Digital PCR (Polymerase Chain Reaction) analysis device and analysis method thereof

Abstract

The invention relates to the technical field of digital PCR analysis, in particular to a digital PCR analysis device and a digital PCR analysis method.The digital PCR analysis device is provided with a microdroplet generation module, a PCR amplification module, a feature extraction module, a feature judgment module, a ventilation module and a fluorescence detection module; denaturation, annealing and extension are carried out on each microdroplet unit through the PCR amplification module, a bubble floating trend characterization value is obtained through the feature extraction module, microdroplet feature areas are screened through the feature judgment module, an annealing adjustment mode is selected, whether PCR amplification continues or not is determined, and the air speed and temperature of inlet air are adjusted through the ventilation module; the micro-droplet unit is adjusted and verified through the adjusting and verifying unit, and the analysis result is output through the output unit, so that annealing is adaptively adjusted according to the actual state of the micro-droplet unit in the denaturation process of PCR amplification, and the accuracy of the digital PCR analysis result is improved.

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 the detection of tumor markers, pathogens, and genotyping, more accurate quantitative analysis of nucleic acid molecules is required to achieve early diagnosis of diseases, formulation of treatment plans, and efficacy monitoring. Traditional PCR technology is usually based on standard curves or reference genes for relative quantification, and its results are affected by many factors. Digital PCR divides the reaction system into tiny droplet units and calculates the absolute content of the target nucleic acid in the sample based on the Poisson distribution principle, which greatly reduces the possibility of non-specific amplification. It is an absolute quantification of the starting sample, not limited by the standard curve and reference genes, and can more accurately determine the content of the target nucleic acid. However, in actual operation, the reliability of each droplet unit often faces many challenges. Temperature changes during PCR amplification will cause abnormal bubbles in the droplet unit, affecting the reliability of the tiny reaction unit, and then affecting the accuracy of the digital PCR analysis results. Therefore, real-time monitoring of the status of each tiny reaction unit during PCR amplification and improving the accuracy of the digital PCR analysis results are technical problems that need to be solved.

[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 arranged inside the box assembly, a clamping assembly is arranged 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 arranged at the bottom of the rotating assembly, a filtering assembly, a temperature control assembly and a blowing assembly are symmetrically arranged on both sides of the box assembly, a control center is also arranged on the box assembly, and a water-absorbing sponge is arranged at the bottom of the box assembly; in the invention, by arranging a temperature control assembly, the temperature inside the box assembly is raised and lowered, thereby fine-tuning the temperature of the PCR tube; by arranging 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 arranging a blowing assembly, the air inside the box assembly and the air outside are exchanged.

[0004] The prior art still has the following problems:

[0005] The prior art does not take into account that changes in the thermal field distribution during PCR amplification may cause abnormal bubbles in the droplet unit, thereby affecting the accuracy of fluorescence signal detection. 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. Summary of the invention

[0006] To this end, the present invention provides a digital PCR analysis device and an 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 object, the present invention provides a digital PCR analysis device, comprising:

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

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

[0010] A feature extraction module, which is connected to the PCR amplification module and is used 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, for screening droplet feature regions according to the bubble floating tendency characterization value of each droplet region, selecting an adjustment method for annealing each droplet unit based on the number of the droplet feature regions, and determining 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 rate to the droplet feature area and adjusting the air inlet temperature of each droplet unit in a step temperature control manner;

[0013] A fluorescence detection module, which is connected to the PCR amplification module and the feature determination module, and includes an adjustment and verification unit for adjusting and verifying the microdroplet unit that has completed a single PCR amplification, and an output unit for performing fluorescence signal statistics on the microdroplet units that have completed several 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] Further, the ventilation module also includes a plurality of ventilation holes arranged below the PCR plate for ventilating the droplet area;

[0016] Wherein, each droplet area corresponds to at least one vent.

