CN108841939B - Multi-digital PCR concentration measuring method and micro-drop type digital PCR system - Google Patents

Multi-digital PCR concentration measuring method and micro-drop type digital PCR system Download PDF

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CN108841939B
CN108841939B CN201810642098.9A CN201810642098A CN108841939B CN 108841939 B CN108841939 B CN 108841939B CN 201810642098 A CN201810642098 A CN 201810642098A CN 108841939 B CN108841939 B CN 108841939B
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李昂
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Beijing Zhiyu Bio Tech Co ltd
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Abstract

The invention discloses a multiple digital PCR concentration measuring method and a micro-drop type digital PCR system, which are suitable for simultaneously detecting the concentration of two or more target spots in a PCR reaction sample, wherein different target spots are marked by different fluorescent groups, and the concentration measuring method comprises the following steps: corresponding to each fluorophore, determining a filtering bandwidth range to determine a corresponding channel unit, and detecting the luminous intensity of each channel unit; obtaining the luminescence contribution ratio of each fluorophore in the channel unit according to the luminescence spectrogram of the fluorophore and the filter bandwidth range corresponding to the fluorophore; and obtaining the concentration of each fluorescent group according to the luminous intensity of the channel unit and the luminous contribution ratio of each fluorescent group in the channel unit. The invention provides a PCR concentration calculation method, which effectively reduces 'gray points' observed in channels through algorithm correction and corrects fluorescence crosstalk among the channels caused by multiple target points.

Description

Multi-digital PCR concentration measuring method and micro-drop type digital PCR system
Technical Field
The invention relates to the technical field of PCR, in particular to a multi-digital PCR concentration measuring method and a micro-drop type digital PCR system.
Background
In the current digital PCR technology, samples are divided into a large number of micro reaction chambers with nanoliter volumes in some way, then PCR cycles are carried out on the micro reaction chambers simultaneously, and when PCR amplification enters a saturation period, an end-point observation method is adopted to distinguish the result of each reaction chamber as a negative reaction or a positive reaction. In practical applications, two or more targets (amplification targets) in a sample are usually detected at the same time, so that different fluorophores (which fluoresce in a specific wavelength band after excitation) are used to label different targets. In order to distinguish fluorophores of different targets, two or more fluorescence band channels are generally used to detect each fluorophore separately.
The ideal design would be to detect only one fluorophore per detection channel. In practice, the emission bands of fluorophores are difficult to avoid from overlapping, as shown in fig. 1, the emission band of the HEX fluorophore is almost covered by the emission band of the FAM fluorophore, so that no matter how to optimize the central wavelength and bandwidth of the detection channel, each channel will detect different fluorophores, which is called fluorescence crosstalk between different channels. This crosstalk can greatly interfere with the negative and positive determinations of the reaction chamber in different observation channels. In addition, the reaction of different target spots in the same reaction chamber can form competition, so that the reaction efficiency of a single target spot is influenced, the end point observation of a fluorescent group for marking the target spot is further influenced, and the negative and positive judgment errors are caused.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a multiple digital PCR concentration measurement method, which overcomes the accuracy of the measurement of the concentration of a fluorescent group caused by a terminal point observation method and fluorescence crosstalk, and adopts the following technical scheme:
in one aspect, the present invention provides a method for measuring the concentration of multiple digital PCR reactions, which is suitable for simultaneously detecting the concentration of two or more targets in a PCR reaction sample, wherein different targets are labeled with different fluorophores, the method for measuring the concentration comprises:
corresponding to each fluorophore, determining a filtering bandwidth range to determine a corresponding channel unit, and detecting the luminous intensity of each channel unit;
obtaining the luminescence contribution ratio of each fluorophore in the channel unit according to the luminescence spectrogram of the fluorophore and the filter bandwidth range corresponding to the fluorophore;
and obtaining the concentration of each fluorescent group according to the luminous intensity of the channel unit and the luminous contribution ratio of each fluorescent group in the channel unit.
