Microfluidic isothermal amplification method and system based on multichannel spectrum detection

Abstract

The invention relates to the technical field of microfluidics, in particular to a microfluidic isothermal amplification method and system based on multichannel spectrum detection, and the method comprises the following steps: adding a sample and a reaction premixed solution into a microfluidic chip to form a reaction liquid; at least two fluorescent probes are added into the reaction premixed solution; carrying out isothermal amplification reaction on the reaction liquid, and collecting spectrum time sequence data in the reaction process; and performing spectral deconvolution on the spectral time sequence data to respectively obtain an independent amplification curve corresponding to each fluorescent probe, and performing quantitative analysis. Aiming at the problem that a reaction amplification quantitative system in the prior art depends on a high-precision quantitative PCR instrument and an optical assembly and is relatively high in cost, a spectral deconvolution mode is introduced to separate independent amplification curves of all fluorescent probes on a calculation level, so that an expensive PCR instrument is omitted, and the cost is reduced. And the requirements on color filtering and detection precision of the optical assembly are reduced, and the method can be applied to detection scenes with relatively wide wave bands.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, and in particular to a microfluidic isothermal amplification method and system based on multi-channel spectral detection. BACKGROUND

[0002] In 2000, Japanese scholar Notomi published a new isothermal nucleic acid amplification technology suitable for genetic diagnosis in the journal Nucleic Acids Res, namely loop-mediated isothermal amplification (LAMP). This method relies on four specific primers (two outer primers and two inner primers) that recognize six conserved sequence regions of the target DNA and a DNA polymerase with strand displacement activity (such as Bst DNA polymerase). The reaction system generally includes four primers, Bst DNA polymerase buffer, Bst DNA polymerase, dNTP, template DNA, betaine, MgSO4, etc. The main principle of LAMP technology is to use four specific primers and a DNA polymerase with strand displacement activity to continuously perform strand displacement synthesis under constant temperature conditions of about 65°C, thereby realizing self-circulating DNA amplification. The amplification of the gene and the detection of the product can be completed in one step, with high amplification efficiency and strong specificity, etc. The copy number of the target DNA sequence can be increased by about 10 9 ~ 10 10 times in 30-60 min.

[0003] In the prior art, there is a microfluidic isothermal amplification system based on the principle of LAMP reaction.

[0004] For example, patent application CN201610879938.4 discloses a portable microfluidic chip LAMP visual detection instrument and its detection method, which includes a box cover and a box body, the box cover and the box body are connected through a lock plate located on the side, a microfluidic chip, a temperature control device and a visual detection system are arranged in the box body; the temperature control device includes a temperature controller and a heating metal block, the microfluidic chip is placed on the heating metal block, a transparent baffle is arranged around the microfluidic chip and the heating metal block, a detachable cover plate is arranged on the transparent baffle, and a visual detection system is arranged on the cover plate; the visual detection system includes a miniature camera and a miniature ultraviolet fluorescent lamp; the detection method of the above-mentioned visual detection instrument includes the preparation of the microfluidic chip, the injection of primers and reaction liquid, constant temperature reaction and the reading of the detection result; and the application of the visual detection instrument is also involved. The instant detection of LAMP reaction is realized, and the time of the user is effectively saved.

[0005] For example, patent application CN201110078450.9 discloses a microfluidic chip for multiple LAMP detection and a preparation method thereof. The chip is made of polymer and prepared by MEMS method. The basic structure of the chip includes: spatially ordered amplification pools, which realize effective differentiation of multiple LAMP signals through spatial differentiation of signals; capillary channels for preventing LAMP primers, amplification products and by-products from mixing with each other between different amplification pools. The amplification pools and capillary channels are connected by connecting pipes to make the fluid flow smoothly and uniformly from the capillary channels into the amplification pools. The chip provides an effective solution for simultaneous detection of multiple pathogens in clinical LAMP method.

[0006] However, in actual implementation, the inventors found that when multiple LAMP reaction signals exist, high-performance molecular diagnostic equipment such as quantitative PCR (qPCR) instruments is usually used to realize multiple detection by relying on complex and expensive optical components, for example, multiple sets of independent excitation / emission filters, dichroic mirrors and photomultiplier tubes, which leads to high cost of hardware platform, and the optical part usually needs to be redesigned after replacing the probe with other fluorescent reactions. SUMMARY

[0007] In view of the above problems in the prior art, the present application provides a microfluidic isothermal amplification method based on multi-channel spectral detection; on the other hand, a system for implementing the microfluidic isothermal amplification method is also provided; on the other hand, a pathogen detection method based on the microfluidic isothermal amplification method is also provided; on the other hand, a genotype analysis method based on the microfluidic isothermal amplification method is also provided; on the other hand, a food safety detection method based on the microfluidic isothermal amplification method is also provided.

[0008] The specific technical solution is as follows: a microfluidic isothermal amplification method based on multi-channel spectral detection, which is suitable for a microfluidic isothermal amplification system; the microfluidic isothermal amplification method comprises the following steps: step S1: adding a sample and a reaction premix to a microfluidic chip to form a reaction liquid; at least two fluorescent probes are added to the reaction premix; step S2: performing an isothermal amplification reaction on the reaction liquid and collecting spectral time series data during the reaction; the spectral time series data covers the exponential growth period and the plateau period of the amplification curve in the time domain; step S3: performing spectral deconvolution on the spectral time series data to obtain independent amplification curves corresponding to each fluorescent probe and performing quantitative analysis.

[0009] On the other hand, the fluorescent probes are selected from 2-6 combinations of FAM, ROX, HEX, CY5, Texas Red, TAMRA, JOE, VIC, NED and PET series fluorescent dyes.

