Multispectral digital PCR detection method and device

Abstract

The present invention discloses a multi-spectrum digital PCR detection method and device. The method includes the following steps: S1. Select n′ dyes that meet the dye spillage performance requirements from n dyes according to the number of待测靶标待翻译; S2. Match and label the dyes with the待测靶标待翻译, and then construct an n′-plex digital PCR reaction system with the待测样品待翻译 and the reaction reagents required for PCR amplification, and perform PCR amplification; S3. Collect spectral data, obtain the fingerprint spectrum data matrix of all droplets, and then obtain the abundance vector of all fluorescent dyes in each droplet through unmixing operation; S4. Calculate the concentrations of the n′待测靶标待翻译 in the待测样品待翻译 respectively. The present invention has a good discrimination effect on fluorescent dyes with similar emission wavelengths, improves the multiplex analysis ability of digital PCR, and can be applied to fields such as pathogen detection, tumor detection, and single-cell sequencing. It should be noted that the terms "待测靶标" and "待测样品" need to be accurately defined in the context for more precise translation. You can provide the specific accurate terms for a more perfect translation.

Description

Technical Field

[0001] The present invention relates to the technical field of digital PCR, and in particular to a multi-spectral digital PCR detection method and device. Background Art

[0002] Digital PCR is a new generation of quantitative PCR analysis technology that has developed rapidly in recent years. It uses microfluidics technology to disperse a large amount of diluted nucleic acid solution into the microreactor of the chip, and the number of nucleic acid templates in each reactor is less than or equal to 1. In this way, after the PCR cycle, the reactor with at least one nucleic acid molecule template will give a fluorescent signal, and the reactor without a template will not have a fluorescent signal. According to the relative proportion and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated. Compared with traditional fluorescent quantitative PCR, it has the advantages of high sensitivity, high specificity, and no need for standard quantitative curves. Since the digital PCR technology was proposed, the relevant technology and industrialization have developed very rapidly. At present, the development trend of digital PCR is to detect more indicators in the same sample, that is, to perform multi-target analysis. However, the mainstream digital PCR on the market mostly has one color of excitation light corresponding to one fluorescent dye. In order to achieve multi-target analysis, it is necessary to increase the number of multiple fluorescent channels. Therefore, it can be said that there are still shortcomings such as insufficient multiple analysis capabilities, the need for multiple sets of fluorescent light paths, and high costs.

[0003] At present, the method of realizing multi-target analysis in dual channels is to use a dye combination between the two channels, such as the published patent "Nucleic acid quantitative detection kit based on multiple digital PCR of dual fluorescent probes" (CN111218501A), which provides a nucleic acid quantitative detection method based on multiple digital PCR of dual fluorescent probes, including a first probe for detecting a first target, a second probe for detecting a second target, and a third probe for detecting a third target; the first probe is labeled with a first fluorescent group, the second probe is labeled with a second fluorescent group, a part of the third probe is labeled with a first fluorescent group, and the other part is labeled with a second fluorescent group, and the first fluorescent group and the second fluorescent group are two different fluorescent groups; and a two-dimensional result map obtained based on the multiple digital PCR of three targets, to achieve the separate quantification of the three targets to be tested. Although this method can improve the multi-target analysis capability to a certain extent, it is limited by the difference in the intensity of fluorescent dyes in different channels, and the improvement of the multi-target analysis capability is limited. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a multi-spectral digital PCR detection method and device in view of the deficiencies in the above-mentioned prior art.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a multi-spectral digital PCR detection method, comprising the following steps:

[0006] S1. Select n dyes from n according to the number of targets to be detected. ′ A total number of candidate dyes that meet the dye spillover performance requirements is equal to the number of targets to be detected;

[0007] S2, n ′ Dyes and n ′ The target to be tested is matched and labeled, and then combined with the sample to be tested and the reaction reagents required for PCR amplification to form n ′ Re-digital PCR reaction system for PCR amplification;

[0008] S3, after PCR amplification is completed, spectral data is collected to obtain the fingerprint spectrum data matrix of all droplets, and then the abundance vector of all fluorescent dyes in each droplet is obtained through unmixing operation;

[0009] S4. Calculate the abundance vector of all fluorescent dyes in each droplet to obtain n ′ The concentration of each target to be detected.