Further, the obtaining the concentration of each fluorophore according to the luminous intensity of the channel unit and the luminous contribution ratio of each fluorophore in the channel unit includes:
the luminous intensity of each fluorophore was obtained by the following formula:
INTENSITYi＝a1i×c1+a2i×c2+......+aji×cjwherein the number of target points and the number of types of fluorophores used for marking the target points are j, i is a channel label, INTENSITYiTo detect the intensity of the emitted light of the ith channel, c1Is the luminous intensity of the first fluorescent group，c2Is the luminous intensity of the second fluorophore, cjThe luminous intensity of the jth fluorophore, a1iAs the ratio of the luminescence contribution of the first fluorophore in the ith channel, a2iThe ratio of the contribution of the luminescence of the second fluorophore in the ith channel, ajiFor the luminescent contribution ratio of the jth fluorophore in the ith channel, i is 2. ltoreq. j.
Further, after obtaining the luminous intensity of each fluorescent group, the method further comprises the following steps:
and obtaining the concentration corresponding to the luminous intensity of the fluorescent group according to a pre-established relation model between the concentration of the fluorescent group and the luminous intensity.
Further, before determining the filter bandwidth range for the fluorophore, the method further comprises: selecting fluorescent groups corresponding to different marked targets, wherein the coincidence degree of the wavelength ranges of the fluorescent groups is larger than a preset first threshold value, and/or the wavelength difference value corresponding to the emission peak value of the fluorescent groups is larger than a preset second threshold value.
Further, fluorophores used for labeling different targets include, but are not limited to, FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, LC RED640, and other fluorophores, each of which is labeled with an emission spectrum.
Further, the ratio of the luminescence contribution of each fluorophore in the channel unit is obtained by:
calculating the integral of different fluorophore emission spectrograms in the corresponding filter bandwidth range of the same channel unit to serve as the light emission contribution of the corresponding fluorophores in the channel unit;
dividing the luminescence contribution of one of the fluorophores in the channel unit by the sum of the luminescence contributions of all the fluorophores in the channel unit to obtain the luminescence contribution ratio of the fluorophore in the channel unit.
Further, said determining a filter bandwidth range for each fluorophore comprises:
correspondingly selecting filtering devices with different frequency bandwidths according to the wavelength characteristics of all the fluorophores, wherein the frequency bandwidths of the filtering devices cover and uniquely cover the emission peak values of the corresponding fluorophores, and/or the overlapping wavelength ranges of a plurality of fluorophore emission spectra in the frequency bandwidths of the filtering devices are larger than a preset third threshold value;
and taking the selected frequency bandwidth of the filter device as the bandwidth of the channel unit corresponding to the fluorophore.
Further, the detecting the luminous intensity of each channel unit comprises:
filtering the PCR reaction samples one by using the selected filtering devices;
and (3) carrying out light intensity detection on the filtered PCR reaction samples to obtain the luminous intensity of the corresponding channel units one by one.
Further, the number of targets in the PCR reaction sample is two, three or more.
In another aspect, the present invention provides a digital PCR system using the above-mentioned measurement method to achieve quantitative observation of each droplet.
The technical scheme provided by the invention has the following beneficial effects:
a. the 'gray point' observed in the channel is effectively reduced through PCR algorithm correction;
b. correcting fluorescence crosstalk between channels caused by multiple target spots;
c. the concentration measurement of a plurality of targets in the same PCR sample can be realized simultaneously;
d. the bandwidth of the filtering channel is not limited specially, so that the number of channels for multiple detection can be increased and far exceeds the number of traditional channels.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph of the emission spectra of FAM fluorophore and HEX fluorophore;
FIG. 2 is a flow chart of a method for measuring the concentration of multiplex digital PCR according to an embodiment of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above 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 is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, apparatus, article, or device that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or device.
In the end-point observation of multiplex digital PCR, due to various interference factors, the fluorescence signals of many micro-reaction chambers fall within the negative and positive intervals, which are generally referred to as "gray spots" in the industry. Of course, the source of the interference factor is complex and not limited to the above reasons (such as the type and shape of the sample template). The algorithm provided by the invention is used for correcting the inter-channel crosstalk caused by observing multiple targets in the droplet type digital PCR and the interference caused by the reaction competition of different targets in the same droplet.