[0010] In another aspect, the emission peak wavelength interval of each of the fluorescent probes is greater than 20 nm; and the emission peak wavelength of each of the fluorescent probes is between 380 nm and 1000 nm.

[0011] In another aspect, a calibration model is constructed before the step S1; the calibration model is used to characterize the fluorescent signal corresponding to each of the fluorescent probes or a combination of fluorescent probe concentrations; and in the step S3, the spectral deconvolution is performed based on the calibration model using a classical least squares method, a partial least squares method, or a principal component regression.

[0012] In another aspect, in the step S3, the spectral deconvolution is performed by any one of an artificial neural network, a support vector machine regression, or a random forest regression.

[0013] In another aspect, in the step S3, the main components of the spectral time series data are first extracted by principal component analysis as core features, and then the spectral deconvolution is performed on the core features using an artificial intelligence model.

[0014] In another aspect, the output fluorescent signal is preprocessed when measuring the fluorescent signal each time; the fluorescent signal preprocessing includes at least one of dark signal correction, baseline correction, spectral normalization, and noise filtering.

[0015] In another aspect, the step S2 includes: a step S21 of heating the reaction liquid to a predetermined temperature and maintaining; a step S22 of, when the predetermined temperature is reached, cyclically collecting the fluorescent signal on a plurality of spectral channels of the reaction liquid and recording until a predetermined time length is reached; the predetermined time length is determined according to the plateau period of the isothermal amplification reaction; and a step S23 of assembling the recorded fluorescent signal to obtain the spectral time series data.

[0016] In another aspect, after the step S3 is performed, a melting curve analysis is further performed to verify the specificity of the amplification product and to distinguish between specific amplification and non-specific amplification.

[0017] A microfluidic isothermal amplification system for implementing the microfluidic isothermal amplification method described above; the microfluidic isothermal amplification system includes: a microfluidic chip, the microfluidic chip is provided with two liquid inlets for placing samples and reaction premix respectively; a reaction area is provided in the microfluidic chip, the reaction area is connected to the liquid inlets through flow channels respectively; a heating platform, the microfluidic chip is arranged above the heating platform, the heating platform heats the reaction area to a predetermined temperature and maintains constant temperature; an optical module, the optical module irradiates the reaction area and collects the fluorescent signal of the reaction area; a processing device, the processing device is connected to and controls the heating platform and the optical module, and the processing device receives and analyzes the fluorescent signal.

[0018] In another aspect, the microfluidic chip is made of at least one of polymethyl methacrylate, polycarbonate, cyclic olefin copolymer, glass or quartz.

[0019] In another aspect, the optical module comprises: an excitation light source, the light source type of which is determined according to the excitation wavelength band of the fluorescent probe in the reaction premix; and a multi-channel spectral sensor, which collects the fluorescent signals on multiple spectral channels of the reaction region.

[0020] In another aspect, the processing device comprises a microcontroller, a storage unit, a communication interface and an algorithm processing module; and the processing device supports two working modes of real-time data processing and offline data analysis.

[0021] In another aspect, the excitation light source is any one of a laser diode, a white light LED or a wide-spectrum light source; the multi-channel spectral sensor has 2-64 independent detection channels; the detection range of the multi-channel spectral sensor covers 350-1000 nm; in another aspect, the microfluidic isothermal amplification system is powered by a battery; and the processing device has a low-power mode.

[0022] In another aspect, the microfluidic isothermal amplification system realizes simultaneous quantitative analysis of at least two fluorescent probes through a single and fixed optical detection path, only by using the spectral resolution capability of the multi-channel spectral sensor for mixed fluorescent signals and the deconvolution algorithm of the processing device.

[0023] A pathogen detection method is realized based on the above microfluidic isothermal amplification method; and the pathogen comprises at least one of a virus, a bacterium and a fungus.

[0024] A genotype analysis method is realized based on the above microfluidic isothermal amplification method; and the genotype analysis method comprises single nucleotide polymorphism detection and gene deletion / insertion mutation detection.

[0025] A food safety detection method is realized based on the above microfluidic isothermal amplification method; and the food safety detection method is used for detecting pathogenic bacteria, allergens and genetically modified ingredients.

[0026] A reverse transcription analysis method is realized based on the above microfluidic isothermal amplification method; and the reverse transcription analysis method is used for RNA detection.

[0027] The technical scheme has the following advantages or beneficial effects: in the prior art, the reaction amplification quantitative system relies on a high-precision quantitative PCR instrument and optical components, and the cost is high; in the embodiment, a spectrum deconvolution method is introduced to separate the independent amplification curves of each fluorescent probe at the calculation level, thereby eliminating the need for expensive PCR instruments and reducing the requirements for color filtering and detection accuracy of the optical components, and the method can be applied to a wide range of detection scenarios. BRIEF DESCRIPTION OF DRAWINGS

[0028] Reference will be made to the accompanying drawings to more fully describe embodiments of the present application. However, the accompanying drawings are only used for illustration and explanation, and do not constitute a limitation on the scope of the present application.

[0029] Figure 1 is a schematic diagram of the embodiment of the present application; Figure 2 is a schematic diagram of the system in the embodiment of the present application; Figure 3 is a schematic diagram of the first calibration process in the embodiment of the present application; Figure 4 is a schematic diagram of step S3 in the embodiment of the present application; Figure 5 is a schematic diagram of the second calibration process in the embodiment of the present application; Figure 6 is a schematic diagram of step S2 in the embodiment of the present application; Figure 7 is a schematic diagram of the optical module in the embodiment of the present application. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0031] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0032] The present application will be further described below with reference to the accompanying drawings and specific embodiments, but is not limited by the present application.