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

[0018] The nucleic acid sample is prepared into a PCR reaction system solution according to a certain proportion;

[0019] The PCR reaction system solution is divided into a plurality of droplet units, which are placed on a PCR plate, and the PCR plate is divided into a plurality of droplet areas, each droplet area comprising a plurality of droplet units;

[0020] Performing several 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 microdroplet unit is selected based on the number of the microdroplet 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 micro-droplet units that have completed a single PCR amplification, and determining whether to continue PCR amplification on each micro-droplet unit according to 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] Further, 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 area, and calculate the bubble contour area according to 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] Further, 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 region;

[0035] Wherein, 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 screened 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] Further, the adjustment method for annealing each droplet unit includes:

[0040] If the number of the droplet characteristic regions meets the overall adjustment condition, a step temperature control method is selected to adjust the inlet air temperature;

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

[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 intake rate is negatively correlated with the bubble floating tendency characterization value.

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

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

[0046] The tilt angle of the PCR plate is adjusted, and the fluorescence signal statistics of the droplet units in the characteristic area under different tilt angles are performed with the same optical path to obtain the fluorescence signal statistics corresponding to the 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 beneficial effect of the present invention lies in that 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 made 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 is used to denature, anneal and extend each droplet unit; the feature extraction module is used to divide the PCR plate into a plurality of droplet areas; the bubble floating tendency characterization value of each droplet area during the denaturation process is obtained; the feature determination module is used to screen the droplet characteristic area according to the bubble floating tendency characterization value of each droplet area; Based on the number of droplet characteristic areas, an adjustment method for annealing each droplet unit is selected, and it is determined whether to continue PCR amplification for each droplet unit. The air inlet rate of the droplet characteristic area is adjusted through the ventilation module, and the air inlet temperature of each droplet unit is adjusted in a step temperature control manner. The droplet unit that has completed a single PCR amplification is adjusted and verified through the adjustment and verification unit. The fluorescence signal of the droplet unit that has completed several PCR amplifications is counted and the analysis results are output through the output unit. Therefore, 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.

[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 the theory of thermodynamics, 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.

[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 larger 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 serious the degree of influence of bubbles on the area is. 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 be 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 for 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.

[0054] In particular, when the number of droplet characteristic areas meets the overall adjustment conditions, the present invention selects to adjust the inlet air temperature in a step-by-step temperature control manner. It can be understood that the number of droplet characteristic areas meets the overall adjustment conditions, indicating 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, condensed water is easily generated due to rapid changes in temperature. The condensed 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 step-by-step temperature control method can regularly adjust the inlet air temperature by setting different temperature steps, so that the temperature change is smoother and more orderly, avoiding the generation of condensed 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 to adjust the inlet air temperature in a step-by-step temperature control manner, 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 temperatures, making the annealing process more in line with the requirements and avoiding unnecessary effects 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 is, the more heat accumulates in the droplet characteristic area, and the smaller the temperature adjustment step amount is needed to make the temperature environment change of the droplet characteristic area more gentle, avoiding the generation of condensed water caused by rapid temperature changes, and further, 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.

[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 in the characteristic area several times to obtain the fluorescence signal statistics corresponding to different optical paths respectively. It can be understood that the presence of bubbles will cause refraction, scattering and other phenomena in the optical path, affecting the transmission and detection of the fluorescence signal. Different optical paths are affected by bubbles to different degrees. By taking statistics of the fluorescence signals of multiple optical paths, the differences in signals under different optical paths can be compared and analyzed, so as to verify 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 is, the more serious the influence of the bubble on the current droplet unit is, and the more serious the influence on the PCR analysis is, and the PCR amplification should be stopped in time; the smaller the difference is, the weaker the influence of the bubble on the current droplet unit is, and the higher the reliability of the PCR analysis is, and each droplet unit can continue to perform PCR amplification, thereby improving the accuracy of the digital PCR analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0062] Figure 2 A step diagram of a digital PCR analysis method according to an embodiment of the present invention;

[0063] Figure 3 A logic flow chart for screening droplet characteristic 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 only used to explain the present invention and are not used 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 protection scope 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 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] In addition, it should be noted that in the description of the present invention, unless otherwise clearly specified and limited, the terms "installation" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be the internal communication of two components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0069] See also Figure 1 As shown, it 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 comprises:

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

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

[0072] A feature extraction module, which is connected to the PCR amplification module and is used 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, for screening droplet feature regions according to the bubble floating tendency characterization value of each droplet region, selecting an adjustment method for annealing each droplet unit based on the number of the droplet feature regions, and determining 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 rate to the droplet feature area and adjusting the air inlet temperature of each droplet unit in a step temperature control manner;

[0075] A fluorescence detection module, which is connected to the PCR amplification module and the feature determination module, and includes an adjustment and verification unit for adjusting and verifying the microdroplet unit that has completed a single PCR amplification, and an output unit for performing fluorescence signal statistics on the microdroplet units that have completed several 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 a number of 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 realized by the cooperation of 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. It will not be repeated 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, which will not be repeated 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. It will not be repeated here.

