Multi-coding method for digital droplet PCR (Polymerase Chain Reaction)

Abstract

The invention provides a multi-coding method for digital droplet PCR (polymerase chain reaction). The multi-coding method comprises the following steps: acquiring at least one target volume and a candidate optical set; matching each candidate optical combination for each target volume to obtain a plurality of first candidate liquid drops marked by each candidate optical combination and corresponding to each target volume; performing photoelectric testing on all the first candidate liquid drops to obtain photoelectric signal characteristics and photoelectric signal distinction degree of each first candidate liquid drop so as to obtain a target optical combination corresponding to each target volume, and taking the target volume and the corresponding target optical combination as a group of coding units; the photoelectric signal characteristics corresponding to each group of coding units are used as target molecule recognition characteristics under the coding units. According to the encoding method, the digital droplet PCR technology is more adaptive to various complex nucleic acid samples, and the problem of poor accuracy or misjudgment in traditional detection is solved.

Description

Technical Field

[0001] The present application relates to the technical field of digital droplet PCR, and in particular to a multiple encoding method for digital droplet PCR. Background Art

[0002] With the continuous deepening of molecular biology research and the increasing requirements for the accuracy and efficiency of nucleic acid detection in clinical diagnosis, digital droplet PCR (Polymerase Chain Reaction) technology has attracted widespread attention as an advanced nucleic acid detection method that can achieve absolute counting of target molecules. This technology distributes nucleic acid samples into a large number of independent and parallel micro-reaction units (i.e. droplets) for amplification and detection, greatly improving the sensitivity and accuracy of detection.

[0003] In the prior art, this technology often uses color marking to distinguish whether different droplets carry target molecules and what kind of target molecules they carry. However, in actual complex nucleic acid samples, the sample may contain multiple similar target molecules, or the concentration of target molecules in the sample is at a low level. This marking method can easily cause insufficient differentiation of the optical signals presented by different droplets, which in turn leads to the inability to accurately determine whether the droplets actually carry target molecules during the detection process, or to mistakenly judge droplets carrying different target molecules as the same, seriously affecting the accuracy of the detection results. Summary of the invention

[0004] In view of the above-mentioned defects or deficiencies in the prior art, the present application aims to provide a multiple encoding method for digital droplet PCR to improve detection accuracy; the method comprises the following steps: Acquire at least one target volume and a candidate optical set; the candidate optical set includes a plurality of candidate optical combinations, each of which includes a visible light color label and a fluorescent probe dye type; each of the visible light color labels corresponds to a dye color; the target volume is a droplet volume whose photoelectric signal intensity of the corresponding droplet is in a first intensity interval; Matching each of the candidate optical combinations to each of the target volumes, respectively, to obtain a plurality of first candidate droplets corresponding to each of the target volumes and marked by each of the candidate optical combinations, wherein the first candidate droplets contain target molecules; Performing photoelectric testing on all the first candidate droplets respectively to obtain a photoelectric signal feature of each of the first candidate droplets; According to the photoelectric signal characteristics of each of the first candidate droplets, the photoelectric signal discrimination of each of the first candidate droplets is obtained, and then the target optical combination corresponding to each of the target volumes is obtained, the target optical combination being a candidate optical combination whose photoelectric signal discrimination is greater than or equal to a first preset threshold; The target volume and the target optical combination corresponding thereto are used as a group of coding units, and the photoelectric signal characteristics corresponding to each group of coding units are used as target molecule recognition characteristics under the coding unit.

[0005] According to the technical solution provided in the embodiment of the present application, obtaining at least one target volume specifically includes the following steps: Acquire a candidate volume set, where the candidate volume set includes a plurality of candidate volumes; Acquire a second candidate droplet corresponding to each of the candidate volumes, and perform a photoelectric test on each of the second candidate droplets to obtain a photoelectric signal intensity of each of the second candidate droplets; A number of target candidate droplets whose photoelectric signal intensities are within the first intensity range are screened out from all the second candidate droplets, and the candidate volume corresponding to each target candidate droplet is used as the target volume.

