CN118090562B - Digital analysis method and device based on stream type single molecule detection - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 34
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Abstract
The invention relates to the field of biological data analysis, and discloses a digital analysis method and a digital analysis device based on flow type single-molecule detection, wherein a flow cytometer is adopted to acquire fluorescent signals of a sample after immune reaction, so as to obtain a sample scatter diagram; dividing the sample scatter diagram into at least 2 areas Q3 and Q4 according to the yin-yang threshold; and calculating a fitting parameter AEB according to specific logic, and fitting to obtain a calibration curve for calculating the sample concentration according to the sample concentration and the fitting parameter AEB. The invention expands the analysis range of the flow type single molecule detection technology based on the existing method for single molecule analysis by adopting the flow type detection technology, ensures that the stability of the detection result is better, and establishes a new algorithm, namely a formula through the deduction of the algorithm; meanwhile, a method for establishing a flow type single-molecule negative and positive judgment threshold value through data calculation is also established.
Description
Technical Field
The invention relates to the field of biological data analysis, in particular to a digital analysis method and device based on stream type single molecule detection.
Background
The single molecule immune analysis technology is to convert the whole immune detection signal into discrete signal, utilize immune reaction under low concentration, make the single molecule of reaction accord with poisson's distribution concept in the whole molecule, carry on certain formula conversion, get the quantity of actually taking place to react, then carry on the curve fitting with concentration of the labeling substance of the number of reaction, reach the purpose of being able to determine the sample of unknown concentration through the curve fitted.
The algorithm is published, and the specific calculation method of "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations", published by Nature in 2010 is as follows:
1. The positive microsphere ratio was below 70% and the concentration was quantified by microsphere counting and poisson statistics.
2. When the proportion of positive microspheres is above 70%, the poisson curve deviates from linearity, and enzyme molecules are quantified by measuring the average fluorescence intensity of the microspheres, which is called a 'simulation' method.
3. The concentration of the calibrator and AEB were fitted to a calibration curve for calculation of sample concentration.
The method is based on space isolation, and in a tiny space, the reacted and unreacted signals can be well distinguished.
However, if the single molecule method is implemented in the flow technique, the immune reaction needs to be limited to each reaction carrier, and since the signal of each reaction carrier is not a fixed size, but is within a range, the positive carrier and the negative carrier cannot be strictly distinguished by using the cross gate, so that the sensitivity and the repeatability of the actual detection result are affected.
Disclosure of Invention
The invention mainly aims to solve the technical problem that the conventional algorithm in the prior art cannot strictly distinguish positive vectors from negative vectors. A digital analysis method based on stream type single molecule detection comprises the following steps:
Obtaining a fluorescence signal of a sample after immune reaction by adopting a flow cytometer to obtain a sample scatter diagram;
Dividing the sample scatter diagram into at least 2 areas Q3 and Q4 according to the yin-yang judging threshold;
The fitting parameters AEB are calculated according to the following logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang)
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
and fitting according to the sample concentration and the fitting parameter AEB to obtain a calibration curve for calculating the sample concentration.
The second aspect of the present invention provides a digital analysis device based on stream type single molecule detection, comprising:
the acquisition unit is used for acquiring fluorescent signals of the sample after immune response by adopting a flow cytometer to obtain a sample scatter diagram;
the region dividing unit is used for dividing the sample scatter diagram into at least 2 regions Q3 and Q4 according to the yin-yang judging threshold value;
an AEB calculation unit for calculating a fitting parameter AEB according to the logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang)
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
And the curve fitting unit is used for fitting to obtain a calibration curve for calculating the sample concentration according to the sample concentration and the fitting parameter AEB.
A third aspect of the present invention provides an electronic device, comprising: a memory and at least one processor, the memory having instructions stored therein, the memory and the at least one processor being interconnected by a line; the at least one processor invokes the instructions in the memory to cause the electronic device to perform the above-described digital analysis method based on streaming single molecule detection.
A fourth aspect of the invention provides a computer readable storage medium having instructions stored therein which, when run on a computer, cause the computer to perform the above described method of digitised analysis based on streaming single molecule detection.
The invention has the following beneficial effects:
The invention expands the analysis range of the flow type single molecule detection technology based on the existing method for single molecule analysis by adopting the flow type detection technology, ensures that the stability of the detection result is better, and establishes a new algorithm, namely a formula through the deduction of the algorithm; meanwhile, a method for establishing a flow type single-molecule negative and positive judgment threshold value through data calculation is also established.
The invention establishes an algorithm for streaming single molecules, and when negative and positive are judged through a threshold value, reaction data of a part lower than the threshold value is also included into the algorithm through calculation;
Meanwhile, the streaming technology mostly adopts a method of manually delineating the threshold value to cause unstable factors, and the invention also establishes an algorithm for establishing the streaming single-molecule negative-positive judgment threshold value through data calculation.
