bubbles are on the droplet unit liquid surface, and the more seriously the area is affected by bubbles. The present invention screens the droplet characteristic area through the bubble floating tendency characterization value of each droplet area, thereby achieving an intuitive reflection of the bubble status in different droplet areas and improving the accuracy of the digital PCR analysis results.

[0114] See also Figure 4 As shown, it is a logic flow chart for selecting an adjustment method for annealing each droplet unit according to an embodiment of the present invention. The adjustment method for annealing each droplet unit includes:

[0115] If the number of the droplet characteristic areas meets the overall adjustment conditions, the inlet air temperature is adjusted in a step-by-step temperature control manner;

[0116] If the number of the droplet characteristic areas does not meet the overall adjustment condition, adjusting the air inlet velocity of the droplet characteristic areas;

[0117] The overall adjustment condition is that the proportion of the number of the droplet characteristic regions to the total number of droplet regions exceeds a preset proportion threshold.

[0118] Specifically, the preset proportion threshold can be set by those skilled in the art according to the accuracy requirements of PCR analysis. The higher the accuracy requirement, the smaller the preset proportion threshold. Preferably, the proportion threshold can be 0.4.

[0119] Specifically, the present invention selects an adjustment method for annealing each droplet unit by the number of droplet characteristic regions. It can be understood that by adopting an adjustment method that is compatible with the proportion of the number of droplet characteristic regions in the total number of droplet regions, over-adjustment or under-adjustment can be avoided. When overall adjustment is required, step temperature control is adopted, which can have a positive impact on a large number of droplet units at the same time and quickly improve reaction conditions. When only local adjustment is required, the air inlet rate is adjusted according to the droplet characteristic region, which can concentrate resources to solve key problems and improve the efficiency of the entire PCR experiment. The present invention selects an adjustment method for annealing each droplet unit by the number of droplet characteristic regions, thereby achieving adaptive adjustment of annealing according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0120] Specifically, when the number of droplet characteristic areas meets the overall adjustment conditions, the present invention selects a stepped temperature control method to adjust the inlet air temperature. It can be understood that the number of droplet characteristic areas meets the overall adjustment conditions, indicating that more droplet areas are affected by bubbles as a whole, and a suitable temperature adjustment method is required to alleviate the impact of bubbles on the accuracy of PCR analysis results. During the PCR amplification process, condensation water is easily generated due to rapid temperature changes. Condensation water will take away some heat, resulting in uneven temperature distribution of the droplet units, affecting the accuracy and reliability of the analysis. The stepped temperature control method can regularly adjust the inlet air temperature by setting different temperature steps, making the temperature change more gentle and orderly, avoiding the generation of condensation water caused by rapid temperature changes, and helping to balance the temperature differences between the droplet units, thereby improving the uniformity and stability of the overall reaction. When the number of droplet characteristic areas meets the overall adjustment conditions, the present invention selects a stepped temperature control method to adjust the inlet air temperature, thereby achieving adaptive adjustment of annealing and improving the accuracy of digital PCR analysis results.

[0121] Specifically, when the number of droplet characteristic areas does not meet the overall adjustment conditions, the present invention selects to adjust the air inlet rate of the droplet characteristic areas. It can be understood that the number of droplet characteristic areas does not meet the overall adjustment conditions, which means that there are fewer droplet areas affected by bubbles as a whole. Targeted adjustment of the air inlet rate of these droplet characteristic areas can more accurately solve local problems and directly change the heat dissipation rate of the droplet units in these areas, thereby quickly and accurately adjusting their temperature, making the annealing process more in line with the requirements and avoiding unnecessary impacts on other normal droplet areas on the entire PCR plate. When the number of droplet characteristic areas does not meet the overall adjustment conditions, the present invention selects to adjust the air inlet rate of the droplet characteristic areas, thereby achieving adaptive adjustment of annealing and improving the accuracy of digital PCR analysis results.

[0122] Specifically, the temperature adjustment step amount of the step temperature control method is negatively correlated with the bubble floating tendency characterizing value, and the air inlet speed is negatively correlated with the bubble floating tendency characterizing value.