[0006] According to the technical solution provided in the embodiment of the present application, each of the candidate optical combinations is matched to each of the target volumes to obtain a plurality of first candidate droplets marked by each of the candidate optical combinations corresponding to each of the target volumes, specifically comprising the following steps: Acquire a marker set for each target volume, wherein the marker set includes a plurality of candidate optical combinations, each group of candidate optical combinations includes a fluorescent probe dye type and a plurality of dye colors corresponding to each group of fluorescent probe dye types; Obtain reference droplets of the target volume, and mark each of the reference droplets with a set of the fluorescent probe dye type and the dye color in the marking set to obtain a plurality of the first candidate droplets; each of the first candidate droplets is marked with a different fluorescent probe dye type and a different dye color.

[0007] According to the technical solution provided in the embodiment of the present application, the photoelectric test is performed on all the first candidate droplets respectively to obtain the photoelectric signal characteristics of each of the first candidate droplets, which specifically includes the following steps: A photoelectric test is performed on each of the first candidate droplets under a dark field to obtain a photoelectric signal characteristic of each of the first candidate droplets under a dark field; the photoelectric signal characteristic under a dark field is related to the type of fluorescent probe dye.

[0008] According to the technical solution provided in the embodiment of the present application, the photoelectric signal characteristics at least include photoelectric intensity; The step of obtaining the photoelectric signal discrimination of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets specifically comprises the following steps: The photoelectric intensity of each of the first candidate droplets under a dark field is used as the photoelectric signal discrimination degree of each of the first candidate droplets.

[0009] According to the technical solution provided in the embodiment of the present application, the photoelectric test is performed on all the first candidate droplets respectively to obtain the photoelectric signal characteristics of each of the first candidate droplets, which specifically includes the following steps: Performing photoelectric tests on each of the first candidate droplets in a dark field and a bright field, respectively, to obtain a dark field feature of an optical signal of each of the first candidate droplets in a dark field and a bright field feature of an optical signal of each of the first candidate droplets in a bright field; the dark field feature is related to the type of the fluorescent probe dye, and the bright field feature is related to the color of the dye; Based on the dark field feature and the bright field feature of each of the first candidate droplets, a photoelectric signal feature of each of the first candidate droplets is obtained.

[0010] According to the technical solution provided in the embodiment of the present application, the dark field feature at least includes the dark field photoelectric intensity, and the bright field feature at least includes the bright field photoelectric intensity; The step of obtaining the photoelectric signal discrimination of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets specifically comprises the following steps: Assigning corresponding dark field weights and bright field weights to the dark field photoelectric intensity and bright field photoelectric intensity respectively; The photoelectric signal discrimination degree of each first candidate droplet is obtained according to the dark field photoelectric intensity and the dark field weight of each first candidate droplet, and the bright field photoelectric intensity and the bright field weight.

[0011] According to the technical solution provided in the embodiment of the present application, after the target volume and the target optical combination corresponding thereto are used as a group of coding units, and the photoelectric signal characteristics corresponding to each group of coding units are used as the target molecule recognition characteristics under the coding unit, the following steps are also included: All the coding units and the target molecule recognition features under each coding unit are stored in a coding database.

[0012] According to the technical solution provided in the embodiment of the present application, a second candidate droplet corresponding to each of the candidate volumes is obtained through a microfluidic chip, and the method further includes constructing a flow channel structure of the microfluidic chip; The construction of the flow channel structure of the microfluidic chip specifically includes the following steps: Constructing a benchmark physical model, and using the benchmark physical model to simulate the sheath flow step emulsification process of the oil-water two-phase to obtain simulation results, wherein the simulation results at least include the flow states of the oil-water two-phase at different times and different positions; Based on the simulation results, key information related to the generation of target droplets by the oil-water two-phase is obtained; According to the key information, structural parameters are obtained, wherein the structural parameters at least include a flow channel shape; According to the structural parameters, a flow channel structure of a microfluidic chip is constructed.

[0013] According to the technical solution provided in the embodiment of the present application, obtaining the second candidate droplet corresponding to each of the candidate volumes through the microfluidic chip specifically includes the following steps: The second candidate droplet corresponding to each candidate volume is obtained by controlling the flow rate of the fluid in the flow channel structure of the microfluidic chip.