In one embodiment of the present invention, a multiplex digital PCR concentration measurement method is provided, which is suitable for simultaneously detecting the concentration of two or more target points in a PCR reaction sample, wherein different target points are labeled with different fluorophores, as shown in fig. 2, and the concentration measurement method comprises the following steps:
and S1, determining the filtering bandwidth range corresponding to each fluorophore to determine the corresponding channel unit, and detecting the luminous intensity of each channel unit.
In an embodiment of the present invention, a method for measuring concentrations of three target points is taken as an example for illustration, three fluorophores are selected for the three target points to represent a first target point, a second target point and a third target point respectively, and a first principle of selecting the fluorophores is that the coincidence degree of the wavelength ranges of the fluorophores is greater than a preset first threshold (for example, 100 nm); in another preferred embodiment, the selection of fluorophores follows the following second principle: the difference of the wavelengths corresponding to the emission peaks of the fluorophores is larger than a preset second threshold (such as 30nm), or the two principles are simultaneously followed. The reason and the advantageous effect of these two principles are explained in connection with the following selection of the frequency bandwidth of the filtering means:
the determining of the filtering bandwidth range is actually selecting a filtering device (filter or filter) with a specific frequency bandwidth, and determining a corresponding channel unit with the specific frequency bandwidth (taking the frequency bandwidth of the selected filtering device as the bandwidth of the channel unit corresponding to the fluorophore), as shown in fig. 1, FAM is displayed as a filling domain enclosed by a curve 1 and a horizontal axis, and HEX is displayed as a filling domain enclosed by a curve 2 and a horizontal axis; the rectangular box with A, B as the vertices represents the filter bandwidth range for the first channel, and the rectangular box with E, F as the vertices represents the filter bandwidth range for the second channel. In the conventional multi-channel design, in order to avoid crosstalk interference as much as possible, the channels should not overlap each other as much as possible, that is, the vertex B must be smaller in wavelength than the vertex E, so the number and design of the channels are limited to a great extent. In the method provided by the application, the limitation is not needed, and the bandwidth of the filtering channel is not specially limited, so that the number of channels for multiple detection can be increased and is far more than the number of traditional channels. Based on the first principle, the overlapping wavelength range (overlapping two by two) of the multiple fluorophore emission spectra within the bandwidth of the filter device is greater than the preset third threshold (e.g. 10nm), which is obviously different from the prior art that avoids the overlapping emission bands of the fluorophores as much as possible, in the prior art, the crosstalk generated by multiple fluorophores in the channel is channel noise, which is not favorable for detecting the main fluorophore in the channel, while in the present invention, the overlapping degree of the wavelength ranges of the fluorophores does not play a role in the concentration measurement, because: in the application, the information of multiple fluorophores in the channel belongs to effective information, the first threshold value is set to facilitate setting of the filtering frequency bandwidth of the coincidence wavelength range larger than the third threshold value during filtering, and the third threshold value is set to facilitate calculating the effective information of the corresponding fluorophores in the range, for example, a curve (especially a curve close to an axis) in a 1nm sampling range is integrated, and the error ratio of the integrated result is larger.
Based on the second principle, the frequency bandwidth of the filter device covers and uniquely covers the wavelength corresponding to the emission peak of the corresponding fluorophore, on one hand, information near the wavelength corresponding to the emission peak of the fluorophore can reflect the luminescence information of the fluorophore more than a valley region, and therefore, the frequency bandwidth of the filter device to be selected covers the wavelength corresponding to the emission peak of the corresponding fluorophore; on the other hand, each selected filter corresponds to one fluorophore, that is, although a plurality of fluorophores are involved in each selected frequency bandwidth range, the corresponding fluorophore is a primary fluorophore, and in order to prevent primary and secondary dissimilarity (for example, covering two peak points), the frequency bandwidth of the selected filter is required to uniquely cover the wavelength corresponding to the emission peak of the corresponding fluorophore. In summary, in selecting the filtering bandwidth range, the selection should be within a certain range from the left and right of the corresponding fluorophore emission peak, so that the adjacent peak is not selected.