[0010] Preferably, the step S1 specifically includes:

[0011] S1-1. The detector collects the individual fingerprint spectra of the n dyes as feature vectors, and the feature vectors form the dye fingerprint matrix O:

[0012]

[0013] Among them, ij represents the intensity of the detected dye i on the jth detector, n is the total number of dye types, m is the length of the feature vector, that is, the number of detectors, and m>n ′ ;

[0014] The fingerprint spectrum of each dye refers to the fluorescence spectrum of the dye under the excitation of each wavelength of excitation light obtained by m detectors;

[0015] S1-2. Evaluation of dye overflow performance:

[0016] S1-2-1, calculating the similarity Xs between the fingerprint spectra of each dye;

[0017] S1-2-2. Condition number K(O) value of dye fingerprint matrix O:

[0018]

[0019] Among them, σ max (O) and σ min (O) are the maximum and minimum singular values ​​of the dye fingerprint matrix O respectively.

[0020] S1-2-3. Screen dyes whose Xs and K(O) are both smaller than the set threshold as candidate dyes.

[0021] Preferably, the step S1-2-1 is specifically as follows: calculating the Pearson correlation coefficient ρ between the fingerprint spectra of each dye ij :

[0022] Among them, i is the characteristic spectrum vector of dye i, is the mean of the characteristic spectrum vector.

[0023] Preferably, the step S1-2-1 is specifically: calculating the cosine similarity cosθ between the fingerprint spectra of each dye:

[0024]

[0025] Among them, i is the characteristic spectrum vector of dye i, is the mean of the characteristic spectrum vector.

[0026] Preferably, the step S3 specifically includes:

[0027] S3-1. After PCR amplification is completed, the fluorescence spectrum data of all droplets are collected through m detectors to obtain the fingerprint spectrum data matrix U of each droplet:

[0028]

[0029] where μ ij represents the value of the i-th fingerprint spectrum channel in the j-th droplet, i = 1, 2, ..., m, j = 1, 2, ..., k, k is the total number of droplets, m is the number of fingerprint spectrum channels, that is, the number of detectors;

[0030] S3-2, perform unmixing operation on the fingerprint spectrum data matrix U:

[0031] remember u i′ = {u i′1 ,u i′2 ...,u i′m} T Indicates the i ′ The number of all features detected in a droplet, whose length is m, i ′ =1,2,...,k,v i′ = {v i′1 ,v i′2 ...,v i′n′} T Indicates the i ′ The abundance vector of all fluorescent dyes after the droplets are unmixed is n in length. ′, then the process of unmixing operation is expressed as:

[0032] R i ′ =u i ′

[0033] Where R represents the mixing matrix. Since the unmixed sample event vector v i′ The number of variables n ′ Less than the measured sample event vector u i′ The number of variables is m, and the least squares algorithm is used to obtain the solution of the above equation, that is, v i′ = {v i′1 ,v i′2 ...,v i′n′} T The solution of is used to construct the abundance vector of the fluorescent dyes collected by each channel of all droplets, which is represented by the abundance matrix V:

[0034]

[0035] Among them, v ij is the fluorescence intensity of the jth droplet in the ith dye channel, i = 1, 2, ..., n ′ , n ′ is the dye channel, that is, the number of dyes; j = 1, 2, ..., k, k is the total number of droplets.

[0036] Preferably, the step S4 specifically includes:

[0037] S4-1. According to the abundance matrix V, for all fluorescence intensities v in the i-th dye channel i = {v i1 ,...,v ik}, using the preset threshold, all the droplets in the channel are divided into positive droplets and negative droplets, and the proportion of positive droplets is calculated, and then according to the Poisson formula:

[0038]

[0039] Calculate the average number of copies of the analyte per droplet in the i-th dye channel;

[0040] in is the average number of copies of the analyte in each droplet, i.e., the concentration of the analyte, p i represents the probability that a droplet is a positive droplet, and its estimated value is That is, the ratio of positive droplets to total droplets. H i is the number of positive droplets, C i is the total number of droplets, Yesi An unbiased estimate of

[0041] S4-2, according to the concentration calculation formula:

[0042]

[0043] Calculate the target sequence concentration of the i-th dye channel, where Con i is the concentration of specific target sequence in the sample, V d is the droplet volume.

[0044] The present invention also provides a multi-spectral digital PCR detection device, characterized in that it adopts the method as described above to realize multi-spectral digital PCR detection.

[0045] Preferably, the device comprises a microfluidic channel, a fluorescence excitation module, a fluorescence detection module and a host computer software module;

[0046] The droplets containing the sample to be tested flow through the microchannel in a single row in sequence, and emit fluorescence after being excited by the excitation light emitted by the fluorescence excitation module. The fluorescence detection module collects the fluorescence and converts it into an electrical signal and transmits it to the host computer software module. The host computer software module uses the method of steps S3-S4 to calculate the n in the sample to be tested. ′ The concentration of each target to be detected.