[0014] Compared with the prior art, the beneficial effect of the present application is that: in the present encoding method, the selection of the target volume is determined based on the photoelectric signal intensity being in a suitable range, which means that the selection of these target volume droplets for subsequent operations can ensure that the photoelectric signal intensity obtained during the detection is relatively large and of better quality, so that the photoelectric signal is clearer and easier to accurately distinguish under complex experimental environments and sample conditions. Combined with the target optical combination, the encoding method makes the digital droplet PCR technology more adaptable when facing various complex nucleic acid samples. Especially when facing situations such as when the concentration of target molecules in the sample is at a low level, a strong and clear photoelectric signal helps to overcome the misjudgment problem that may be caused by weak signals, improves the ability to accurately judge whether the droplet carries the target molecule and the specific target molecule type carried, and thus significantly improves the accuracy of the entire digital droplet PCR detection; in addition, the droplets generated by this encoding method have a total amount of information, and the information load of the same droplet includes three dimensions of "bright field visible light + dark field fluorescent probe dye + multiple volumes", and the droplets generated in this way have ultra-high information density and load. Functionally, this super-multiplex encoding method of digital droplet PCR combines the multiple fluorescence and multiple volume digital PCR detection functions, which to a certain extent increases the amount of information contained in the droplets, reduces the detection cost, and is expected to promote the development of instruments in the corresponding medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A flowchart of the steps of the multiple encoding method for digital droplet PCR provided in an embodiment of the present application; Figure 2 Photoelectric signal diagram provided for the embodiment of the present application; Figure 3 A microscopic photograph of a droplet under a green dye provided in an embodiment of the present application; Figure 4 A microscopic photograph of a droplet under a red dye provided in an embodiment of the present application; Figure 5A microscopic photograph of a droplet under a blue dye provided in an embodiment of the present application; Figure 6 A schematic diagram of multiple visible light encoding provided in an embodiment of the present application. DETAILED DESCRIPTION

[0016] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0017] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0018] Example 1 As mentioned in the background technology, in response to the problems in the prior art, this application proposes a multiple encoding method for digital droplet PCR, please refer to Figure 1 As shown, the following steps are included: S1, acquiring at least one target volume and a candidate optical set; the candidate optical set includes a plurality of candidate optical combinations, each of which includes a visible light color label and a fluorescent probe dye type; each of the visible light color labels corresponds to a dye color; the target volume is a droplet volume whose photoelectric signal intensity of the corresponding droplet is in a first intensity interval; Specifically, the fluorescent probe dye type is a fluorescent base liquid type available on the market, such as ethidium bromide (EB) and propidium iodide (PI). The visible light color label is a label corresponding to the dye color that can obviously display color, such as a red label corresponding to a red dye, a green label corresponding to a green dye, and a blue label corresponding to a blue dye. The candidate optical combinations are obtained by permutation and combination of fluorescent probe dye types and visible light color labels. For example, ethidium bromide can form a candidate optical combination with a red label, and can also form a candidate optical combination with a green label, and can also form a candidate optical combination with a blue label. Propidium iodide can form a candidate optical combination with a red label, and can also form a candidate optical combination with a green label, and can also form a candidate optical combination with a blue label. In this way, a candidate optical set containing 6 candidate optical combinations is obtained.

[0019] Further, the obtaining of at least one target volume specifically comprises the following steps: Acquire a candidate volume set, where the candidate volume set includes a plurality of candidate volumes; Specifically, the candidate volumes are a plurality of volumes obtained randomly, and all candidate volumes constitute a candidate volume set.

[0020] Acquire a second candidate droplet corresponding to each of the candidate volumes, and perform a photoelectric test on each of the second candidate droplets to obtain a photoelectric signal intensity of each of the second candidate droplets; A number of target candidate droplets whose photoelectric signal intensities are within the first intensity range are screened out from all the second candidate droplets, and the candidate volume corresponding to each target candidate droplet is used as the target volume.

[0021] Exemplarily, [60 mV, 100 mV] is the first intensity interval. After analyzing the photoelectric signal intensities of all second candidate droplets, the target candidate droplets whose photoelectric signal intensities are in the first intensity interval are screened out. Assuming that among the multiple candidate volumes of the second candidate droplets generated above, the photoelectric signal intensities of the target candidate droplets with volumes of 10μm³ and 12μm³ are 70 mV and 85 mV respectively, which are in the [60 mV, 100 mV] interval, then the candidate volumes 10μm³ and 12μm³ corresponding to these two target candidate droplets are determined as target volumes.

