WO2022057584A1 - 一种高效的核酸检测和基因测序方法及其装置 - Google Patents

一种高效的核酸检测和基因测序方法及其装置 Download PDF

Info

Publication number
WO2022057584A1
WO2022057584A1 PCT/CN2021/114636 CN2021114636W WO2022057584A1 WO 2022057584 A1 WO2022057584 A1 WO 2022057584A1 CN 2021114636 W CN2021114636 W CN 2021114636W WO 2022057584 A1 WO2022057584 A1 WO 2022057584A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
spatial
signal
gene sequencing
fluorescence
Prior art date
Application number
PCT/CN2021/114636
Other languages
English (en)
French (fr)
Inventor
王中阳
李文文
肖康
高琪
孙静
Original Assignee
中国科学院上海高等研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海高等研究院 filed Critical 中国科学院上海高等研究院
Priority to US18/026,950 priority Critical patent/US20230257807A1/en
Publication of WO2022057584A1 publication Critical patent/WO2022057584A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06F—ELECTRIC DIGITAL DATA PROCESSING
    • G06F17/00—Digital computing or data processing equipment or methods, specially adapted for specific functions
    • G06F17/10—Complex mathematical operations
    • G06F17/16—Matrix or vector computation, e.g. matrix-matrix or matrix-vector multiplication, matrix factorization
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics
    • G01N2021/6471—Special filters, filter wheel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • G01N2201/129—Using chemometrical methods
    • G01N2201/1296—Using chemometrical methods using neural networks

Definitions

  • the present invention relates to the field of molecular diagnosis, and more particularly to an efficient nucleic acid detection and gene sequencing method and device thereof.
  • PCR polymerase chain reaction
  • qPCR real-time quantitative PCR
  • dPCR digital PCR
  • Gene sequencing is based on the principle of base complementary pairing to detect the nucleic acid sequence of living organisms, including DNA sequencing and RNA sequencing.
  • the method of fluorescent labeling is generally used for gene sequencing.
  • Four-color fluorescence imaging is achieved to identify bases.
  • the gene detector has also undergone three generations of development.
  • the technical basis of the second-generation sequencer is the fluorescence imaging of high-density gene chips, which has the advantages of high throughput and low cost.
  • PCR polymerase chain reaction
  • the third-generation sequencing technology does not require the process of PCR library building, it can directly sequence the DNA molecules in the sample. , the error rate of sequencing is still high, and the throughput and cost cannot be compared with the second-generation sequencing technology in a short period of time.
  • Multi-channel detection efficiency By labeling with different fluorescent dyes, data information of different channels can be obtained, and the detection category of a single sample can be improved.
  • the current multi-channel implementation methods on the market mainly include the following two detection methods: 1.
  • the multi-channel switching sequential exposure detection method is used, and only one fluorescent reagent can be detected at a time.
  • the detection efficiency is low and cannot meet the purpose of detecting multiple fluorescent reagents at the same time;
  • 2 The single-channel system is mechanically superimposed or integrated, and each independent detection system and each separate reagent to be detected are connected by optical fibers. This detection efficiency is relatively Compared with the single-channel system, it is improved, but the volume of the whole system is larger and the cost is higher.
  • the information acquisition efficiency is low, thus restricting the detection sensitivity and throughput, resulting in low detection efficiency.
  • the purpose of the present invention is to provide an efficient nucleic acid detection and gene sequencing method and device thereof, thereby solving the problems of low detection sensitivity, low throughput and low detection efficiency of the nucleic acid detection and gene sequencing methods in the prior art.
  • the present invention adopts the following technical solutions:
  • target nucleic acid sequences are labeled with different fluorescent probes, excited by a light source, and multicolor fluorescent signals are processed and collected, and the collected signals are reconstructed by optical correlation imaging method.
  • the spatial, spectral and intensity distribution information of probes enables high-throughput, high-sensitivity, rapid, multiplex nucleic acid detection and gene sequencing.