[0033] The present application includes: a microfluidic isothermal amplification method based on multi-channel spectrum detection, suitable for a microfluidic isothermal amplification system; such as Figure 1As shown, the microfluidic isothermal amplification method comprises: step S1: adding a sample and a reaction premix to a microfluidic chip to form a reaction liquid; at least two fluorescent probes are added to the reaction premix; step S2: performing an isothermal amplification reaction on the reaction liquid, and collecting spectral time series data during the reaction; the spectral time series data covers the exponential growth period and the plateau period of the amplification curve in the time domain; step S3: performing spectral deconvolution on the spectral time series data to obtain independent amplification curves corresponding to each fluorescent probe and performing quantitative analysis.

[0034] Specifically, to solve the problem of high cost of the existing isothermal amplification reaction amplification quantitative system relying on high-precision quantitative PCR instruments and optical modules, in the embodiment, the spectral deconvolution method is introduced to separate the independent amplification curves of each fluorescent probe at the calculation level, thereby eliminating the expensive PCR instrument and reducing the requirement for the color filter and detection accuracy of the optical module, which can be applied to a wide range of detection scenarios.

[0035] Specifically, the microfluidic isothermal amplification method described above is mainly applicable to a microfluidic isothermal amplification system.

[0036] Among them, the "sample" is used to refer to the target sample in the detection, which can be a DNA-containing sample or an RNA-containing sample. The common sample detection object is a DNA sample, which is amplified by adding a reaction premix containing corresponding primers, DNA polymerase and fluorescent probes, and heating to a predetermined temperature.

[0037] However, according to the needs of detection, the sample can also be replaced by an RNA sample, at which time an RNA reverse transcriptase needs to be additionally added, and the components in the reaction premix need to be adjusted accordingly, and then heated to a predetermined temperature for amplification.

[0038] Taking DNA sample detection as an example, Figure 2 A simple system schematic diagram of a microfluidic isothermal amplification system is shown. The system at least includes a group of microfluidic chips A1 and a heating platform A2. The microfluidic chip A1 is fixed on the heating platform A2, which is used to heat the DNA sample and the reaction premix to the reaction temperature.

[0039] The isothermal amplification reaction is selected from any one of LAMP (Loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), HAD (Helicase-dependent Amplification), and SDA (Strand Displacement Amplification).

[0040] The heating platform A2 is also integrated with an optical module A3 for providing an excitation light source for a fluorescent probe in the isothermal amplification reaction and collecting a fluorescent signal for quantitative analysis by the processing device A4.

[0041] In view of the problem of needing to set a color filter in the prior art, in the present scheme, a calculation mode of deconvolving the spectral channels is introduced, the spectral channels corresponding to multiple fluorescent signals are separated at the calculation level, thereby forming independent amplification curves corresponding to each fluorescent probe, and then quantitative analysis is performed, thereby reducing the demand for precision at the hardware level, and the complexity of calculation replaces the expensive and bulky physical optical module (such as multiple sets of optical filters or grating spectrometers) in the traditional detection equipment, and the precise thermal cycler in the PCR instrument, thereby greatly reducing the system cost and volume while ensuring the analysis performance.

[0042] Taking the LAMP reaction system as an example, the biochemical basis of the reaction system is the loop-mediated isothermal amplification (LAMP) technology. The reaction process is roughly to inject the DNA sample and the LAMP reaction premix into the microfluidic chip at the same time and heat to the amplification temperature, so that the DNA sample is rapidly amplified.

[0043] Among them, the main components of the LAMP reaction premix are primers and biomarkers of isothermal amplification reactions, such as fluorescent probes. A typical reaction premix includes Bst polymerase, primers, dNTPs, and fluorescent probes. Among them, the fluorescent probe determines the staining method according to the item to be measured and the DNA species.

[0044] When the DNA sample and the LAMP reaction premix are heated to the amplification temperature, usually 65 ℃, the DNA sample will rapidly amplify, and the positive antibody in it will react with the fluorescent probe and produce a fluorescent signal in the excitation process. Since the LAMP reaction speed is fast, significant reaction usually occurs within 40 minutes, so the overall reaction time can be controlled within 60 minutes.

[0045] On this basis, the reaction process of the LAMP reaction is inserted with corresponding sampling points at multiple time points. The sampling operation at each sampling point includes exciting the fluorescent probe by a light source of a specific wavelength, collecting light signal data of multiple spectral channels as sampling data at the current sampling point, and assembling the sampling data in time sequence to finally obtain spectral time sequence data. The start time point and the end time point of the sampling point should cover the exponential growth period and the plateau period of the amplification curve in the time domain.

[0046] A typical design is to start sampling after heating to a predetermined temperature, insert a set of sampling points every 30 seconds, return multi-channel spectral data, and stop until the reaction time is reached. According to a typical isothermal amplification reaction time of 40 minutes, 80 sampling points will be generated in 40 minutes, which is enough to reconstruct the exponential growth period and the plateau period of the amplification curve, so as to accurately calculate the "time to threshold" and perform related analysis.

[0047] The number of spectral channels should be determined according to the spectral range of the fluorescent reaction. The typical number of spectral channels is 12. The light intensity data of 12 channels at each sampling point is collected and combined to obtain the spectral vector at the sampling point.

[0048] Finally, the spectral vectors are added to the spectral time sequence data in the order of collection to form a matrix data. Spectral deconvolution is performed on the data matrix to separate the independent amplification curves, and quantitative analysis is performed.

[0049] When other isothermal amplification reactions are used, the primers in the reaction premix can be adjusted according to the corresponding isothermal amplification reaction type, and the reaction temperature of the heating platform can be adjusted.