[0022] Further, obtaining a second candidate droplet corresponding to each of the candidate volumes through a microfluidic chip, the method further includes constructing a flow channel structure of the microfluidic chip; The construction of the flow channel structure of the microfluidic chip specifically includes the following steps: Constructing a benchmark physical model, and using the benchmark physical model to simulate the sheath flow step emulsification process of the oil-water two-phase to obtain simulation results, wherein the simulation results at least include the flow states of the oil-water two-phase at different times and different positions; Specifically, the chip size, interfacial tension, and two-phase flow rate ratio of the sheath flow step emulsification are collected, and these key parameters are input into the COMSOL software environment. In this environment, the physical modeling function provided is used to build a physical model that can simulate the sheath flow step emulsification process. The established physical model is run to obtain the simulation results of the sheath flow step emulsification process. The simulation results include the flow state of the oil-water two phases at different times and positions, the flow state at different times and positions includes the flow velocity distribution, pressure changes, and a detailed set of the droplet formation process, including the initial generation position, generation frequency, and size change trend of the droplets.

[0023] Based on the simulation results, key information related to the generation of target droplets by the oil-water two-phase is obtained; Specifically, the target droplets are droplets with uniform size and good stability.

[0024] According to the key information, structural parameters are obtained, wherein the structural parameters at least include a flow channel shape; Specifically, the key information includes at least the type of flow channel and the local specific shape of the flow channel. The flow channel type includes multiple shape types. The structural parameters are the flow channel shapes corresponding to the shape types. The local specific shape of the flow channel can be an increase or decrease in the size of the flow channel at a certain position. The structural parameters can also include changes in width of different areas, the design of inlets and outlets, and possible special functional areas (such as buffer areas for regulating flow rate, etc.).

[0025] According to the structural parameters, a flow channel structure of a microfluidic chip is constructed.

[0026] For example, the overall height of the microfluidic chip used in the present application is 40 μm, the width of the chip flow channel structure is 40 μm, and the inlet and outlet designs of the flow channel structure are as follows: the size of the sample inlet and the oil outlet is 1.5 mm.

[0027] In a preferred embodiment, the step of obtaining the second candidate droplet corresponding to each of the candidate volumes by means of a microfluidic chip specifically comprises the following steps: The second candidate droplet corresponding to each candidate volume is obtained by controlling the flow rate of the fluid in the flow channel structure of the microfluidic chip.

[0028] Specifically, the continuous phase and the discrete phase reagent containing biological pigment used in the present invention are configured according to the experimental requirements, and are placed in the centrifuge tube used in the experiment according to the storage requirements for experimental use. By using an air pump to squeeze the centrifuge tube containing the continuous phase and the continuous phase reagent containing the pigment, the liquid flows into the microfluidic chip through the catheter under the action of air pressure, and the microfluidic chip controls and transports the continuous phase and discrete phase introduced therein. There are stepped holes drilled in advance on the flow channel structure of the microfluidic chip. When the sample flows through here, it is emulsified to generate droplets under the action of the stepped holes, and enters the detection area according to the flow channel structure designed by the microfluidic chip. A series of droplets of different volumes can be generated by adjusting the flow rate, that is, a second candidate droplet is obtained.

[0029] S2, matching each of the candidate optical combinations to each of the target volumes, respectively, to obtain a plurality of first candidate droplets corresponding to each of the target volumes and marked by each of the candidate optical combinations, wherein the first candidate droplets contain target molecules; Furthermore, matching each of the candidate optical combinations to each of the target volumes to obtain a plurality of first candidate droplets corresponding to each of the target volumes and marked by each of the candidate optical combinations specifically includes the following steps: Acquire a marker set for each target volume, wherein the marker set includes a plurality of candidate optical combinations, each group of candidate optical combinations includes a fluorescent probe dye type and a plurality of dye colors corresponding to each group of fluorescent probe dye types; Obtain reference droplets of the target volume, and mark each of the reference droplets with a set of the fluorescent probe dye type and the dye color in the marking set to obtain a plurality of the first candidate droplets; each of the first candidate droplets is marked with a different fluorescent probe dye type and a different dye color.