  • the spatial and spectral calibration matrix A is obtained by experimental calibration or ray tracing and wave optics calculation, or deep learning training, and the point light sources of different spatial positions and different wavelengths on the calibration surface are imaged by the imaging module The light intensity distribution imaged onto the area array detector, thereby constructing the spatial and spectral scaling matrix A.
  • the imaging module includes a projection lens group and a multi-channel filter group, and the area array detector detects the image based on the point spread function, Gaussian spot or Airy disk. Multicolor fluorescence two-dimensional intensity measurement matrix.
  • the imaging module includes a projection lens group, a multi-channel filter group and a spatial modulation module, wherein the spatial modulation module adopts a spatial random phase modulator to realize the optical field.
  • the speckle image of the fluorescence signal is obtained by random modulation, and the area array detector detects a multi-color fluorescence two-dimensional intensity measurement matrix based on the speckle pattern.
  • the imaging module includes a projection lens group, a multi-channel filter group and a spatial encoding module, wherein the spatial encoding module adopts a liquid crystal spatial light modulator or DMD to construct a specific Two-dimensional coding matrix, the area array detector detects the coded multi-color fluorescence two-dimensional intensity measurement matrix.
  • the imaging module includes a projection lens group, a multi-channel filter group and a dispersive element, and the dispersive element disperses the spectrum of the multi-color fluorescent signal, and the area array detects it.
  • the detector detects a multicolor fluorescence two-dimensional intensity measurement matrix based on spectral signals.
  • the correlation reconstruction algorithm is selected from any of the following methods A sort of:
  • the compressed sensing algorithm combined with the matrix mapping theory and the optical correlation imaging algorithm, can quickly recover the target signal space and spectral intensity information by finding the optimal solution of the signal;
  • Deep learning algorithm by building a neural network model, using the weak fluorescence signal at different photon number levels to continuously train and optimize the network, so as to achieve the recovery of fluorescence weak signal images;
  • the maximum likelihood estimation algorithm through the statistical probability relationship between the weak signal and the strong signal, establishes the likelihood function between the weak signal and the signal that needs to be restored, and uses the likelihood function to combine with the external prior information of the weak signal. Construct the objective function, optimize the likelihood function through the optimization method, so as to complete the recovery of the weak fluorescence signal, or combine with the compressed sensing algorithm to realize the sparse Poisson-based compressed sensing algorithm;
  • the image reconstruction algorithm based on sparse constraints, by marking the fluorescent signal with sparse characteristics, combined with the characteristics of noise that cannot be sparsely expressed, impose sparse constraints on the signal to be restored and combine the noise variance distribution to construct an optimization problem, and then use the optimization algorithm to restore The original fluorescent weak signal, or combined with the compressed sensing algorithm, realizes the compressed sensing algorithm based on sparse constraints.
  • an efficient nucleic acid detection and gene sequencing device which is used to realize the above-mentioned efficient nucleic acid detection and gene sequencing method.
  • the device includes: an excitation light source module, which is excited by a single excitation light source or multiple excitation light sources according to the requirements of single or multiple target fluorescent labeling of nucleic acid samples; an imaging module, including: a projection lens group and a multi-channel filter group; and Area array detector; wherein, the nucleic acid sample to be detected emits one-color or multi-color fluorescence signal after the sample is excited by single-channel excitation light source or multi-channel excitation light source, modulated and encoded by the imaging module, and then sampled by the area array detector, and finally adopted
  • the correlation reconstruction algorithm recovers the spatial, spectral and intensity distribution information of one-color or multi-color fluorescent molecules in the sample, and realizes high-throughput, high-sensitivity, rapid, multiple nucleic acid detection and sequencing of nucleic acid samples.
  • the nucleic acid sample to be detected may be a real-time quantitative PCR (qPCR) sample for nucleic acid detection, or a digital PCR (dPCR) chip, or a gene chip for gene sequencing, and the like.