In the prior art, as shown in table 1, fluorophores used for labeling different targets include, but are not limited to, FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, LC Red640, and other fluorophores (such as CY5, LC Red705, and the like), each fluorophore is labeled with an emission spectrum corresponding to the target, it should be noted that the above is only a part of commonly used fluorophores in the prior art, and does not represent all fluorophores, many different fluorophores from 520 to 800nm can be freely combined, and the selection of the type of the fluorophore is not the inventive point of the present invention, but the idea of selecting the fluorophore and the idea of selecting the filter device are to better detect the concentration of the fluorophore subsequently, and have certain inventive innovations.
TABLE 1
Figure BDA0001702661850000061
The detecting of the luminous intensity of each channel unit specifically comprises: in the experiment fluorescence imaging detection, filtering the PCR reaction samples one by using a selected filtering device; and (3) carrying out light intensity detection on the filtered PCR reaction samples to obtain the luminous intensity of the corresponding channel units one by one. For example, the first filtering device is used for filtering and detecting the light intensity, so as to obtain the luminous intensity INTENSITY of the first channel unit1By analogy, INTENSITY is obtained by detection respectively2And INTENSITY3。
And S2, obtaining the luminous contribution ratio of each fluorophore in the channel unit according to the luminous spectrum of the fluorophore and the filter bandwidth range corresponding to the fluorophore.
As can be seen from fig. 1, the main contribution of AB channel imaging is FAM fluorophore emission spectra, while the contribution of HEX fluorophore emission spectra is also included; the main contribution of EF channel imaging is the HEX fluorophore emission spectrum, while the contribution of FAM fluorophore emission spectrum, i.e. the magnitude of the fluorophore contribution, is not only related to the emission spectrum but also to the bandwidth of the filtering means.
In the imaging detection of the AB channel, the contribution of the FAM fluorophore is the integral of its emission spectrum within the AB channel filter bandwidth (AB), denoted famInAB, and the contribution of the HEX fluorophore is the integral of its emission spectrum within the AB channel filter bandwidth (AB), denoted hexInAB. In the imaging detection of the EF channel, the contribution of the FAM fluorophore is the integral of its luminescence spectrum within the bandwidth of the EF channel filter (EF), denoted as famInEF, and the contribution of the HEX fluorophore is the integral of its luminescence spectrum within the bandwidth of the EF channel filter (EF), denoted as hexInEF. In the absence of the third fluorophore, the contribution ratio of the FAM fluorophore in the AB channel is famInAB/(famInAB + hexInAB), and the contribution ratio of the HEX fluorophore in the AB channel is hexInAB/(famInAB + hexInAB); the contribution ratio of FAM fluorophores in the EF channel is famInEF/(famInEF + hexInEF), and the contribution ratio of HEX fluorophores in the EF channel is hexInEF/(famInEF + hexInEF).
Similarly, in the embodiment of the present invention, taking three kinds of fluorophores as an example and taking the first channel unit as an example for explanation, the light emission contribution ratio of each fluorophore is obtained by the following steps:
calculating the integral of the corresponding calibrated luminescence spectrogram of the three fluorophores in a first channel unit (corresponding to the filtering bandwidth range) to serve as the luminescence contribution of the corresponding fluorophores in the channel unit;
dividing the luminescence contribution of the first fluorophore in the first channel unit by the sum of the luminescence contributions of the three fluorophores in the first channel unit to obtain the luminescence contribution ratio a1 of the first fluorophore in the first channel unit1Similarly, the light emission contribution ratio a2 of the second fluorescent group in the first channel unit is obtained1And the ratio of the luminescence contribution of the third fluorescent group in the first channel unit a31. The same method is used to obtain the light emission contribution ratio a1 of the first fluorescent group in the second channel unit2The ratio of the emission contribution of the second fluorescent group in the second channel unit, a22And the light emission contribution ratio a3 of the third fluorescent group in the second channel unit2(ii) a And the luminous contribution ratio a1 of the first fluorescent group in the third channel unit3The ratio of the luminescence contribution of the second fluorophore in the third channel unit, a23And the luminous contribution ratio a3 of the third fluorophore in the third channel unit3。
And S3, obtaining the concentration of each fluorophore according to the luminous intensity of the channel unit and the luminous contribution ratio of each fluorophore in the channel unit.