[0030] For example, referring to the RGB color system or the CMYK color system, select a visible light pigment dye with biocompatibility, please refer to Figure 6 As shown, the pigments selected in the present invention are red, green, blue and orange; candidate optical combination 11 is (red visible light color label, fluorescent probe dye A), candidate optical combination 12 is (green visible light color label, fluorescent probe dye A), candidate optical combination 13 is (blue visible light color label, fluorescent probe dye A); candidate optical combination 21 is (red visible light color label, fluorescent probe dye B), etc. The dye color and fluorescent probe dye type corresponding to each combination have different optical properties and can be used for subsequent droplet marking.

[0031] Exemplarily, the target molecule is a virus, the sample liquid is blood or body fluid infected with the virus, and a device that can generate a fixed volume is used (this application selects a microfluidic chip model to generate droplets, but is not limited to this method, and any method that can generate fixed volume droplets can be used) to generate a number of reference droplets of target volume from the sample liquid, and then each reference droplet is marked with a corresponding candidate optical combination to obtain a first candidate droplet. For example, for a reference droplet with a target volume of 10μm³, it is marked with fluorescent probe dye ethidium bromide and red dye to obtain a first candidate droplet, and then the first candidate droplet can be subjected to photoelectric testing.

[0032] S3, performing photoelectric testing on all the first candidate droplets respectively to obtain a photoelectric signal feature of each of the first candidate droplets; S4. Obtaining the photoelectric signal discrimination of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets, and then obtaining a target optical combination corresponding to each of the target volumes, wherein the target optical combination is a candidate optical combination whose photoelectric signal discrimination is greater than or equal to a first preset threshold; Exemplarily, it is found that for a target volume of 10 μm³, if the photoelectric signal differentiation between the first candidate droplet marked with (blue visible light color label, fluorescent probe dye A) and the droplets marked with other combinations of optical combination 13 is greater than or equal to a pre-set first preset threshold, then the candidate optical combination 13 is the target optical combination corresponding to the target volume of 10 μm³.

[0033] S5. The target volume and the target optical combination corresponding thereto are used as a group of coding units, and the photoelectric signal characteristics corresponding to each group of coding units are used as target molecule recognition characteristics under the coding unit.

[0034] Specifically, for a coding unit with a target volume of 10 μm³ and a corresponding target optical combination 13 (blue visible light color label, fluorescent probe dye A), it is assumed that after the photoelectric test of the first candidate droplet corresponding to the coding unit, its photoelectric signal characteristic is obtained as the photoelectric signal intensity in a specific interval [ , ], the pulse amplitude is , the pulse width is , the optical power is These photoelectric signal characteristics will be used as the target molecule recognition characteristics under this coding unit. In the actual digital droplet PCR detection, when an unknown droplet of 10μm³ and marked by the target optical combination 13 is detected, if the photoelectric signal intensity is detected to be [ , ], the pulse amplitude is close to , the pulse width is close to , the optical power is close to , then it can be determined that the droplet carries a specific target molecule corresponding to this set of coding units. Each different target molecule can find its corresponding coding unit through the same process as above, and use the photoelectric signal feature corresponding to the coding unit as the identification feature of the target molecule.

[0035] It should be noted that if Figure 2 As shown in the figure, it is a typical photoelectric signal diagram. According to these photoelectric signal diagrams, the light signal characteristics can be obtained as the target molecule recognition characteristics. In addition to using the photoelectric test through the photoelectric signal characteristics as the screening condition, the microscopic photos can also be used as the screening condition, and the gray value in the microscopic photos can be used as the target molecule recognition feature, such as Figure 3 , 4 ,5, Figure 3 This is a micrograph of a droplet under green dye. Figure 4 This is a microscopic photo of a droplet under red dye. Figure 5 Micrograph of a droplet under blue dye.

[0036] It should be noted that this method can also be used in isothermal amplification nucleic acid detection (a type of technology that performs nucleic acid amplification at a constant temperature) and non-amplification nucleic acid detection technology. For example, the patent publication number CN117683616A mentions that m*n types of labeling combinations can be achieved through a combination method of m dye probes and n concentrations. Although this method improves the flux of target molecules to a certain extent, it does not improve the problem of low information density of PCR detection technology. However, this solution greatly improves the information density of PCR detection technology.