[0123] Specifically, the temperature adjustment step amount of the step temperature control method of the present invention is negatively correlated with the bubble floating tendency characterization value. It can be understood that the larger the bubble floating tendency characterization value, the more heat accumulation in the droplet characteristic area, and the smaller the temperature adjustment step amount is required to make the temperature environment change in the droplet characteristic area more gentle, avoiding the generation of condensed water caused by sudden temperature changes, and thus achieving adaptive adjustment of annealing according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0124] Specifically, the air inlet rate of the droplet characteristic area of the present invention is negatively correlated with the bubble floating tendency characterization value. It can be understood that the air inlet rate affects the heat exchange rate. The larger the bubble floating tendency characterization value, the more heat accumulates in the droplet characteristic area, and the smaller the air inlet rate is needed to reduce the heat exchange rate to avoid condensation caused by rapid temperature changes, which affects the accuracy of the digital PCR analysis results. Furthermore, the annealing is adaptively adjusted according to the actual state of the droplet unit during the denaturation process of PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0125] Specifically, the adjustment and verification of the droplet unit that completes a single PCR amplification includes:

[0126] Performing fluorescence signal statistics of the droplet unit in the characteristic area for several times under different light paths, and obtaining fluorescence signal statistics corresponding to the different light paths;

[0127] The tilt angle of the PCR plate is adjusted, and fluorescence signal statistics of the droplet units in the characteristic area at different tilt angles are performed using the same optical path to obtain fluorescence signal statistics corresponding to different tilt angles.

[0128] In this embodiment, three different light paths may be selected to perform fluorescence signal statistics on the droplet units within the characteristic area.

[0129] In this embodiment, three different tilt angles of the PCR plate may be selected to perform fluorescence signal statistics on the droplet units within the characteristic region. The tilt angle may range from [5, 15], with the interval unit being degrees.

[0130] Specifically, the present invention performs fluorescence signal statistics of different optical paths for the droplet units in the characteristic area several times to obtain the fluorescence signal statistics corresponding to different optical paths. It can be understood that the presence of bubbles will cause the optical path to undergo refraction, scattering and other phenomena, affecting the transmission and detection of the fluorescence signal. Different optical paths are affected by bubbles to different degrees. By performing statistics on the fluorescence signals of multiple optical paths, the differences in signals under different optical paths can be compared and analyzed, thereby verifying whether the current annealing adjustment method is effective, thereby improving the accuracy of the digital PCR analysis results.

[0131] Specifically, the present invention adjusts the tilt angle of the PCR plate, and uses the same optical path to perform fluorescence signal statistics on the droplet units in the characteristic area under different tilt angles, so as to respectively obtain the fluorescence signal statistics corresponding to different tilt angles. It can be understood that adjusting the tilt angle of the PCR plate can remove the bubbles remaining in the droplet units, and different tilt angles have different degrees of influence on the bubbles. By obtaining the fluorescence signal statistics corresponding to different tilt angles, comparing and analyzing the differences in fluorescence signal statistics at different tilt angles, it is possible to verify whether the current annealing adjustment method is effective, thereby improving the accuracy of the digital PCR analysis results.

[0132] Specifically, determining whether to continue PCR amplification for each droplet unit includes:

[0133] Calculate the difference between each fluorescence signal statistic and any other fluorescence signal statistic;

[0134] If the difference does not exceed a preset difference threshold, determining to continue PCR amplification for each droplet unit;

[0135] If the difference exceeds a preset difference threshold, it is determined not to continue PCR amplification on each droplet unit and a warning signal is issued.

[0136] Specifically, the preset difference threshold can be set by those skilled in the art according to the accuracy requirements of PCR analysis. The higher the accuracy requirements, the smaller the preset difference threshold. Preferably, the difference threshold can be 5%.

[0137] Specifically, the present invention determines whether to continue PCR amplification for each droplet unit by calculating the difference between the statistics of each fluorescence signal and the statistics of any other fluorescence signal. It can be understood that the larger the difference, the more serious the impact of the bubble in the current droplet unit, and the more serious the impact on the PCR analysis, and the PCR amplification should be stopped in time. The smaller the difference, the weaker the impact of the bubble in the current droplet unit, and the higher the reliability of the PCR analysis. Each droplet unit can continue PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0138] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