  • Nucleic acid samples can be labeled with various fluorophores according to the detection requirements.
  • the excitation light source module adopts multiple or single excitation light sources according to fluorescence detection requirements.
  • the excitation light source is a high-power, narrow-band LED light source or a laser.
  • the excitation light source module has no mechanical device, simple structure, small volume and high wavelength utilization rate.
  • the above imaging modules are divided into the following four types:
  • the multi-channel filter set includes a dichroic mirror and a filter in front of the detection module, the dichroic mirror is used to reflect the light source into the lens set, and the reflected fluorescence collected by the lens set
  • the filter is a multi-channel filter, which suppresses the interference of the excitation light source and obtains multi-color fluorescent signals with high signal-to-noise ratio.
  • the area array detector detects the fluorescence signal Gaussian spot or Airy disk signal.
  • the spatial modulation module uses a spatial random phase modulator to realize random modulation of the light field of the fluorescent signal, and obtains the speckle image of the fluorescent signal.
  • the area array detector detects the speckle pattern on the entire imaging surface.
  • the spatial random phase modulator is ground glass with a certain aspect ratio range and random distribution of particles, or a phase modulator that generates random phases under computer programming control.
  • the spatial encoding module uses a liquid crystal spatial light modulator or a digital micromirror device (DMD) to construct a specific two-dimensional encoding matrix to encode the intensity of the fluorescent signal.
  • DMD digital micromirror device
  • the area array detector detects the intensity information encoded by the fluorescent signal.
  • the dispersive element adopts a grating or a prism to realize spectral dispersion and light separation, and the area array detector detects the spatial and spectral information of the fluorescent signal.
  • the area array detector adopts a single-photon camera composed of a combination of an image intensifier and a high-speed CMOS camera, or a two-dimensional array of photomultiplier tubes (PMT)/avalanche diodes (APD), with nanosecond high-speed electronic shutter and photoelectric sensor.
  • Second-level high-precision timing control can achieve high-speed detection with single-photon sensitivity, while effectively suppressing the interference of background light.
  • Other highly sensitive CMOS or CCD detectors can also be used.
  • the present invention provides an efficient nucleic acid detection and gene sequencing method.
  • a fluorescent probe is used to label a target nucleic acid sequence
  • a light source is used to excite and a fluorescent signal is processed and collected
  • an optical correlation imaging method is used to reconstruct the labeled fluorescent probe from the collected signal.
  • the spatial, spectral and intensity distribution information of needles enables high-throughput, high-sensitivity, rapid, multiplex nucleic acid, gene detection and sequencing. Based on the method, an efficient nucleic acid detection and gene sequencing device is provided.
  • the invention applies the image reconstruction method and algorithm based on photolinkage imaging to nucleic acid and gene detection and sequencing, and utilizes prior information to greatly improve the efficiency of information acquisition, as well as the signal-to-noise ratio, reconstruction accuracy and speed of image restoration, thereby Shorten nucleic acid, gene detection and sequencing time, and improve nucleic acid detection efficiency, detection sensitivity, throughput and accuracy.
  • the efficient nucleic acid detection and gene sequencing method and device of the present invention have the following advantages:
  • High-sensitivity and rapid detection using the correlation imaging method, using prior information, can greatly improve the image restoration signal-to-noise ratio and reconstruction accuracy, combined with single-photon sensitivity cameras, improve detection sensitivity and image information acquisition efficiency; at the same time, it can Efficiently detect fluorescent signals in fewer PCR cycles, reducing detection time.
  • the present invention adopts a highly sensitive and high-throughput detection method, which breaks through the limitations of traditional nucleic acid detection and gene sequencing on low-concentration nucleic acid samples, and has revolutionary advantages in low-concentration nucleic acid detection.
  • the optical system of the present invention has a simple structure and no mechanical transmission device, which simplifies the complex opto-mechanical device required for multi-channel fluorescence switching of traditional PCR instruments and gene sequencing devices and the huge optical system designed to achieve high throughput.