Specifically in the three-fluorophore embodiment of the present invention, the following equations are associated:
INTENSITY1＝a11×c1+a21×c2+a31×c3；
INTENSITY2＝a12×c1+a22×c2+a32×c3；
INTENSITY3＝a13×c1+a23×c2+a33×c3(ii) a Wherein c to be solved1Is the luminous intensity of the first fluorophore, c2Is the luminous intensity of the second fluorophore, c3Luminous intensity of the third fluorophore, INTENSITY1、INTENSITY2And INTENSITY3To measure the resulting luminous intensity of each channel, a11、a21、a31、a12、a22、a32、a13、a23And a33As already obtained in the above-mentioned S2, the system of equations is solved to obtain c1、c2And c3I.e. the luminescence intensity of the respective fluorophore.
It should be noted that, when the number of targets (i.e. the number of types of fluorophores) is two or more than three, the method is the same as the method for measuring the concentration of three fluorophores, and the luminous intensity of each fluorophore is obtained by the following formula:
INTENSITYi＝a1i×c1+a2i×c2+......+aji×cjwherein the number of target points and the number of types of fluorophores used for marking the target points are j, i is a channel label, INTENSITYiTo detect the intensity of the emitted light of the ith channel, c1Is the luminous intensity of the first fluorophore, c2Is the luminous intensity of the second fluorophore, cjThe luminous intensity of the jth fluorophore, a1iIn the ith channelThe ratio of the luminescence contribution of the first fluorophore in, a2iThe ratio of the contribution of the luminescence of the second fluorophore in the ith channel, ajiFor the luminescent contribution ratio of the jth fluorophore in the ith channel, i is 2. ltoreq. j. The other difference between the method and the prior art is that in order to prevent fluorescence crosstalk in the prior art, certain errors exist when the concentrations of two fluorescent groups are detected, when three fluorescent groups are detected, large errors can be generated, in the prior art, more than three fluorescent groups can not be measured basically by using a fluorescence labeling detection method, the measuring method has no limit on the number of target spots in a PCR reaction sample, and the light intensity of each fluorescent group can be accurately calculated through accurate calculation. After the correction is carried out by the PCR algorithm provided by the invention, the gray points observed in different channels can be effectively reduced.
And after obtaining the luminous intensity of each fluorescent group, the method further comprises the following steps: and obtaining the concentration corresponding to the luminous intensity of the fluorescent group according to a pre-established relation model between the concentration of the fluorescent group and the luminous intensity. For example, the light intensity of the fluorophore is measured according to the known concentration of the fluorophore, a concentration/light intensity relationship model (concentration/light intensity curve) of the fluorophore is established, and the obtained luminous intensity of the fluorophore is converted into the corresponding concentration according to the relationship model. The concentration of target was calculated by counting the proportion of all negative and positive droplets. The algorithm calculates the concentration of the groups in a specific droplet, and the calculation of the concentration helps us to judge whether the droplet is negative or positive. This application is through the concentration of accurate interior fluorophore of calculation liquid drop, judges whether certain fluorophore in the liquid drop is negative or positive according to group concentration, and this compares with traditional way promptly through the negative/positive of liquid drop in the light intensity judgement certain passageway, and the measuring result of this application has more the reliability.
In another embodiment of the present invention, a digital PCR system is provided, which uses the measurement method described in the above embodiments to achieve quantitative observation of each droplet.