[0037] Furthermore, after the target volume and the target optical combination corresponding thereto are used as a group of coding units, and the photoelectric signal characteristics corresponding to each group of coding units are used as the target molecule recognition characteristics under the coding unit, the following steps are also included: All the coding units and the target molecule recognition features under each coding unit are stored in a coding database.

[0038] Specifically, the coding database includes several groups of coding units, and the photoelectric signal characteristics corresponding to each group of coding units, and the target molecule category. In actual use, the sample liquid to be detected of the target population is collected, and the sample liquid to be detected is divided into several small sample liquids, and the detection of the target molecule Q, the target molecule W, the target molecule E, and the target molecule R are respectively performed, and the process of performing the targeted molecule Q is described exemplarily, and the coding unit Q of the targeted molecule Q is obtained by calling the coding database, and a small sample liquid is transferred to the microfluidic chip through the targeted molecule Q to form a droplet of the target volume in the coding unit Q, and then the droplet is marked by the target optical combination in the coding unit Q, and the marked droplet is sent to the detection area for photoelectric testing. If the obtained photoelectric signal result matches the photoelectric signal characteristic corresponding to the coding unit Q, it indicates that the targeted molecule Q exists in the sample liquid to be detected, and the sample liquid Q to be detected is identified as positive.

[0039] In a preferred embodiment, performing photoelectric testing on all the first candidate droplets to obtain the photoelectric signal characteristics of each of the first candidate droplets specifically includes the following steps: A photoelectric test is performed on each of the first candidate droplets under a dark field to obtain a photoelectric signal characteristic of each of the first candidate droplets under a dark field; the photoelectric signal characteristic under a dark field is related to the type of fluorescent probe dye.

[0040] In a preferred embodiment, the photoelectric signal characteristics include at least photoelectric intensity; The step of obtaining the photoelectric signal discrimination of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets specifically comprises the following steps: The photoelectric intensity of each of the first candidate droplets under a dark field is used as the photoelectric signal discrimination degree of each of the first candidate droplets.

[0041] Specifically, place the first candidate droplet samples one by one in the dark field detection area, turn on the dark field excitation light source, make the fluorescent probe dye in the droplet generate fluorescence, collect and convert the fluorescent signal into an electrical signal, and record the dark field photoelectric intensity value corresponding to each droplet. For example, for the first sample of the first candidate droplet with a target volume of 12μm³ and marked as the target optical combination 13 (blue visible light color label, fluorescent probe dye A), the first sample with a target volume of 12μm³ and marked as the candidate optical combination 11 (red visible light color label, fluorescent probe dye A), the third sample with a target volume of 12μm³ and marked as the candidate optical combination 13 (blue visible light color label, fluorescent probe dye A)... respectively, detect them and record the dark field photoelectric intensity value (unit: mV) of each sample.

[0042] This embodiment only considers the dark field detection method, which provides a simpler method for obtaining the photoelectric signal discrimination of each of the first candidate droplets.

[0043] In a preferred embodiment, performing photoelectric testing on all the first candidate droplets to obtain the photoelectric signal characteristics of each of the first candidate droplets specifically includes the following steps: Performing photoelectric tests on each of the first candidate droplets in a dark field and a bright field, respectively, to obtain a dark field feature of an optical signal of each of the first candidate droplets in a dark field and a bright field feature of an optical signal of each of the first candidate droplets in a bright field; the dark field feature is related to the type of the fluorescent probe dye, and the bright field feature is related to the color of the dye; Based on the dark field feature and the bright field feature of each of the first candidate droplets, a photoelectric signal feature of each of the first candidate droplets is obtained.

[0044] In a preferred embodiment, the dark field characteristics at least include dark field photoelectric intensity, and the bright field characteristics at least include bright field photoelectric intensity; The step of obtaining the photoelectric signal discrimination of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets specifically comprises the following steps: Assigning corresponding dark field weights and bright field weights to the dark field photoelectric intensity and bright field photoelectric intensity respectively; The photoelectric signal discrimination degree of each first candidate droplet is obtained according to the dark field photoelectric intensity and the dark field weight of each first candidate droplet, and the bright field photoelectric intensity and the bright field weight.