  • the system is conducive to realizing miniaturization.
  • the present invention changes the traditional optical detection method, develops an optical correlation imaging method for nucleic acid detection and gene sequencing, and based on this, proposes a small and efficient nucleic acid detection and gene sequencing device, which can realize high-throughput detection. Quantitative, highly sensitive, rapid, multiplex nucleic acid detection and gene sequencing.
  • FIG. 1 is a schematic diagram of the principle and process flow of an efficient nucleic acid detection and gene sequencing method proposed by the present invention.
  • FIG. 2 is a schematic diagram of the principle of a small and efficient nucleic acid detection and gene sequencing device based on a spatial phase modulator according to an embodiment of the present invention.
  • FIG. 3 is a schematic diagram of the principle of a small and efficient nucleic acid detection and gene sequencing device based on a dispersive element according to another embodiment of the present invention.
  • FIG. 4 is a schematic diagram of the principle of a small and efficient nucleic acid detection and gene sequencing device based on a spatial coding module according to another embodiment of the present invention.
  • FIG. 5 is a schematic diagram of the principle of a small and efficient nucleic acid detection and gene sequencing device according to another embodiment of the present invention.
  • an efficient nucleic acid detection and gene sequencing method includes the following steps:
  • Step S1 The single wavelength ⁇ 1 fluorescence signal emitted by the point light source 102 on the sample surface 101 is modulated or encoded by the imaging module or directly imaged on the detector, and the two-dimensional fluorescence intensity information 103 detected by the detector is used as a column of the calibration matrix A104 A(i,1); then move the point light source 102 to the next position on the sample surface 101, obtain the intensity information of this position as A(i,2), continue to repeat this operation until each position on the sample surface 101 is obtained
  • the intensity information 103 of the point light source is used as the calibration matrix A(i,1) ⁇ A(i,n); another point light source with wavelength ⁇ 2 is selected, and the above operation is repeated to obtain ⁇ 1 ⁇ m under different wavelengths and different spatial positions.
  • a spatial and spectral scaling matrix Ai ⁇ j is constructed, where i is the number of pixels of the two-dimensional detector and j is m ⁇ n. This process can be obtained through experimental calibration or ray tracing and wave optics calculations, or deep learning training.
  • Step S2 label the target nucleic acid sequence 106 with different fluorescent probes, prepare a nucleic acid chip 105 with spatial distribution, excite the nucleic acid chip 105 to emit multi-color fluorescent signals, and use the imaging module and the area array detector to sequentially modulate, encode and collect them , obtain multicolor fluorescence intensity information 107, and construct a two-dimensional intensity measurement matrix Y108;
  • the imaging modules can be divided into 4 types:
  • the area array detector detects a multi-color fluorescence two-dimensional intensity measurement matrix based on point spread function, Gaussian spot or Airy disk.
  • It includes a projection lens group, a multi-channel filter group and a spatial modulation module, wherein the spatial modulation module uses a spatial random phase modulator to realize random modulation of the light field, thereby obtaining a speckle image of the fluorescent signal, and the area detected by the area array detector.
  • the spatial modulation module uses a spatial random phase modulator to realize random modulation of the light field, thereby obtaining a speckle image of the fluorescent signal, and the area detected by the area array detector. is a speckle pattern-based multicolor fluorescence two-dimensional intensity measurement matrix.
  • the spatial encoding module uses a liquid crystal spatial light modulator or DMD to construct a specific two-dimensional encoding matrix, and the area array detector detects the encoded multicolor fluorescence Two-dimensional intensity measurement matrix.
  • the dispersive element disperses the spectrum of the polychromatic fluorescence signal, and the area array detector detects a two-dimensional intensity measurement matrix of the polychromatic fluorescence based on the spectral signal.