In conclusion, the invention provides a PCR concentration calculation method and a digital PCR system for measuring the concentration by using the algorithm, which effectively reduce the 'grey point' observed in a channel and correct the fluorescence crosstalk among the channels caused by multiple target spots through algorithm correction; in addition, by using the calculation method of the application, the bandwidth of the filtering channel can not be specially limited, so that the number of channels for multiple detection can be increased.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

1. A multiple digital PCR concentration measurement method is characterized in that the method is suitable for simultaneously detecting the concentrations of a plurality of target spots in a micro-droplet PCR reaction sample, the number of the target spots is more than or equal to two, different target spots are marked by different fluorescent groups, and the concentration measurement method comprises the following steps:
corresponding to each fluorophore, determining a filtering bandwidth range to determine a corresponding channel unit, and detecting the luminous intensity of each channel unit; the same microdroplet is provided with a plurality of channel units, crosstalk generated by more than two fluorophores exists in one or more channel units, and the number of the channel units is more than or equal to two;
obtaining the luminescence contribution ratio of each fluorophore in the channel unit according to the luminescence spectrum of the fluorophore and the filter bandwidth range corresponding to the fluorophore, including: calculating the integral of different fluorophore emission spectrograms in the corresponding filter bandwidth range of the same channel unit to serve as the light emission contribution of the corresponding fluorophores in the channel unit; dividing the luminescence contribution of one of the fluorophores in the channel unit by the sum of the luminescence contributions of all the fluorophores in the channel unit to obtain the luminescence contribution ratio of the fluorophore in the channel unit;
obtaining the concentration of each fluorescent group according to the luminous intensity of the channel unit and the luminous contribution ratio of each fluorescent group in the channel unit, and obtaining the luminous intensity of each fluorescent group through the following formula:
INTENSITYi＝a1i×c1+a2i×c2+......+aji×cjwherein the number of target points and the number of types of fluorophores used for marking the target points are j, i is a channel label, INTENSITYiTo detect the intensity of the emitted light of the ith channel, c1Is the luminous intensity of the first fluorophore, c2Is the luminous intensity of the second fluorophore, cjThe luminous intensity of the jth fluorophore, a1iAs the ratio of the luminescence contribution of the first fluorophore in the ith channel, a2iThe ratio of the contribution of the luminescence of the second fluorophore in the ith channel, ajiFor the luminescent contribution ratio of the jth fluorophore in the ith channel, i is 2. ltoreq. j.
2. The multiplex digital PCR concentration measurement method according to claim 1, further comprising, after obtaining the luminescence intensity of each fluorophore:
and obtaining the concentration corresponding to the luminous intensity of the fluorescent group according to a pre-established relation model between the concentration of the fluorescent group and the luminous intensity.
3. The multiplexed digital PCR concentration measurement method of claim 1, further comprising, prior to determining a filter bandwidth range for a fluorophore: selecting fluorophores corresponding to different marked targets, wherein the coincidence degree of the wavelength ranges of the multiple fluorophores is larger than a preset first threshold value, and/or the wavelength difference value corresponding to the emission peak values of the multiple fluorophores is larger than a preset second threshold value.
4. The method for multiplex digital PCR concentration measurement according to claim 1, wherein the fluorophores used for labeling different targets include FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, and LC RED640, and each fluorophore is labeled with an emission spectrum chart.
5. The multiplexed digital PCR concentration measurement method of claim 1, wherein the determining a filter bandwidth range for each fluorophore comprises:
correspondingly selecting filtering devices with different frequency bandwidths according to the wavelength characteristics of all the fluorophores, wherein the frequency bandwidths of the filtering devices cover and uniquely cover the emission peak values of the corresponding fluorophores, and/or the superposition wavelength ranges of a plurality of fluorophores in the frequency bandwidths of the filtering devices are larger than a preset third threshold;
and taking the selected frequency bandwidth of the filter device as the bandwidth of the channel unit corresponding to the fluorophore.
6. The multiplex digital PCR concentration measurement method according to claim 5, wherein the detecting the luminescence intensity of each channel unit comprises:
filtering the PCR reaction samples one by using the selected filtering devices;
and (3) carrying out light intensity detection on the filtered PCR reaction samples to obtain the luminous intensity of the corresponding channel units one by one.
7. The multiplex digital PCR concentration measurement method according to claim 1, wherein the number of target spots in the PCR reaction sample is greater than or equal to two.
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Denomination of invention: Multiple digital PCR concentration measurement method and droplet digital PCR system

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