[0139] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A digital PCR analysis device, characterized in that: include: A droplet generation module, which is used to prepare a nucleic acid sample into a PCR reaction system solution in proportion and divide the PCR reaction system solution into a plurality of droplet units; a PCR amplification module connected to the droplet generation module, comprising a PCR plate for placing a plurality of droplet units, and a temperature control component for denaturing, annealing, and extending each droplet unit; a feature extraction module connected to the PCR amplification module, configured to divide the PCR plate into a plurality of droplet regions and obtain a bubble floating tendency characterization value of each droplet region during the denaturation process; Wherein, obtaining the bubble characterization value corresponding to the droplet region at different temperature values includes obtaining liquid surface images corresponding to each droplet unit in the droplet region at different temperature values, calculating the bubble contour area based on the liquid surface image, calculating the average value of the bubble contour area corresponding to each droplet unit, and determining the average value as the bubble characterization value of the droplet region; The process of determining the bubble floating tendency characterization value of each droplet region during the denaturation process includes obtaining a bubble characterization value corresponding to a first denaturation temperature value of the droplet region within a preset temperature range, determining the bubble characterization value as a first bubble characterization value, obtaining a bubble characterization value corresponding to a second denaturation temperature value of the droplet region within the preset temperature range, determining the bubble characterization value as a second bubble characterization value, calculating a difference between the first bubble characterization value and the second bubble characterization value, and determining the difference as the bubble floating tendency characterization value corresponding to the droplet region; The first denaturation temperature value is the upper limit of the preset temperature range, and the second denaturation temperature value is the lower limit of the preset temperature range; a feature determination module, connected to the feature extraction module, configured to screen droplet feature regions based on a bubble rising tendency characteristic value of each droplet region, select an adjustment method for annealing each droplet unit based on the number of droplet feature regions, and determine whether to continue PCR amplification for each droplet unit; If the bubble floating tendency characterization value corresponding to the droplet region meets the droplet bubble characterization condition, the droplet region is selected as a droplet characteristic region; The droplet bubble characterization condition is that the bubble floating tendency characterization value corresponding to the droplet area exceeds the average value of the bubble floating tendency characterization values corresponding to each droplet area; If the number of the droplet characteristic areas meets the overall adjustment conditions, the inlet air temperature is adjusted in a step-by-step temperature control manner; If the number of the droplet characteristic areas does not meet the overall adjustment condition, adjusting the air inlet velocity of the droplet characteristic areas; The overall adjustment condition is that the proportion of the number of the droplet characteristic regions to the total number of the droplet regions exceeds a preset proportion threshold; a ventilation module connected to the PCR amplification module and the feature determination module, for adjusting the air inlet velocity to the droplet feature area and adjusting the air inlet temperature of each droplet unit in a step-by-step temperature control manner; a fluorescence detection module connected to the PCR amplification module and the feature determination module, comprising an adjustment and verification unit for adjusting and verifying a droplet unit that has completed a single PCR amplification, and an output unit for performing fluorescence signal statistics on droplet units that have completed multiple PCR amplifications and outputting analysis results; Wherein, the adjustment and verification unit also includes a control component for adjusting the tilt angle of the PCR plate.

2. The digital PCR analysis device according to claim 1, characterized in that The ventilation module further includes a plurality of ventilation holes arranged below the PCR plate for ventilating the droplet area; Each droplet area corresponds to at least one vent.

3. A digital PCR analysis method, using the digital PCR analysis device according to any one of claims 1 to 2, characterized in that: include: Prepare the nucleic acid sample into a PCR reaction system solution according to the proportion; Dividing the PCR reaction system solution into a plurality of droplet units, placing the units on a PCR plate, and dividing the PCR plate into a plurality of droplet areas, each droplet area comprising a plurality of droplet units; Performing multiple PCR amplifications on the droplet units of each droplet region, wherein a single PCR amplification includes sequential denaturation, annealing, and extension; Obtaining a bubble floating tendency characterization value of each droplet region during the denaturation process, and screening droplet characteristic regions according to the bubble floating tendency characterization value of each droplet region; Wherein, the bubble floating tendency characterization value is determined according to the bubble characterization values corresponding to different temperature values of the droplet area; The adjustment method for annealing each droplet unit is selected based on the number of the droplet characteristic regions, including: Adjusting the inlet air temperature in a step-by-step temperature control manner, or adjusting the inlet air velocity for the droplet characteristic area; performing adjustment and verification on the droplet units that have completed a single PCR amplification, and determining whether to continue PCR amplification on each droplet unit based on the result of the adjustment and verification; The adjustment verification includes performing fluorescence signal statistics on the characteristic area of the droplet under different detection light paths and different PCR plate tilt states; The fluorescence signal statistics of the droplet units that have completed several PCR amplifications are performed and the PCR analysis results are output.

4. The digital PCR analysis method according to claim 3, characterized in that The temperature adjustment step amount of the step temperature control method is negatively correlated with the bubble floating tendency characterization value, and the air inlet rate is negatively correlated with the bubble floating tendency characterization value.

5. The digital PCR analysis method according to claim 4, characterized in that Adjustment and verification of the droplet unit that completes a single PCR amplification includes: Performing fluorescence signal statistics of the droplet unit in the characteristic area for several times under different light paths, and obtaining fluorescence signal statistics corresponding to the different light paths; The tilt angle of the PCR plate is adjusted, and fluorescence signal statistics of the droplet units in the characteristic area at different tilt angles are performed using the same optical path to obtain fluorescence signal statistics corresponding to different tilt angles.

6. The digital PCR analysis method according to claim 5, characterized in that Determining whether to continue PCR amplification for each droplet unit includes: Calculate the difference between each fluorescence signal statistic and any other fluorescence signal statistic; If the difference does not exceed the preset difference threshold, it is determined to continue PCR amplification for each droplet unit.

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