[0047] Preferably, the fluorescence detection module includes m detectors, and the detectors are APD arrays, PMT arrays, or linear array CCDs.

[0048] Preferably, the fluorescence excitation module comprises m excitation light units, which are used to emit excitation lights of m different wavelengths and irradiate the excitation lights onto the droplets in the microfluidic channel.

[0049] The beneficial effects of the present invention are as follows: in the present invention, the fluorescence intensity of the dye under multiple lasers and multiple spectrum bands is collected as a feature, and an algorithm is used to unmix the components and their contents in the sample, thereby distinguishing different substances to be tested;

[0050] The present invention has a good distinguishing effect on fluorescent dyes with similar emission wavelengths, improves the multiple analysis capability of digital PCR, and can be applied to the fields of pathogen detection, tumor detection, single-cell sequencing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a flow chart of the multi-spectral digital PCR detection method in Example 1 of the present invention;

[0052] Figure 2 It is a schematic diagram of the multi-spectral digital PCR detection device in Example 2 of the present invention;

[0053] Figure 3 This is a light path diagram of the fluorescence excitation module and the fluorescence detection module in Example 2 of the present invention. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0055] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0056] Example 1

[0057] Reference Figure 1 This embodiment provides a multi-spectral digital PCR detection method, comprising the following steps:

[0058] S1. Select n dyes from n according to the number of targets to be detected. ′ A total number of candidate dyes that meet the dye spillover performance requirements is equal to the number of targets to be detected;

[0059] Specifically include:

[0060] S1-1. The detector collects the individual fingerprint spectra of the n dyes as feature vectors, and the feature vectors form the dye fingerprint matrix O:

[0061]

[0062] Among them, ij represents the intensity of the detected dye i on the jth detector, n is the total number of dye types, m is the length of the feature vector, that is, the number of detectors, and m>n ′ ;

[0063] The fingerprint spectrum of each dye refers to the fluorescence spectrum of the dye under the excitation of each wavelength of excitation light obtained by m detectors;

[0064] S1-2. Evaluation of dye overflow performance:

[0065] S1-2-1, calculating the similarity Xs between the fingerprint spectra of each dye;

[0066] Calculate the Pearson correlation coefficient ρ between the fingerprint spectra of each dye ij Or cosine similarity cosθ:

[0067]

[0068]

[0069] Among them,i is the characteristic spectrum vector of dye i, is the mean of the characteristic spectrum vector.

[0070] S1-2-2. Condition number K(O) value of dye fingerprint matrix O:

[0071]

[0072] Among them, σ max (O) and σ min (O) are the maximum and minimum singular values ​​of the dye fingerprint matrix O respectively.

[0073] S1-2-3. Screen dyes whose Xs and K(O) are both less than the set threshold as candidate dyes, so that better results can be obtained in multi-target experiments.

[0074] S2, matching and labeling n' kinds of dyes with n' kinds of targets to be tested, and then constructing an n'-multiple digital PCR reaction system with the samples to be tested and the reaction reagents required for PCR amplification to perform PCR amplification;

[0075] S3, after PCR amplification is completed, spectral data is collected to obtain the fingerprint spectrum data matrix of all droplets, and then the abundance vector of all fluorescent dyes in each droplet is obtained through unmixing operation;

[0076] Specifically include:

[0077] S3-1. After PCR amplification is completed, the fluorescence spectrum data of all droplets are collected through m detectors to obtain the fingerprint spectrum data matrix U of each droplet:

[0078]

[0079] where μ ij represents the value of the i-th fingerprint spectrum channel in the j-th droplet, i=1,2,...,m, j=1,2,...,k, k is the total number of droplets, m is the number of fingerprint spectrum channels, that is, the number of detectors;

[0080] S3-2, perform unmixing operation on the fingerprint spectrum data matrix U:

[0081] remember u i′ = {u i′1 ,u i′2 ...,u i′m} T represents the number of all features detected in the i′th droplet, whose length is m, i′=1,2,...,k, v i′ = {v i′1 ,v i′2 ...,v i′n′} Trepresents the abundance vector of all fluorescent dyes after the i′th droplet is unmixed, and its length is n′. The unmixing operation process is expressed as:

[0082] R i′ =u i′

[0083] Where R represents the mixing matrix. Since the unmixed sample event vector v i′ The number of variables n′ is less than the measured sample event vector u i′ The number of variables is m, and the least squares algorithm is used to obtain the solution of the above equation, that is, v i′ = {v i′1 ,v i′2 ...,v i′n′} T The solution of is used to construct the abundance vector of the fluorescent dyes collected by each channel of all droplets, which is represented by the abundance matrix V:

[0084]

[0085] Among them, v ij is the fluorescence intensity of the jth droplet in the ith dye channel, i = 1, 2, ..., n ′ , n ′ is the dye channel, that is, the number of dyes; j = 1, 2, ..., k, k is the total number of droplets.