  • the detected two-dimensional fluorescence intensity information 103 and polychromatic fluorescence intensity information 107 may be polychromatic fluorescence two-dimensional intensity information based on point spread function, Gaussian spot or Airy disk, or Speckle pattern-based multicolor fluorescence 2D intensity information, or encoded multicolor fluorescence 2D intensity information, or multicolor fluorescence 2D intensity information based on spectral signals.
  • the associated imaging algorithm specifically includes the following four types:
  • Compressed sensing algorithm making full use of the developed compressed sensing theory and algorithm, combined with matrix mapping theory and algorithm, can quickly restore the measured fluorescence molecular space and spectral intensity information.
  • Deep learning algorithm by building neural network models, including convolutional neural networks, fully connected networks, generative adversarial networks and their combinations, and by using weak fluorescence signals at different photon number levels to continuously train and optimize the network, so as to achieve fluorescence Recovery of weak signal images.
  • the maximum likelihood estimation algorithm through the statistical probability relationship between the weak signal and the strong signal, establishes the likelihood function between the weak signal and the signal that needs to be restored, and uses the likelihood function to combine with the external prior information of the weak signal
  • the objective function is constructed to maximize the likelihood function through the optimization method, and then combined with the compressed sensing algorithm to complete the restoration of the original signal.
  • the image reconstruction algorithm based on sparse constraints, by marking the fluorescent signal with sparse characteristics, combined with the characteristics of noise that cannot be sparsely expressed, impose sparse constraints on the signal to be restored and combine the noise variance distribution to construct an optimization problem, and then use the optimization algorithm to restore
  • the raw fluorescent weak signal is combined with the compressed sensing algorithm to realize the compressed sensing algorithm based on sparse constraints.
  • a small and efficient nucleic acid detection and gene sequencing device includes an excitation light source module 201 , imaging modules 202 to 206 , and an area array detector 207 .
  • the multi-channel or single-channel excitation light source 201 is reflected by the dichroic mirror 202, and irradiates the nucleic acid sample 204 to be detected through the projection lens group 203, and the fluorescent signal generated by the excitation sample passes through the projection lens group 203, and then passes through the dichroic mirror.
  • the multi-channel filter set 205 further filters out the interference of the excitation light source, and the spatial phase modulator 206 performs random light field modulation on the fluorescence signal to obtain a speckle image of the fluorescence signal. Then use the area array detector 207 to sample the speckle image on the entire imaging surface, and finally use the correlation imaging reconstruction algorithm to recover the spatial, spectral and intensity distribution information of the fluorescent molecules in the sample, and realize high-throughput and rapid multiple nucleic acid detection. and gene sequencing.
  • the excitation light source module 201 uses multiple or single-channel LED light sources or lasers.
  • the nucleic acid sample 204 to be detected may be a real-time quantitative PCR (qPCR) sample for nucleic acid detection, or a digital PCR (dPCR) chip, or a gene chip for gene sequencing, or the like.
  • Nucleic acid samples can be labeled with various fluorophores according to the detection requirements.
  • the projection lens group 203 can use a compound lens with a large aperture and short focal length, or a high numerical aperture objective lens or a projection objective lens, or a microlens array to achieve a large field of view and high-efficiency fluorescence signal collection.
  • the spatial random phase modulator 206 is ground glass with a certain aspect ratio range and random distribution of particles, or a phase modulator that generates random phases under computer programming control.
  • the area array detector 207 can be a single-photon camera composed of a combination of an image intensifier and a high-speed CMOS camera, or a two-dimensional array of photomultiplier tubes (PMT)/avalanche diodes (APD) with nanosecond-scale High-speed electronic shutter and picosecond-level high-precision timing control.
  • PMT photomultiplier tubes
  • APD avalanche diodes
  • the fluorescence weak signal image reconstruction algorithm is mainly based on the correlation imaging reconstruction algorithm of the speckle field, and the specific algorithm refers to step S3 in the first embodiment.