[0045] Specifically, each first candidate droplet sample is placed in the bright field detection area again, and under the irradiation of a stable bright field light source, the light signals generated by the droplets due to reflection, transmission, etc. are collected and converted into electrical signals, and the bright field photoelectric intensity value corresponding to each droplet is recorded. According to the actual application requirements and the importance of dark field and bright field photoelectric intensities in distinguishing droplets, corresponding dark field weights and bright field weights are assigned to the dark field photoelectric intensity and the bright field photoelectric intensity, respectively. For example, it is believed that the dark field photoelectric intensity is more critical for distinguishing droplets with different labels, so the dark field weight is set to 0.6 and the bright field weight is set to 0.4 (the total weight is 1). For each first candidate droplet, the sum of the product of its dark field photoelectric intensity and dark field weight and the product of the bright field photoelectric intensity and bright field weight is used as the photoelectric signal discrimination degree.

[0046] Exemplarily, the calculated photoelectric signal discrimination of the first candidate droplet of each candidate optical combination marker is compared with a first preset threshold (assuming that the first preset threshold is set to 60). If the photoelectric signal discrimination of the droplet of a candidate optical combination marker is greater than or equal to 60, then the candidate optical combination can be determined as the target optical combination.

[0047] Based on the color label and the fluorescent probe dye with the optimal wavelength as the optimal probe dye, this scheme also adds the volume coding of the droplet to the bright field color-fluorescent probe coding to form a super multiplex coding of digital droplet PCR. Following the above idea, the volume information of the droplet, the bright field visible light color information, and the dark field fluorescence information are combined to construct a high-dimensional information matrix, which can be addressable for the droplet. Similarly, according to the logical relationship between the above encoding process and the actual PCR reaction, it can be decoded. In this method, it can be seen that the droplets generated by this encoding method have a total amount of information. The information load of the same droplet includes three dimensions of "bright field visible light + dark field fluorescent probe dye + multiple volumes", and the droplets generated in this way have ultra-high information density and load. Functionally, this super multiplex coding method of digital droplet PCR has both multiple fluorescence and multiple volume digital PCR detection functions, which increases the amount of information contained in the droplets to a certain extent, reduces the detection cost, and is expected to promote the development of instruments in the corresponding medical field.

[0048] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A multiple encoding method for digital droplet PCR, characterized in that: The following steps are involved: Acquire at least one target volume and a candidate optical set; the candidate optical set includes a plurality of candidate optical combinations, each of which includes a visible light color label and a fluorescent probe dye type; each of the visible light color labels corresponds to a dye color; the target volume is a droplet volume whose photoelectric signal intensity of the corresponding droplet is in a first intensity interval; Matching each of the candidate optical combinations to each of the target volumes, respectively, to obtain a plurality of first candidate droplets corresponding to each of the target volumes and marked by each of the candidate optical combinations, wherein the first candidate droplets contain target molecules; Performing photoelectric testing on all the first candidate droplets respectively to obtain a photoelectric signal feature of each of the first candidate droplets; According to the photoelectric signal characteristics of each of the first candidate droplets, the photoelectric signal discrimination of each of the first candidate droplets is obtained, and then the target optical combination corresponding to each of the target volumes is obtained, the target optical combination being a candidate optical combination whose photoelectric signal discrimination is greater than or equal to a first preset threshold; The target volume and the target optical combination corresponding thereto are used as a group of coding units, and the photoelectric signal characteristics corresponding to each group of coding units are used as target molecule recognition characteristics under the coding unit.

2. The multiple encoding method for digital droplet PCR according to claim 1, characterized in that: The obtaining of at least one target volume specifically comprises the following steps: Acquire a candidate volume set, where the candidate volume set includes a plurality of candidate volumes; Acquire a second candidate droplet corresponding to each of the candidate volumes, and perform a photoelectric test on each of the second candidate droplets to obtain a photoelectric signal intensity of each of the second candidate droplets; A number of target candidate droplets whose photoelectric signal intensities are within the first intensity range are screened out from all the second candidate droplets, and the candidate volume corresponding to each target candidate droplet is used as the target volume.