[0050] The following are the reaction temperatures of common amplification reactions: Recombinase Polymerase Amplification (RPA): usually at 37-42 ℃.

[0051] Nucleic Acid Sequence-Based Amplification (NASBA): the standard reaction temperature is 41 ℃.

[0052] Helicase-Dependent Amplification (HDA): the reaction temperature is usually set at 65 ℃.

[0053] Strand Displacement Amplification (SDA): the reaction temperature range is wide, usually between 37-60°C.

[0054] After replacing the amplification reaction, the corresponding sampling interval and duration should also be adjusted according to the type of the amplification reaction, so that the spectral time series data covers the exponential growth period and the plateau period of the amplification curve in the time domain.

[0055] In one embodiment, the fluorescent probes are selected from 2-6 combinations of FAM, ROX, HEX, CY5, Texas Red, TAMRA, JOE, VIC, NED, and PET series fluorescent dyes.

[0056] In another embodiment, the fluorescent probes consist of FAM and ROX.

[0057] Specifically, in selecting the types of fluorescent probes, multiple fluorescent probes are usually added to achieve better measurement results. However, when the emission wavelength bands of two or more fluorescent probes are similar, they will cause spectral overlap and are difficult to separate effectively.

[0058] Therefore, when selecting fluorescent probes, the emission wavelength bands of the fluorescent probes should be controlled as much as possible to avoid overlap.

[0059] Based on this, it can be found through analysis of the fluorescent probes that when FAM (emission peak about 517 nm) and ROX (emission peak about 604 nm) are used as fluorescent probes, the fluorescence emission will be mainly detected by the F4 (center wavelength 515 nm), FY (center wavelength 555 nm), and F5 (center wavelength 550 nm) channels of the sensor. The fluorescence emission of ROX will mainly fall within the response range of the FXL (center wavelength 600 nm) and F6 (center wavelength 640 nm) channels, and FAM will only produce a slight spectral crosstalk phenomenon in the detection channel of ROX, so selecting the above two dyes will achieve better analysis results.

[0060] However, in actual detection, different combinations of fluorescent probes may be selected according to the different genotypes to be detected, such as 2-6 combinations of FAM, ROX, HEX, CY5, Texas Red, TAMRA, JOE, VIC, NED, and PET series fluorescent dyes.

[0061] Generally, to achieve better spectral deconvolution results, the emission peak wavelength interval of each fluorescent probe needs to be controlled to be greater than 20 nm to reduce crosstalk.

[0062] At the same time, it is also necessary to control the emission peak wavelength within the detection range of the multispectral sensor, such as a typical range of 380-1000 nm.

[0063] Based on the design of the above process, the optical deconvolution process can be realized by using methods such as classical least squares method, partial least squares method or principal component regression.

[0064] Taking the classical least squares method as an example, in this embodiment, before executing step S1, a first calibration model is constructed for the microfluidic chip; in step S3, the spectral time series data is deconvolved based on the first calibration model; Figure 3 As shown, the first calibration process for generating the first calibration model includes: step A01: for each fluorescent probe, the corresponding positive template DNA is used to react in the microfluidic chip, and after reaching the plateau phase, the first multi-channel spectral sample signal is measured for the fluorescence signal emitted by the fluorescent probe; the first multi-channel spectral sample signal and the spectral time series data have the same spectral channel; step A02: the first calibration model is obtained by assembling all the first multi-channel spectral sample signals; in the first calibration model, each multi-channel spectral sample signal is used as a row of the matrix, and the spectral channel is used as a column of the matrix.

[0065] Specifically, due to the broad peak characteristics of the fluorescent dye emission spectrum and the limited spectral resolution of the sensor channel, the signal measured by this system is a mixed signal. , the signal vector measured by the system (a vector of 12 channel intensity values) is not the response of a single dye, but rather a linear superposition of the signals of all fluorescent components in the sample (in this case, FAM and ROX).

[0066] This problem can be accurately described by a linear algebra model. The data collected during the entire reaction process can be represented as a data matrix , where each row represents a measurement vector at a time point , each column represents the signal change of a specific spectral channel at all time points. This mixed data matrix can be modeled as the product of two (or more) more fundamental matrices, plus an error term: ; Where, represents the spectral time series data actually measured, represents the actual concentration matrix of each fluorescent probe, yes The pure component spectral matrix, also called the calibration matrix, Represents the transposed matrix of the pure component spectra matrix. Each row of the matrix represents the response vector of a pure component (e.g. FAM) on the 12 spectral channels, i.e. its "spectral fingerprint". The total number of rows is , the number of different pure components. The last row is the error term of the measurement. This matrix needs to be determined in advance by a calibration experiment.

[0067] Before performing step S1, a first calibration model needs to be constructed in advance.

[0068] The construction process of the first calibration model usually includes obtaining the spectral fingerprint under a single fluorescent probe. The spectral fingerprint may vary depending on the actual model of the dye, the measurement system, etc., thus it needs to be calibrated separately.

[0069] Specifically, for each fluorescent probe, an isothermal amplification reaction system containing only the type of labeled probe needs to be prepared in advance. A sufficient amount of positive template DNA is added, and the reaction is allowed to proceed to the plateau stage, at which time the FAM fluorescence signal reaches a maximum and is stable. In this state, the spectral signals of the 12 channels are measured by the device to obtain a 12-dimensional vector. This vector can be used as the corresponding spectral fingerprint after signal correlation processing.

[0070] The spectral fingerprint of each fluorescent probe is obtained by repeating the process, and then the calibration matrix is constructed.

[0071] For example, the calibration matrix of FAM and ROX probes in a 12-channel detection system is a 2x12 matrix: .