Drawings
FIG. 1 is a flow cytometer sample scatter plot.
Detailed Description
The terms "first," "second," "third," "fourth," and the like in the description and in the claims and in the above drawings, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments described herein may be implemented in other sequences than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus.
Flow Cytometry (FCM)
Flow cytometry is used to count and sort small particles suspended in a fluid. Specifically, it is a technique for realizing high-speed, cell-by-cell quantitative analysis and sorting of single cells or other biological particles in suspension by detecting fluorescent signals of markers. The flow cytometry can carry out multiparameter and rapid quantitative analysis on single cells or other biological particles through monoclonal antibodies on the cellular molecular level, and has the advantages of high speed, high precision and good accuracy.
As one embodiment of the present invention, a digital analysis method based on stream single molecule detection includes the steps of:
Obtaining a fluorescence signal of a sample after immune reaction by adopting a flow cytometer to obtain a sample scatter diagram;
Dividing the sample scatter diagram into at least 2 areas Q3 and Q4 according to the yin-yang judging threshold;
The fitting parameters AEB are calculated according to the following logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang)
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
and fitting according to the sample concentration and the fitting parameter AEB to obtain a calibration curve for calculating the sample concentration.
Specifically:
1. Flow cytometer obtaining fluorescent signal and sample scatter diagram
First, the immunoreactive samples were prepared as suspensions suitable for flow cytometry detection. The sample is then passed through a flow cytometer in which the cells or particles emit a fluorescent signal as they pass through the laser beam. These fluorescent signals are captured by the instrument and converted into electrical signals, thereby generating a scattergram of the sample. Each point on the scatter plot represents a cell or particle whose position is determined by the intensity of the fluorescent signal and the particle size/surface complexity.
2. Dividing regions according to yin-yang determination threshold
Next, as shown in fig. 1, the sample scattergram is divided into two areas Q3 and Q4 according to a preset yin-yang determination threshold. These thresholds are typically determined based on the experimental background fluorescence, the fluorescence intensity of the negative control and the positive control. The Q3 region generally represents a positive event (i.e., a cell or particle with a particular fluorescent signal), while the Q4 region may represent other types of events or background noise.
3. Calculating fitting parameters AEB
And calculating fitting parameters AEB by adopting the algorithm according to the proportion of all carriers in the Q3 region to all carriers.
When the positive rate of the Q3 region is 2% -10%, the fluorescence signal intensity in this range can generally better distinguish between positive and negative, while also ensuring a sufficient sample size to calculate the average fluorescence intensity. Such a selection helps to ensure the stability and accuracy of I Yang (Yang) , thereby reflecting the fluorescence characteristics of positive events more accurately.
4. Fitting a calibration curve
And finally, fitting according to the sample concentration and the calculated fitting parameter AEB to obtain a calibration curve for calculating the sample concentration. The calibration curve describes the relationship between sample concentration and AEB for accurate determination of sample concentration in subsequent experiments.
Through the flow, the fluorescence signal of the sample after immune reaction is obtained by using a flow cytometer, and the sample concentration is obtained through data analysis. Such information is of great importance for biological research and medical diagnostics.
According to the average fluorescence intensity of the to-be-detected object at zero concentration, the average fluorescence intensity when the positive rate of the Q3 area is 2% -10%; the proportion of all vectors in the Q4 region to all vectors; the proportion of all vectors in the Q3 region to all vectors; testing by a flow cytometer to obtain the average fluorescence intensity of the Q4 region; the flow cytometer tests to obtain the average fluorescence intensity of the Q3 region, calculates the fitting parameter AEB, is closer to the true value compared with the prior art,
As a preferred embodiment, the method for calculating the yin-yang determination threshold value includes:
Obtaining the fluorescence intensity of all carriers under a zero-concentration sample to be detected;
Calculating standard deviation and average value of fluorescence intensity of all carriers under a zero-concentration sample to be detected;
the negative-positive determination threshold C is calculated by the following formula:
C=I0+K×SD
Wherein I 0 is the average value of fluorescence intensity when no reaction occurs on all carriers, SD is the standard deviation of fluorescence intensity of all carriers under a zero-concentration sample to be detected, K is a coefficient, and the value is 2-3.
The initial positive rate P 0 ranged from 2% to 10%. There may be different selection methods of the initial positive rate P 0 for different reaction systems, and as a preferred embodiment, the specific value of the initial positive rate P 0 is determined experimentally.