[0086] S4. Calculate the abundance vector of all fluorescent dyes in each droplet to obtain n ′ The concentration of each target to be detected.

[0087] Step S4 refers to patent CN106596489B - a method for processing fluorescence intensity data in fluorescent droplet detection, and specifically includes:

[0088] S4-1. According to the abundance matrix V, for all fluorescence intensities v in the i-th dye channel i = {v i1 ,...,v ik}, using the preset threshold, all the droplets in the channel are divided into positive droplets and negative droplets, and the proportion of positive droplets is calculated, and then according to the Poisson formula:

[0089]

[0090] Calculate the average number of copies of the analyte per droplet in the i-th dye channel;

[0091] in is the average number of copies of the analyte in each droplet, i.e., the concentration of the analyte, p irepresents the probability that a droplet is a positive droplet, and its estimated value is That is, the ratio of positive droplets to total droplets. H i is the number of positive droplets, C i is the total number of droplets, Yes i An unbiased estimate of

[0092] S4-2, according to the concentration calculation formula:

[0093]

[0094] Calculate the target sequence concentration of the i-th dye channel, where Con i is the concentration of specific target sequence in the sample (copy number / uL), V d is the droplet volume.

[0095] For example, in one embodiment, the above method is used to perform 20-plex digital PCR detection, that is, n ′ =20, firstly, 20 dyes with dye overflow performance that meet the requirements need to be screened, and then the 20 dyes are used to match the 20 target sequences to be tested, and a PCR reaction system is constructed with the test sample, polymerase, buffer, etc. Each dye is used to dye the amplification primer and template of a target, and the PCR reaction system is generated into droplets for PCR amplification, and then sent to the full-spectrum digital PCR to collect spectral data. After that, the concentration of each of the 20 targets in the test sample can be analyzed according to the above method.

[0096] Example 2

[0097] This embodiment provides a multi-spectral digital PCR detection device, which uses the method of embodiment 1 to implement multi-spectral digital PCR detection. Figure 2 ,The device includes a microfluidic channel, a fluorescence excitation module, a fluorescence detection module and a host computer software module;

[0098] The droplets containing the sample to be tested flow through the microchannel in a single row in sequence, and emit fluorescence after being excited by the excitation light emitted by the fluorescence excitation module. The fluorescence detection module collects the fluorescence and converts it into an electrical signal and transmits it to the host computer software module. The host computer software module uses the method of steps S3-S4 to calculate the n in the sample to be tested. ′ The concentration of each target to be detected.

[0099] The fluorescence detection module includes m detectors, which are APD arrays, PMT arrays or linear CCD arrays. The fluorescence excitation module includes m excitation light units, which are used to emit excitation lights of m different wavelengths and irradiate the droplets in the microchannel.

[0100] Reference Figure 3 In one embodiment, three detectors and three excitation light units (including lasers, collimators, dichroic mirrors, etc.) are used as an example for illustration. The central wavelengths of the three excitation light units are 488nm, 561nm, and 638nm, respectively. Each laser is synthesized through its own optical path and irradiated to the objective lens and focused on the droplet. The fluorescence emitted by the droplet passes through the fluorescence transmission component and reaches the three detectors to realize the collection of the fluorescence spectrum.