  • the advantage of the optical correlation imaging algorithm based on the random measurement of the speckle field is that the random measurement method improves the random characteristics of the signal by performing random modulation of the light field on the fluorescent signal, and can better meet the compression requirements. Perceives the requirements of random measurement, which greatly improves the positioning accuracy and density of signal reconstruction, and has spectral resolution capability to achieve single-exposure multi-color imaging. This method greatly improves the efficiency of information acquisition and can achieve fast, high-throughput. , highly sensitive nucleic acid detection and gene sequencing.
  • a small and efficient nucleic acid detection and gene sequencing device includes an excitation light source module 301 , imaging modules 302 to 306 , and an area array detector 307 .
  • the multi-channel or single-channel excitation light source 301 is reflected by the dichroic mirror 302, and illuminates the nucleic acid sample 304 to be detected through the projection lens group 303, and the fluorescent signal generated by the excitation sample passes through the projection lens group 303, and then passes through the dichroic mirror.
  • the multi-channel filter set 305 further filters out the interference of the excitation light source, and the dispersive element 306 performs spectral dispersion on the fluorescence signal to obtain the spectral information of the fluorescence signal.
  • the area array detector 307 is used to sample the spatial and spectral information of the fluorescence signal on the entire imaging surface, and finally the correlation imaging reconstruction algorithm is used to recover the spatial, spectral and intensity distribution information of the fluorescent molecules in the sample, to achieve high-throughput, fast multiplex nucleic acid detection and gene sequencing.
  • the laser light source module 301 the multi-channel filter group 302 , the projection lens group 303 , the nucleic acid sample to be tested 304 , and the area array detector 307 are the same as those in the first embodiment.
  • dispersive element 306 may be a grating or a prism.
  • the optical link imaging reconstruction algorithm is mainly based on the correlation imaging calculation of the spatial and spectral signals.
  • the specific algorithm refer to step S3 in the first embodiment.
  • a small and highly sensitive multiple nucleic acid rapid detector includes an excitation light source module 401 , imaging modules 402 to 406 , and an area array detector 407 .
  • the multi-channel or single-channel excitation light source 401 is reflected by the dichroic mirror 402, and irradiates the nucleic acid sample 404 to be detected through the projection lens group 403, and the fluorescent signal generated by the excitation sample passes through the projection lens group 403, and then passes through the dichroic mirror.
  • 402 transmits
  • the multi-channel filter set 405 further filters out the interference of the excitation light source
  • the spatial encoding module 406 performs spatial intensity encoding on the fluorescence signal.
  • the laser light source module 401 the multi-channel filter group 402 , the projection lens group 403 , the nucleic acid sample to be tested 404 , and the area array detector 407 are the same as those in the first embodiment.
  • the spatial encoding module 406 employs a liquid crystal spatial light modulator or DMD to construct a specific two-dimensional encoding matrix.
  • the optical link imaging reconstruction algorithm is mainly based on the correlation imaging calculation of the encoded spatial signal.
  • the specific algorithm refer to step S3 in the first embodiment.
  • a small and highly sensitive multiple nucleic acid rapid detector includes an excitation light source module 501 , imaging modules 502 to 505 , and an area array detector 506 .
  • the multi-channel or single-channel excitation light source 501 is reflected by the dichroic mirror 502, and illuminates the nucleic acid sample 504 to be detected through the projection lens group 503, and the fluorescent signal generated by the excitation sample passes through the projection lens group 503, and then passes through the dichroic mirror.
  • 502 transmits, and the multi-channel filter set 505 further filters out the interference of the excitation light source.
  • the laser light source module 501 the projection lens group 503 , the multi-channel filter group 502 , the nucleic acid sample to be tested 504 , and the area array detector 506 are the same as those in the first embodiment.
  • the correlation imaging reconstruction algorithm is mainly based on using the point spread function (PSF).