For easy understanding, the following describes a specific flow of an embodiment of the present invention, and a first embodiment of a digital analysis method based on stream single molecule detection in the embodiment of the present invention includes:
Firstly preparing to-be-detected objects with different concentrations (including zero concentration), carrying out immune reaction on the to-be-detected objects and the carriers coated with the corresponding antibodies, and respectively detecting the to-be-detected objects and the carriers by using a flow analyzer, wherein the obtained data at least comprise the proportion of negative carriers, the average fluorescence intensity, the proportion of positive carriers, the average fluorescence intensity and the like in a to-be-detected sample;
Obtaining a fluorescence signal of a sample after immune reaction by adopting a flow cytometer to obtain a sample scatter diagram;
Dividing the sample scatter diagram into at least 2 areas Q3 and Q4 according to the yin-yang judging threshold;
The fitting parameters AEB are calculated according to the following logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang)
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
and fitting according to the sample concentration and the fitting parameter AEB to obtain a calibration curve for calculating the sample concentration.
The calculation method of the yin-yang judgment threshold value comprises the following steps:
Obtaining the fluorescence intensity of all carriers under a zero-concentration sample to be detected;
Calculating standard deviation and average value of fluorescence intensity of all carriers under a zero-concentration sample to be detected;
the negative-positive determination threshold C is calculated by the following formula:
C=I0+K×SD
wherein, I 0 is the average value of fluorescence intensity when no reaction occurs on all carriers, SD is the standard deviation of fluorescence intensity of all carriers under the sample to be measured with zero concentration, K is the coefficient, and the value of the sample is 2.
The range of initial positive rate P 0 was 5%.
Comparative data 1
The results of 10 replicates at 20pg/ml sample concentration were compared, the average fluorescence intensity in the Q3 region at 5% positive rate for I Yang (Yang) in this example was 3150, and the average fluorescence intensity in the Q4 region at zero concentration for I Yin type vagina in this example was 1580, P compensation was calculated using equation ①②, AEB was calculated using equation ③, AEB correction was calculated using equation ④, and the specific results are shown in Table 1.
TABLE 1
As can be seen from this example, the reproducibility obtained with the new algorithm is better at a sample concentration of 20 pg/ml.
Comparative data 2
The results of 10 replicates at 50pg/ml sample concentration were compared, in this example, the average fluorescence intensity of the Q3 region at 5% positive rate for I Yang (Yang) , the average fluorescence intensity of the Q4 region for zero concentration sample for I Yin type vagina , in this example, the median value was 856, P compensation was calculated using equation ①②, AEB was calculated using equation ③, AEB correction was calculated using equation ④, and the specific results are shown in Table 2.
TABLE 2
As can be seen from this example, the reproducibility obtained with the new algorithm is better at a sample concentration of 50 pg/ml.
TABLE 3 Table 3
See table 3. In this example, when repeated detection is performed by using various sample concentrations, the repeatability obtained by using the new algorithm is better than that of the existing algorithm, but when the concentration is higher, the advantage of the new algorithm becomes smaller, and the reason is that the percentage of the Q4 region is smaller than 1%, and the compensation effect on the whole data becomes smaller accordingly.
The above description is made on the digital analysis method based on the flow type single molecule detection in the embodiment of the present invention, and the following description is made on the digital analysis device based on the flow type single molecule detection in the embodiment of the present invention, where the first embodiment of the digital analysis device based on the flow type single molecule detection in the embodiment of the present invention includes:
the acquisition unit is used for acquiring fluorescent signals of the sample after immune response by adopting a flow cytometer to obtain a sample scatter diagram;
the region dividing unit is used for dividing the sample scatter diagram into at least 2 regions Q3 and Q4 according to the yin-yang judging threshold value;
an AEB calculation unit for calculating a fitting parameter AEB according to the logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang)
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
And the curve fitting unit is used for fitting to obtain a calibration curve for calculating the sample concentration according to the sample concentration and the fitting parameter AEB.
An electronic device provided by embodiments of the present invention may vary considerably in configuration or performance, and may include one or more processors (central processing units, CPUs) (e.g., one or more processors) and memory, one or more storage media (e.g., one or more mass storage devices) that store applications or data. The memory and storage medium may be transitory or persistent. The program stored on the storage medium may include one or more modules (not shown), each of which may include a series of instruction operations in the electronic device. Still further, the processor may be configured to communicate with a storage medium and execute a series of instruction operations in the storage medium on an electronic device.
The electronic device may also include one or more power supplies, one or more wired or wireless network interfaces, one or more input/output interfaces, and/or one or more operating systems, such as Windows Serve, mac OS X, unix, linux, freeBSD, and the like. It will be appreciated by those skilled in the art that the electronic device structure of the present invention is not limited to electronic devices, and may include more or fewer components than shown, or may be combined with certain components, or may have different arrangements of components.