[0082] Specifically, the present invention does not limit the specific structure of the adjustment 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, which will not be described here.

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

[0086] Wherein, 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 inlet air duct, and control the inlet air rate by controlling the blower unit on the inlet air duct. Preferably, it can be a programmable logic controller. The control of functional devices by a programmable logic controller is a prior art 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 droplets on a PCR plate, and dividing the PCR plate into a plurality of droplet areas, each droplet area comprising 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, adjusting and verifying the micro-droplet units that have completed a single PCR amplification, and determining whether to continue PCR amplification for each micro-droplet unit according to the result of the adjustment and 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 PCR amplifications, 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 area, and calculate the bubble contour area according to 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 region;

[0106] Wherein, 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 technicians in this field according to the accuracy requirements of PCR analysis. The higher the accuracy requirement, the larger the temperature range is set. Preferably, the temperature range can be [94, 98], and the interval unit is ℃.

[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 the theory of thermodynamics, 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 characteristic region of the droplet according to an embodiment of the present invention. Screening the characteristic region of the droplet includes:

[0110] If the bubble floating tendency characterization value corresponding to the droplet region meets the droplet bubble characterization condition, the droplet region is screened 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 larger 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 serious the degree of influence of bubbles on the area is. 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 state 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 of selecting an adjustment method for annealing each micro-droplet unit in an embodiment of the present invention. The adjustment method for annealing each micro-droplet unit includes:

[0115] If the number of the droplet characteristic regions meets the overall adjustment condition, a step temperature control method is selected to adjust the inlet air temperature;

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

[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 may be set by a technician in this field according to the accuracy requirement of the PCR analysis. The higher the accuracy requirement, the smaller the preset proportion threshold. Preferably, the proportion threshold may be 0.4.

[0119] Specifically, the present invention selects an adjustment method for annealing each droplet unit by the number of droplet characteristic areas. It can be understood that by adopting an adjustment method that is compatible with the proportion of the number of droplet characteristic areas in the total number of droplet areas, 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 for the droplet characteristic area, 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 areas, 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 to adjust the inlet air temperature in a step-by-step temperature control manner. It can be understood that the number of droplet characteristic areas meets the overall adjustment conditions, indicating 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, condensed water is easily generated due to rapid changes in temperature. The condensed water will take away part of the heat, resulting in uneven temperature distribution of the droplet unit, affecting the accuracy and reliability of the analysis. The step-by-step temperature control method can regularly adjust the inlet air temperature by setting different temperature steps, so that the temperature change is smoother and more orderly, avoiding the generation of condensed 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 to adjust the inlet air temperature in a step-by-step temperature control manner, 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 temperatures, making the annealing process more in line with the requirements and avoiding unnecessary effects 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 characterization value, and the air intake rate is negatively correlated with the bubble floating tendency characterization 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 is, the more heat accumulates in the droplet characteristic area, and the smaller the temperature adjustment step amount is needed to make the temperature environment change of the droplet characteristic area more gentle, avoiding the generation of condensed water caused by sudden temperature changes, and further, realizing 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 different light paths for a plurality of times on the droplet unit in the characteristic area, and obtaining fluorescence signal statistics corresponding to the different light paths respectively;

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

[0129] In this embodiment, three different tilt angles of the PCR plate may be selected to perform fluorescence signal statistics on the droplet units in 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 unit in the characteristic area several times to obtain the fluorescence signal statistics corresponding to different optical paths respectively. It can be understood that the presence of bubbles will cause refraction, scattering and other phenomena in the optical path, affecting the transmission and detection of the fluorescence signal. Different optical paths are affected by bubbles to different degrees. By taking statistics of the fluorescence signals of multiple optical paths, the differences in signals under different optical paths can be compared and analyzed, so as to verify 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 for each droplet unit and a warning signal is issued.