3. The multiple encoding method for digital droplet PCR according to claim 1, characterized in that: The step of matching each of the candidate optical combinations to each of the target volumes to obtain a plurality of first candidate droplets corresponding to each of the target volumes and marked by the candidate optical combinations specifically includes the following steps: Acquire a marker set for each target volume, wherein the marker set includes a plurality of candidate optical combinations, each group of candidate optical combinations includes a fluorescent probe dye type and a plurality of dye colors corresponding to each group of fluorescent probe dye types; Obtain reference droplets of the target volume, and mark each of the reference droplets with a set of the fluorescent probe dye type and the dye color in the marking set to obtain a plurality of the first candidate droplets; each of the first candidate droplets is marked with a different fluorescent probe dye type and a different dye color.

4. The multiple encoding method for digital droplet PCR according to claim 1, characterized in that: The performing photoelectric testing on all the first candidate droplets respectively to obtain the photoelectric signal characteristics of each of the first candidate droplets specifically includes the following steps: A photoelectric test is performed on each of the first candidate droplets under a dark field to obtain a photoelectric signal characteristic of each of the first candidate droplets under a dark field; the photoelectric signal characteristic under a dark field is related to the type of fluorescent probe dye.

5. The multiple encoding method for digital droplet PCR according to claim 4, characterized in that: The photoelectric signal characteristics at least include photoelectric intensity; obtaining the photoelectric signal discrimination degree of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets; The specific steps include: The photoelectric intensity of each of the first candidate droplets under a dark field is used as the photoelectric signal discrimination degree of each of the first candidate droplets.

6. The multiple encoding method for digital droplet PCR according to claim 1, characterized in that: The performing photoelectric testing on all the first candidate droplets respectively to obtain the photoelectric signal characteristics of each of the first candidate droplets specifically includes the following steps: Performing photoelectric tests on each of the first candidate droplets in a dark field and a bright field, respectively, to obtain a dark field feature of an optical signal of each of the first candidate droplets in a dark field and a bright field feature of an optical signal of each of the first candidate droplets in a bright field; the dark field feature is related to the type of the fluorescent probe dye, and the bright field feature is related to the color of the dye; Based on the dark field feature and the bright field feature of each of the first candidate droplets, a photoelectric signal feature of each of the first candidate droplets is obtained.

7. The multiple encoding method for digital droplet PCR according to claim 6, characterized in that: The dark field feature at least includes dark field photoelectric intensity, and the bright field feature at least includes bright field photoelectric intensity; obtaining the photoelectric signal discrimination degree of each of the first candidate droplets according to the photoelectric signal characteristics of each of the first candidate droplets; The specific steps include: Assigning corresponding dark field weights and bright field weights to the dark field photoelectric intensity and bright field photoelectric intensity respectively; The photoelectric signal discrimination degree of each first candidate droplet is obtained according to the dark field photoelectric intensity and the dark field weight of each first candidate droplet, and the bright field photoelectric intensity and the bright field weight.

8. The multiple encoding method for digital droplet PCR according to claim 1, characterized in that: After the target volume and the target optical combination corresponding thereto are used as a group of coding units, and the photoelectric signal characteristics corresponding to each group of coding units are used as the target molecule recognition characteristics under the coding unit, the following steps are also included: All the coding units and the target molecule recognition features under each coding unit are stored in a coding database.

9. The multiple encoding method for digital droplet PCR according to claim 2, characterized in that: Acquiring a second candidate droplet corresponding to each of the candidate volumes through a microfluidic chip, the method further comprising constructing a flow channel structure of the microfluidic chip; The construction of the flow channel structure of the microfluidic chip specifically includes the following steps: Constructing a benchmark physical model, and using the benchmark physical model to simulate the sheath flow step emulsification process of the oil-water two-phase to obtain simulation results, wherein the simulation results at least include the flow states of the oil-water two-phase at different times and different positions; Based on the simulation results, key information related to the generation of target droplets by the oil-water two-phase is obtained; According to the key information, structural parameters are obtained, wherein the structural parameters at least include a flow channel shape; According to the structural parameters, a flow channel structure of a microfluidic chip is constructed.

10. The multiple encoding method for digital droplet PCR according to claim 9, characterized in that: The method of obtaining the second candidate droplet corresponding to each of the candidate volumes by using the microfluidic chip specifically comprises the following steps: The second candidate droplet corresponding to each candidate volume is obtained by controlling the flow rate of the fluid in the flow channel structure of the microfluidic chip.

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