The present invention also provides a computer readable storage medium, which may be a non-volatile computer readable storage medium, and may also be a volatile computer readable storage medium, in which instructions are stored which, when executed on a computer, cause the computer to perform the steps of a digital analysis method based on stream single molecule detection.
It will be clearly understood by those skilled in the art that, for convenience and brevity of description, the specific working process of the system or apparatus and unit described above may refer to the corresponding process in the foregoing method embodiment, which is not repeated herein.
The integrated units, if implemented in the form of software functional units and sold or used as stand-alone products, may be stored in a computer readable storage medium. Based on such understanding, the technical solution of the present invention may be embodied essentially or in part or all of the technical solution or in part in the form of a software product stored in a storage medium, including instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the method according to the embodiments of the present invention. And the aforementioned storage medium includes: a U-disk, a removable hard disk, a read-only memory (ROM), a random access memory (random access memory, RAM), a magnetic disk, or an optical disk, or other various media capable of storing program codes.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (4)
1. A digital analysis method based on stream type single molecule detection, characterized in that the method comprises the following steps:
Obtaining a fluorescence signal of a sample after immune reaction by adopting a flow cytometer to obtain a sample scatter diagram;
Dividing the sample scatter diagram into at least 2 areas Q3 and Q4 according to the yin-yang judging threshold;
The fitting parameters AEB are calculated according to the following logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)；
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)；
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang) ;
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
fitting to obtain a calibration curve for calculating the sample concentration according to the sample concentration and fitting parameters AEB;
The calculation method of the yin-yang judgment threshold value comprises the following steps:
Obtaining the fluorescence intensity of all carriers under a zero-concentration sample to be detected;
Calculating standard deviation and average value of fluorescence intensity of all carriers under a zero-concentration sample to be detected;
the negative-positive determination threshold C is calculated by the following formula:
C=I0+K×SD；
wherein, I 0 is the average value of fluorescence intensity when all carriers do not react, SD is the standard deviation of fluorescence intensity of all carriers under a zero concentration sample to be detected, K is a coefficient, and the value is 2-3;
The initial positive rate P 0 ranged from 2% to 10%.
2. A digital analysis device based on flow type single molecule detection, characterized in that the device comprises:
the acquisition unit is used for acquiring fluorescent signals of the sample after immune response by adopting a flow cytometer to obtain a sample scatter diagram;
the region dividing unit is used for dividing the sample scatter diagram into at least 2 regions Q3 and Q4 according to the yin-yang judging threshold value;
an AEB calculation unit for calculating a fitting parameter AEB according to the logic:
when P Q3 is smaller than the initial positive rate P 0, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln(1-PQ3)；
When P Q3 is between the initial positive rate P 0 and 70%, the calculation formula of the fitting parameter AEB is:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]-ln(1-PQ3)；
When P Q3 is greater than 70%, the calculation formula of the fitting parameter AEB is as follows:
AEB=-ln[1-(I to be measured -I Yin type vagina )/(I Yang (Yang) -I Yin type vagina )×PQ4]+PQ3×IQ3/I Yang (Yang) ;
wherein, I Yin type vagina is the average fluorescence intensity of the object to be detected at zero concentration, and I Yang (Yang) is the average fluorescence intensity when the positive rate of the Q3 region is 2% -10%; p Q4 is the proportion of all vectors in the Q4 region to all vectors; p Q3 is the proportion of all vectors in the Q3 region to all vectors; i Q4 is the average fluorescence intensity of the Q4 region obtained by flow cytometry test; i Q3 is the average fluorescence intensity of the Q3 region obtained by flow cytometry test;
The curve fitting unit is used for fitting to obtain a calibration curve for calculating the sample concentration according to the sample concentration and fitting parameters AEB;
The calculation method of the yin-yang judgment threshold value comprises the following steps:
Obtaining the fluorescence intensity of all carriers under a zero-concentration sample to be detected;
Calculating standard deviation and average value of fluorescence intensity of all carriers under a zero-concentration sample to be detected;
the negative-positive determination threshold C is calculated by the following formula:
C=I0+K×SD；
wherein, I 0 is the average value of fluorescence intensity when all carriers do not react, SD is the standard deviation of fluorescence intensity of all carriers under a zero concentration sample to be detected, K is a coefficient, and the value is 2-3;
The initial positive rate P 0 ranged from 2% to 10%.
3. An electronic device comprising a memory and at least one processor, the memory having instructions stored therein, characterized by:
The at least one processor invokes the instructions in the memory to cause the electronic device to perform the steps of the digital analysis method based on streaming single molecule detection as claimed in claim 1.
4. A computer readable storage medium having instructions stored thereon, which when executed by a processor, implement the steps of the stream single molecule detection based digital analysis method of claim 1.
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