[0101] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A multi-spectrum digital PCR detection device, characterized in that, it realizes multi-spectrum digital PCR detection by the following method: S1. Select n' alternative dyes that meet the dye spillage performance requirements from n dyes according to the number of target analytes to be detected, and the total number of alternative dyes is equal to the number of target analytes to be detected; S2. Match and label the n' dyes with the n' target analytes to be detected, and then construct an n'-plex digital PCR reaction system with the sample to be detected and the reaction reagents required for PCR amplification, and perform PCR amplification; S3. After PCR amplification, collect spectral data, obtain the fingerprint spectrum data matrix of all droplets, and then obtain the abundance vector of all fluorescent dyes in each droplet through unmixing operation; S4. Calculate the respective concentrations of the n' target analytes to be detected in the sample to be detected according to the abundance vector of all fluorescent dyes in each droplet; The specific steps of S1 include: S1-1. Collect the individual fingerprint spectra of the n dyes as feature vectors through a detector, and form a dye fingerprint spectrum matrix O from the feature vectors: where, o ij represents the intensity of the detected dye i on the j-th detector, n is the total number of dye types, m is the length of the feature vector, i.e., the number of detectors, and m > n; Among them, the fingerprint spectrum of each dye refers to the fluorescence spectrum of the dye excited by the excitation light at each wavelength collected by m detectors; S1-2. Evaluate the dye spillage performance: S1-2-1. Calculate the similarity Xs between the fingerprint spectra of the dyes; S1-2-2. The condition number K(O) value of the dye fingerprint spectrum matrix O; Among them, σ max (O) and σ min (O) are the maximum and minimum singular values of the dye fingerprint spectrum matrix O, respectively; S1-2-3. Screen the dyes with both Xs and K(O) less than the set threshold as alternative dyes; The specific steps of S3 include: S3-1. After PCR amplification, collect the fluorescence spectrum data of all droplets through m detectors to obtain the fingerprint spectrum data matrix U of each droplet; where μ ij represents the value of the i-th fingerprint spectrum channel in the j-th droplet, where i = 1, 2, ..., m, j = 1, 2, ..., k, k is the total number of droplets, and m is the number of fingerprint spectrum channels, i.e., the number of detectors; S3-2. Perform unmixing operation on the fingerprint spectrum data matrix U; Denote u i' ={u i'1 , u i'2 ..., u i'm} T which represents all the detected feature quantities in the \(i'\)-th droplet, with a length of \(m\), where \(i' = 1, 2, \cdots, k\), and \(v i' ={v i'1 , v i'2 ..., v i'n'} T represents the abundance vector of all the fluorescent dyes after unmixing the \(i'\)-th droplet, with a length of \(n'\). Then the process of the unmixing operation is expressed as: Rv i' = u i' Among them, R represents the mixing matrix. Since the number of variables n' in the demixed sample event vector v i' is less than the number of variables m in the measured sample event vector u i' , the solution of the above equation is obtained using the least squares algorithm, that is, v i' ={v i'1 , v i'2 ..., v i'n'} T is obtained, and thus the abundance vector of the fluorescent dyes collected by each channel of all droplets is constructed, which is represented by the abundance matrix V: where v ij is the fluorescence intensity of the j-th droplet in the i-th dye channel, where i = 1, 2, ..., n', n' being the number of dye channels, i.e., the number of dyes; and j = 1, 2, ..., k, k being the total number of droplets; The specific steps of S4 include: S4-1. According to the abundance matrix V, for all fluorescence intensities v in the i-th dye channel i = {v i1 ,..., v ik}, using a preset threshold, all droplets in this channel are divided into two categories: positive droplets and negative droplets, and the proportion of positive droplets is calculated. Then, according to the Poisson formula: Calculate the average number of copies of the analyte per droplet in the i-th dye channel; wherein is the copy number of the analyte in each average droplet, i.e., the analyte concentration, p i represents the probability that the droplet is a positive droplet, and its estimated value is i.e., the proportion of positive droplets in the total droplets, H i is the number of positive droplets, C i is the total number of droplets, is an unbiased estimate of p i ; S4-2. According to the concentration calculation formula: Calculate the concentration of the target sequence in the i-th dye channel, where Con i is the concentration of a specific target sequence in the sample, and V d is the droplet volume.

2. The multi-spectrum digital PCR detection device according to claim 1, characterized in that, The specific content of the step S1-2-1 is as follows: calculate the Pearson correlation coefficient ρ between the fingerprint spectra of each dye ij : Among them, o i is the characteristic spectral vector of dye i, is the mean value of the characteristic spectral vectors.

3. The multi-spectrum digital PCR detection device according to claim 1, characterized in that, The specific step of S1-2-1 is: calculate the cosine similarity cosθ between the fingerprint spectra of the dyes; Among them, o i is the characteristic spectral vector of dye i, is the mean value of the characteristic spectral vectors.

4. The multi-spectrum digital PCR detection device according to claim 1, characterized in that, the device includes a microchannel, a fluorescence excitation module, a fluorescence detection module and a host computer software module; The droplets containing the sample to be detected flow through the microchannel in a single row in sequence, emit fluorescence after being excited by the excitation light emitted by the fluorescence excitation module, the fluorescence detection module collects the fluorescence and converts it into an electrical signal and transmits it to the host computer software module, and the host computer software module calculates the respective concentrations of the n' target analytes to be detected in the sample to be detected by the method of steps S3-S4.

5. The multi-spectrum digital PCR detection device according to claim 4, characterized in that, the fluorescence detection module includes m detectors, and the detectors are APD arrays or PMT arrays or linear CCDs.

6. The multi-spectrum digital PCR detection device according to claim 4, characterized in that, The fluorescence excitation module includes m excitation light units, which are used to emit m kinds of excitation light with different wavelengths and irradiate the droplets in the microchannel.

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