[0072] In an embodiment, as shown in Figure 4 , step S3 includes: step A31, performing least squares calculation on the first calibration model and the spectral time series data to obtain a first concentration matrix; step A32, splitting the column vectors of the first concentration matrix to respectively serve as amplification curves of each fluorescent probe; and step A33, performing quantitative analysis on the amplification curves.

[0073] Specifically, to achieve a more convenient calculation effect, in the present embodiment, the first concentration matrix is obtained by performing least squares calculation on the first calibration model and the spectral time series data, specifically: ; wherein, is the first concentration matrix to be solved, is the spectral time series data, is the first calibration model, is the transpose matrix of the first calibration model.​

[0074] After the first concentration matrix is solved, matrix calculation is performed on each column of the first concentration matrix to obtain the amplification curve of each fluorescent probe, and finally quantitative analysis is performed to obtain the corresponding results.

[0075] For example, in this embodiment, before step S1 is performed, a second calibration model is constructed for the microfluidic chip; in step S3, the partial least squares method is used to perform deconvolution on the spectral time series data based on the second calibration model; as shown in Figure 5 The second calibration process for generating the second calibration model includes: step B01: constructing a plurality of concentration combinations for all fluorescent probes, mixing the fluorescent probes based on the concentration combinations to obtain a test mixture; step B02: reacting the positive template DNA with the test mixture in the microfluidic chip, and after the plateau period, measuring the second multi-channel spectral sample signal from the mixed fluorescence signal; the second multi-channel spectral sample signal has the same spectral channels as the spectral time series data; step B03: calculating the second calibration model from all the second multi-channel spectral sample signals; the second calibration model is a regression model for characterizing the dye concentration from the second multi-channel spectral sample signal.

[0076] Specifically, to achieve better model robustness, in this embodiment, the partial least squares method (PLS) is selected as an alternative solution in the deconvolution process. Unlike the least squares method, the partial least squares method is a supervised regression method that actively seeks the most relevant spectral variation patterns with known concentrations when building the model, and can better handle the collinearity and noise between variables.

[0077] To achieve this processing step, in the process of constructing the calibration model, a calibration set containing a series of standard samples is constructed based on the combination of a plurality of fluorescent probes, and the concentration combinations of the fluorescent probes in these samples are known and can cover any numerical range that may occur during actual measurement.

[0078] On this basis, the fluorescent probes are mixed according to the pre-designed concentration combinations to obtain a test mixture, and the corresponding positive template DNA is reacted with the test mixture in the microfluidic chip, and after the plateau period, the second multi-channel spectral sample signal is measured from the mixed fluorescence signal, which is consistent with the concentration combination. Subsequently, a regression model from the 12-channel spectral data to the concentration of each fluorescent probe is constructed as a second calibration model.

[0079] According to the second calibration model, the partial least squares method is used to calculate to achieve better deconvolution effect.

[0080] In another embodiment, the principal component analysis (PCA) method is used to implement spectral deconvolution.

[0081] For the principal component analysis process, a calibration set containing a series of standard samples is first constructed based on the combination of multiple fluorescent probes. The concentration combinations of each fluorescent probe in these samples are known and can cover any numerical range that may appear in actual measurements.

[0082] On this basis, fluorescent probes are mixed according to pre-designed concentration combinations to create a test mixture. The corresponding positive template DNA is then reacted with the test mixture in a microfluidic chip. After reaching the plateau phase, the emitted mixed fluorescence signal is measured to obtain a third multi-channel spectral sample signal. A known concentration matrix is ​​constructed based on these multiple third multi-channel spectral signals as a priori parameters.

[0083] In actual detection, principal component analysis is performed on the preprocessed spectral time series data to decompose it into a few uncorrelated principal components (PCs). These principal components capture the main variation variance in the original spectral data.

[0084] Then, a multiple linear regression model was established with the extracted principal components as independent variables and the previously known concentration matrix based on the standard samples as the dependent variable.

[0085] For unknown samples, the spectral data is first projected into the established PCA space to obtain principal component scores, and then the component concentrations are predicted using a regression model. Compared to PLS, PCR only considers the variations in the spectral data itself when extracting principal components, without considering its correlation with concentration. However, it can also provide stable and reliable deconvolution results in many application scenarios.

[0086] In addition, in some other embodiments, in step S3, spectral deconvolution is performed by any one of artificial neural network, support vector machine regression, and random forest regression.

[0087] This type of deconvolution method mainly relies on pre-collecting samples, including adjusting different concentration combinations and collecting corresponding fluorescence spectral data and adding annotations based on given probe types and corresponding amplification reactions, thereby forming a large-scale calibration data set.

[0088] Then use the spectral data as input features and the known concentrations as output labels to train a machine learning regression model, such as: Artificial Neural Network: By building a network structure consisting of input layer, hidden layer and output layer, it can learn the complex nonlinear relationship between spectrum and concentration.

[0089] Support Vector Machine Regression: By finding an optimal hyperplane such that the error of all sample points to the plane is minimized.

[0090] Random Forest Regression: By constructing multiple decision trees and averaging their results.

[0091] In some embodiments, ensemble learning and hybrid algorithm strategies can also be used for deconvolution.

[0092] Taking the combination strategy of principal component analysis and artificial neural network as an example, this strategy needs to collect samples in advance, including adjusting different concentration combinations and collecting corresponding fluorescence spectrum data and adding labels on the basis of given probe types and corresponding amplification reactions, so as to form a large-scale calibration data set.

[0093] Based on the calibration data set, the trained artificial neural network is obtained by training the artificial neural network.

[0094] In actual processing, for the collected spectrum time series data, first, pretreatment, denoising and other operations are performed, then principal component analysis is used to reduce the dimension of the pretreated spectrum data, and the first 5-10 principal components are extracted as core features.