[0136] Specifically, the preset difference threshold may be set by a person skilled in the art according to the accuracy requirement of PCR analysis. The higher the accuracy requirement, the smaller the preset difference threshold. Preferably, the difference threshold may 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 bubble influence of the current droplet unit, the more serious the influence on the PCR analysis, and the PCR amplification should be stopped in time; the smaller the difference, the weaker the bubble influence of the current droplet unit, the higher the reliability of the PCR analysis, and each droplet unit can continue to perform PCR amplification, thereby improving the accuracy of the digital PCR analysis results.

[0138] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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 according to a proportion, and divide the PCR reaction system solution into a plurality of droplet units; A PCR amplification module, which is connected to the microdroplet generation module and includes a PCR plate for placing a plurality of microdroplet units, and a temperature control component for denaturing, annealing and extending each microdroplet unit; A feature extraction module, which is connected to the PCR amplification module and is used 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; a feature determination module connected to the feature extraction module, for screening droplet feature regions according to the bubble floating tendency characterization value of each droplet region, selecting an adjustment method for annealing each droplet unit based on the number of the droplet feature regions, and determining whether to continue PCR amplification for each droplet unit; A ventilation module, connected to the PCR amplification module and the feature determination module, for adjusting the air inlet rate to the droplet feature area and adjusting the air inlet temperature of each droplet unit in a step temperature control manner; A fluorescence detection module, which is connected to the PCR amplification module and the feature determination module, and includes an adjustment and verification unit for adjusting and verifying the microdroplet unit that has completed a single PCR amplification, and an output unit for performing fluorescence signal statistics on the microdroplet units that have completed several 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 also includes a plurality of ventilation holes arranged below the PCR plate for ventilating the droplet area; Wherein, each droplet area corresponds to at least one vent.

3. A digital PCR analysis method, for use with the digital PCR analysis device according to any one of claims 1 to 2, characterized in that: include: The nucleic acid sample is prepared into a PCR reaction system solution according to a certain proportion; The PCR reaction system solution is divided into a plurality of droplet units, which are placed on a PCR plate, and the PCR plate is divided into a plurality of droplet areas, each droplet area comprising a plurality of droplet units; Performing several 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 microdroplet unit is selected based on the number of the microdroplet 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 micro-droplet units that have completed a single PCR amplification, and determining whether to continue PCR amplification on each micro-droplet unit according to 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: Obtaining bubble characterization values ​​corresponding to the droplet region at different temperature values ​​includes: Acquire liquid surface images corresponding to different temperature values ​​of each droplet unit in the droplet area, and calculate the bubble contour area according to the liquid surface images; 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.

5. The digital PCR analysis method according to claim 4, characterized in that: The process of determining the bubble uplift tendency characterizing the 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, and 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 a preset temperature range, and 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 a bubble floating tendency characterization value corresponding to the droplet region; Wherein, 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.

6. The digital PCR analysis method according to claim 5, characterized in that: Screening the droplet characteristic region includes, If the bubble floating tendency characterization value corresponding to the droplet region meets the droplet bubble characterization condition, the droplet region is screened as a droplet characteristic region; 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.

7. The digital PCR analysis method according to claim 6, characterized in that: The adjustment methods selected for annealing each droplet unit include: If the number of the droplet characteristic regions meets the overall adjustment condition, the step temperature control method is selected to adjust the inlet air temperature; If the number of the droplet characteristic regions does not meet the overall adjustment condition, adjusting the air inlet velocity of the droplet characteristic regions; 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.

8. The digital PCR analysis method according to claim 7, 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 intake rate is negatively correlated with the bubble floating tendency characterization value.

9. The digital PCR analysis method according to claim 8, characterized in that: Adjustment and verification of the droplet unit that completes a single PCR amplification includes: Performing fluorescence signal statistics of different light paths for a number of times on the droplet unit in the characteristic area, and obtaining fluorescence signal statistics corresponding to the different light paths respectively; The tilt angle of the PCR plate is adjusted, and the fluorescence signal statistics of the droplet units in the characteristic area under different tilt angles are performed with the same optical path to obtain the fluorescence signal statistics corresponding to the different tilt angles.

10. The digital PCR analysis method according to claim 9, 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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