[0095] Subsequently, the core features are input into the trained artificial neural network, and the deconvoluted component concentration curves are finally output.

[0096] In one embodiment, before measuring the fluorescence signal each time, the dark signal of the microfluidic isothermal amplification system is measured in advance; and after step S2 is performed, before step S3 is performed, the measured value of the spectrum time series data is subtracted by the dark signal; and the spectrum time series data is deducted by the drift baseline using the asymmetric least squares method.

[0097] Specifically, considering the problems of noise and baseline drift that may exist in the measurement system, in this embodiment, for the spectrum time series data output by the measurement system each time, including the fluorescence signal output when modeling in advance and the spectrum time series data output when actually measuring, the above method is used for signal pretreatment.

[0098] Specifically, for the originally measured signal, since the system itself has an ambient light background and a dark current, before each measurement, the dark signal of the microfluidic isothermal amplification system is measured in advance, and the dark signal is directly deducted when the original measurement data is output to avoid the influence of the noise floor.

[0099] During the isothermal amplification reaction, slow, nonlinear baseline drift may occur due to reagent degradation or slight temperature fluctuations. To address this, an advanced baseline correction algorithm, such as Asymmetric Least Squares (AsLS), is applied to each column of the spectral time series data matrix (i.e., the time series data for each channel). AsLS intelligently identifies and subtracts drifting baselines while preserving the true amplification signal peaks, making it well-suited for automated processing.

[0100] Furthermore, data can be normalized according to spectral channels as needed to eliminate physical differences between reaction wells. Common methods include Standard Normal Variate (SNV) or Multiplicative Scatter Correction (MSC). These methods effectively eliminate multiplicative interference effects and enhance consistency between experimental data from different samples or batches.

[0101] And, remove signal noise in the sampling system through noise filtering.

[0102] In one embodiment, Figure 6 As shown, step S2 includes: step S21: heating the reaction liquid to a predetermined temperature and maintaining it; step S22: when the predetermined temperature is reached, cyclically collecting and recording the fluorescence signals on multiple spectral channels of the reaction liquid until a predetermined time is reached; the predetermined time is determined according to the plateau phase of the isothermal amplification reaction; step S23: assembling the recorded fluorescence signals to obtain spectral time series data.

[0103] Specifically, to achieve optimal reaction results, this example also uses a PTC (Positive Temperature Coefficient) heating device as the heating platform. The PTC device operates in open or closed loop control, automatically stopping and maintaining the temperature when heating reaches 65°C. Then, according to a pre-configured timer, the reaction liquid is circulated to collect fluorescence signals from multiple spectral channels and added to the data matrix for recording until the predetermined duration is reached.

[0104] In one embodiment, after executing step S3, the method further includes: verifying the specificity of the amplified product by melting curve analysis to distinguish specific amplification from non-specific amplification.

[0105] Specifically, by slowly decreasing the temperature of the PTC heater while continuously monitoring the fluorescence signal, a melting curve of the amplified product can be obtained. Specific and nonspecific products typically have different melting temperatures (Tm values), which can be used to distinguish true-positive from false-positive results.

[0106] Specifically, after the standard isothermal amplification reaction (e.g. LAMP reaction for 45 minutes) is completed, the system automatically enters the melting curve analysis stage.

[0107] Then, the heating platform needs to be controlled to increase the temperature at a slow and precise rate, for example, linearly increase the temperature from 65 ℃ to 95 ℃ at a rate of 0.5 ℃ / s. The upper limit of the temperature is determined according to the highest melting temperature of the specific product expected to appear in the current reaction.

[0108] At each temperature point during the temperature increase, the optical detection module synchronously collects full-channel spectral data as melting spectral data.

[0109] For the collected data, the data processing unit processes the collected data in real time. It selects a spectral channel most sensitive to the signal of double-stranded DNA intercalating dye (e.g. SYBR Green, which can be added additionally in the premix) or specific probe, and draws the original melting curve of "fluorescence intensity vs. temperature".

[0110] By calculating the negative first derivative (-dF / dT) of the original melting curve, one or more peaks can be obtained. The temperature corresponding to the peak top of each peak is the melting temperature (Tm value).

[0111] Specific product: usually shows a single, sharp melting peak in the expected temperature range (e.g. 85-90 ℃). Non-specific product (e.g. primer dimer): usually has a lower Tm value (e.g. <80 ℃) and a wider or multiple peak shape.

[0112] In this way, specific products and non-specific products are distinguished.

[0113] A microfluidic isothermal amplification system for implementing the above-mentioned microfluidic isothermal amplification method; as shown in Figure 2 The microfluidic isothermal amplification system includes: a microfluidic chip A1, which is provided with two liquid inlets for placing DNA samples and reaction premixes, respectively; a reaction area is arranged in the microfluidic chip A1, and the reaction area is connected to the liquid inlets through flow channels; a heating platform A2, the microfluidic chip A1 is arranged above the heating platform A2, and the heating platform A2 heats the reaction area to a predetermined temperature and maintains a constant temperature; an optical module A3, the optical module A3 irradiates the reaction area and collects the fluorescence signal of the reaction area; a processing device A4, the processing device A4 is connected to and controls the heating platform A2 and the optical module A3, and the processing device A4 receives and analyzes the fluorescence signal.

[0114] Specifically, to achieve better measurement results, in the embodiment, the microfluidic isothermal amplification system is constructed. The microfluidic chip A1 is a reaction chip made of polymer material, which is etched with flow channels and multiple chambers for injecting DNA samples and reaction premix, eliminating bubbles of liquid and uniformly mixing, and finally introducing into the reaction area.

[0115] The reaction area is a chamber with a certain volume and optical path. When the detection system is assembled, the reaction area will accurately fall in the center of the heating surface of the heating platform A2, so that the reaction area is uniformly heated to the accurate predetermined temperature; and the reaction area will also be located on the focal point of the optical module A3.

[0116] The heating platform A2 is a heating device that can uniformly heat the microfluidic chip A1 to a predetermined temperature and automatically maintain, and has small temperature fluctuations during the overall reaction process. Common designs include PTC heating modules, nichrome wire heaters, and peltier elements, which achieve good temperature control through open-loop or closed-loop control.

[0117] The optical module A3 mainly includes light source and sensor two parts, which is used for exciting and collecting light signals of fluorescent probes at specific nodes in the reaction process. The control process is realized by the processing device A4.

[0118] The processing device A4 is a computer device that can run specific programs to execute the above-mentioned process. Microprocessors, single-chip microcomputers, etc. can be used, or can be connected to an external computer in the form of a capture card. Common designs are based on STM32 single-chip microcomputers or Raspberry Pi microcomputers.

[0119] The microfluidic isothermal amplification system uses a single, fixed optical detection path, and only uses the spectral resolution capability of the multi-channel optical spectrum sensor for mixed fluorescence signals and the deconvolution algorithm of the processing device to realize the simultaneous quantitative analysis of at least two fluorescent probes.

[0120] In one embodiment, the microfluidic chip A1 is made of polymethyl methacrylate material.

[0121] Specifically, to achieve good heat conduction and optical performance, in the embodiment, polymethyl methacrylate is selected as the material of the microfluidic chip. The selection factors include: Optical transparency: Especially for the material used to construct the detection area, it must have high light transmittance in the excitation light and emission light wavelength range (about 450-650 nm) of the system, and its own background fluorescence (autofluorescence) should be as low as possible to ensure high signal-to-noise ratio.

[0122] Biocompatibility: The inner surface of the chip must be chemically inert and cannot adsorb or inhibit key biological molecules (such as DNA polymerase, primers, and template DNA) in the isothermal amplification reaction.

[0123] Thermal conductivity: The bottom of the chip needs to have good thermal contact with the PTC heater to ensure that heat can be efficiently and uniformly transferred to the reaction liquid.

[0124] In other embodiments, at least one of polycarbonate, cyclic olefin copolymer, glass, or quartz can also be used.

[0125] In one embodiment, as shown in Figure 7 The optical module A3 includes a laser light source A31, and the type of light source of the laser light source A31 is determined according to the excitation wavelength band of the fluorescent probe in the reaction premix. The optical module A3 also includes a multi-channel spectral sensor A32 that collects fluorescent signals on multiple spectral channels of the reaction area.

[0126] Specifically, to achieve better measurement results, in this embodiment, the corresponding laser light source is first determined according to the excitation wavelength band of the fluorescent probe in the reaction premix. In portable devices, a constant-wavelength LED laser light source is usually used for excitation. Taking the case of FAM (maximum excitation wavelength about 495 nm) and ROX (maximum excitation wavelength about 578 nm) fluorescent probes as an example, a typical high-power blue LED (center wavelength about 470 nm) can achieve better excitation efficiency. According to needs, two different wavelength LED laser light sources can also be provided for time-sharing excitation, etc.

[0127] In addition, a white light LED or a wide-spectrum light source can also be provided for excitation according to needs.

[0128] The multi-channel spectral sensor A32 is a sensor integrated with multiple spectral channels, which is lower in cost than a grating spectrometer, but based on the above calculation process, the same effect can be achieved.

[0129] A typical design is the AMS AS7343 multi-channel spectral sensor, which integrates 12 spectral channels covering the visible to near-infrared wavelength band, as well as a full-transmission channel and a flicker detection channel. The 12 spectral channels provide enough data points to effectively separate the mixed spectra of 2-3 fluorescent dyes.

[0130] In other embodiments, different detection channels can be provided according to the selection of the device, but generally, 2-64 independent detection channels are provided, and the detection range covers 350-1000 nm.

[0131] Due to the spectrum deconvolution method, the multi-channel spectral sensor can directly collect the spectrum without adding additional color filters.

[0132] In addition, the processing device comprises a microcontroller, a storage unit, a communication interface and an algorithm processing module; the processing device supports two working modes of real-time data processing and offline data analysis, and provides higher detection flexibility.

[0133] The processing device has a low-power mode, and in combination with battery power supply, the detection system can be applied to on-site rapid detection.

[0134] In addition, a pathogen detection method is also provided, which is realized based on the microfluidic isothermal amplification method; the pathogen comprises at least one of a virus, a bacterium and a fungus.

[0135] A genotype analysis method is also provided, which is realized based on the microfluidic isothermal amplification method; the genotype analysis method comprises single nucleotide polymorphism (SNP) detection and gene deletion / insertion mutation detection.

[0136] A food safety detection method is also provided, which is realized based on the microfluidic isothermal amplification method; the food safety detection method is used for detecting pathogenic bacteria, allergens and genetically modified components.

[0137] A reverse transcription analysis method is also provided, which is realized based on the microfluidic isothermal amplification method; the reverse transcription analysis method is used for RNA detection.

[0138] The above are only the preferred embodiments of the present application, and do not limit the embodiments and protection scope of the present application; for those skilled in the art, it should be realized that any equivalent replacement and obvious change made according to the content of the present application should be included in the protection scope of the present application.

Claims

1. A microfluidic isothermal amplification method based on multi-channel spectral detection, characterized in that: Suitable for a microfluidic isothermal amplification system; the microfluidic isothermal amplification method comprises: step S1: adding a sample and a reaction premix to a microfluidic chip to form a reaction liquid; adding at least two fluorescent probes to the reaction premix; step S2: performing an isothermal amplification reaction on the reaction liquid, and collecting spectral time series data during the reaction process; the spectral time series data covers the exponential growth period and the plateau period of the amplification curve in the time domain; step S3: performing spectral deconvolution on the spectral time series data to obtain independent amplification curves corresponding to each of the fluorescent probes and perform quantitative analysis.

2. The microfluidic isothermal amplification method according to claim 1, characterized in that The fluorescent probe is selected from a combination of 2 to 6 fluorescent dyes in the series of FAM, ROX, HEX, CY5, Texas Red, TAMRA, JOE, VIC, NED, and PET.

3. The microfluidic isothermal amplification method according to claim 1, characterized in that The emission peak wavelength interval of each fluorescent probe is greater than 20 nm; the emission peak wavelength of the fluorescent probe is between 380-1000 nm.

4. The microfluidic isothermal amplification method according to claim 1, characterized in that: A calibration model is constructed before step S1; the calibration model is used to characterize the fluorescence signals corresponding to each fluorescent probe or fluorescent probe concentration combination; in step S3, spectral deconvolution is performed based on the calibration model using classical least squares method, partial least squares method or principal component regression.

5. The microfluidic isothermal amplification method according to claim 1, characterized in that: In step S3, spectral deconvolution is performed by any one of artificial neural network, support vector machine regression, and random forest regression.

6. The microfluidic isothermal amplification method according to claim 1, characterized in that: In step S3, the main components of the spectral time series data are first extracted as core features through principal component analysis, and then the core features are spectrally deconvolved using an artificial intelligence model.

7. The microfluidic isothermal amplification method according to claim 1, characterized in that: Each time the fluorescence signal is measured, the output fluorescence signal is preprocessed; the fluorescence signal preprocessing includes at least one of dark signal correction, baseline correction, spectrum normalization, and noise filtering.

8. The microfluidic isothermal amplification method according to claim 1, characterized in that: The step S2 includes: step S21: heating the reaction liquid to a predetermined temperature and maintaining it; step S22: when the predetermined temperature is reached, cyclically collecting and recording fluorescence signals on multiple spectral channels of the reaction liquid until a predetermined time is reached; the predetermined time is determined according to the plateau phase of the isothermal amplification reaction; step S23: assembling the spectral time series data based on the recorded fluorescence signals.

9. The microfluidic isothermal amplification method according to claim 1, characterized in that: After executing step S3, the method further includes: verifying the specificity of the amplified product by melting curve analysis to distinguish specific amplification from non-specific amplification.

10. A microfluidic isothermal amplification system, characterized in that: Used to implement the microfluidic isothermal amplification method according to any one of claims 1 to 9; the microfluidic isothermal amplification system comprises: a microfluidic chip, the microfluidic chip is provided with two liquid inlets, respectively used to place a sample and a reaction premix; a reaction area is provided in the microfluidic chip, and the reaction area is connected to the liquid inlets through flow channels; a heating platform, the microfluidic chip is arranged above the heating platform, and the heating platform heats the reaction area to a predetermined temperature and maintains a constant temperature; an optical module, the optical module irradiates the reaction area and collects the fluorescence signal of the reaction area; a processing device, the processing device is connected to and controls the heating platform and the optical module, and the processing device receives the fluorescence signal and analyzes it.

11. The microfluidic isothermal amplification system according to claim 10, characterized in that: The microfluidic chip is made of at least one of polymethyl methacrylate, polycarbonate, cycloolefin copolymer, glass or quartz.

12. The microfluidic isothermal amplification system according to claim 10, characterized in that: The optical module includes: an excitation light source, the type of which is determined according to the excitation wavelength band of the fluorescent probe in the reaction premix; and a multi-channel spectral sensor, which collects the fluorescence signals on multiple spectral channels of the reaction area.

13. The microfluidic isothermal amplification system according to claim 12, characterized in that: The processing device includes a microcontroller, a storage unit, a communication interface and an algorithm processing module; the processing device supports two working modes: real-time data processing and offline data analysis.

14. The microfluidic isothermal amplification system according to claim 12, characterized in that: The excitation light source is any one of a laser diode, a white light LED or a broadband light source; the multi-channel spectral sensor has 2-64 independent detection channels; the detection range of the multi-channel spectral sensor covers 350-1000 nm.

15. The microfluidic isothermal amplification system according to claim 12, characterized in that: The microfluidic isothermal amplification system is powered by a battery; the processing device has a low power consumption mode.

16. The microfluidic isothermal amplification system according to claim 12, characterized in that: The microfluidic isothermal amplification system can achieve simultaneous quantitative analysis of at least two fluorescent probes through a single, fixed optical detection path and only utilizes the spectral resolution capability of the multi-channel spectral sensor for mixed fluorescent signals and the deconvolution algorithm of the processing device.

17. A method for detecting pathogens, characterized in that: The method is based on the microfluidic isothermal amplification method according to any one of claims 1 to 9; the pathogen comprises at least one of a virus, a bacterium, and a fungus.

18. A genotyping method, characterized in that: The method is based on the microfluidic isothermal amplification method according to any one of claims 1 to 9; the genotype analysis method includes single nucleotide polymorphism detection and gene deletion / insertion mutation detection.

19. A food safety detection method, characterized in that: The method is based on the microfluidic isothermal amplification method described in any one of claims 1 to 9; the food safety detection method is used to detect pathogens, allergens, and genetically modified ingredients.

20. A reverse transcription analysis method, characterized in that: The method is based on the microfluidic isothermal amplification method according to any one of claims 1 to 9; the reverse transcription analysis method is used for RNA detection.

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