  • PSF point spread function

Abstract

本发明提供一种高效的核酸检测和基因测序方法及其装置，该方法包括步骤：S1：构建空间和光谱定标矩阵A作为先验信息；S2：采用荧光探针标记目标核酸序列，制备具有空间分布的核酸芯片，光源激发核酸芯片发出多色荧光信号，采用成像模块和面阵探测器依次对多色荧光信号进行调制、编码和采集，获得荧光二维强度测量矩阵Y；S3：通过关联重构算法将定标矩阵A与测量矩阵Y进行关联计算，求解Y＝AX，重构出目标信号X，即标记核酸序列的荧光分子空间、光谱和强度分布信息，实现高效的核酸检测和基因测序。本发明通过改变传统光学检测方式，提供一种核酸检测和基因测序方法及其装置。

Description

一种高效的核酸检测和基因测序方法及其装置 技术领域
本发明涉及分子诊断领域，更具体地涉及一种高效的核酸检测和基因测序方法及其装置。
背景技术
核酸、基因检测主要采用聚合酶链式反应(PCR)技术，是一种在体外特异性扩增靶DNA序列的技术，通过多个循环的变性、退火和延伸，能够使微量的遗传物质在几小时内得到几百万倍的扩增，然后通过检测PCR扩增后的荧光信号来定性或定量。PCR技术已经成为了生命科学研究和临床分子诊断领域最重要的支撑技术和核心驱动力。PCR技术主要包括实时荧光定量PCR(qPCR)技术和数字PCR(dPCR)技术。但目前PCR技术在高准确率、低浓度的和快速检测方面仍无法满足快速高效的核酸检测市场需求。商用的dPCR为了提高核酸检测效率，采用样本分割技术，通过制备了数万个乃至数百万个并行PCR反应单元，或是优化PCR反应体系、增加荧光通道数(多重PCR)来实现高通量、高灵敏的检测。但仍然无法解决长时间的PCR反应导致的检测效率低的问题。
基因测序是根据碱基互补配对原理来检测生命体的核酸序列，包括DNA测序和RNA测序，目前普遍使用荧光标记的方法进行基因测序，通过对四类碱基标记四种不同的荧光基团，实现四色荧光成像，以此识别碱基。为了提高基因测序的效率，基因检测仪也经历了三代的发展，第二代测序仪的技术基础是高密度基因芯片的荧光成像，其优点是通量高、成本低，缺点是在测序之前都要通过聚合酶链式反应(PCR)实现DNA扩增的建库过程，这就可能引入外源的碱基突变，且二代测序技术普遍读长较短。第三代测序技术由于不需要经PCR建库的过程，直接对样本中的DNA分子进行测序，其潜在优点是速度快、准确率高，且有望大幅度降低成本，但限于目前的技术发展水平，测序的错误率还较高，且通量和成本在短时间内也无法与第二代测序技术相比。
因此，目前的核酸、基因检测和测序技术因传统光学检测技术的限制仍然存在不可克服的瓶颈，主要体现在以下两个方面：
多通道检测效率：通过不同荧光染料进行标记，可获得不同通道的数 据信息，提高单一样本的检测类别。但是由于光学检测手段的限制，目前市场上多通道的实现方式主要包括以下两种检测方式：一：采用多通道切换依次曝光检测方式，一次只能检测一种荧光试剂。检测效率较低，不能满足同时检测多种荧光试剂的目的；二：将单通道系统机械的进行叠加或集成，用光纤连接各个独立的检测系统和分开的各待检测试剂，这个检测效率相较于单通道系统有所提升，但整个系统的体积较大，成本较高。但是，以上两种方式都需要对多个荧光通道进行分步检测，样品被反复照射或者照射的时间有差异，荧光的淬灭性影响很难测定。并且不同荧光通道之间存在较大串扰和干扰，多色荧光检测技术存在技术不足。
2)检测时间：目前的光学检测手段由于探测灵敏度的限制，无法避免长时间的PCR的扩增反应来实现荧光累积，也就是说，光学检测能力决定了PCR的所需循环数。目前检测时间较长也是由于光学检测能力不足，导致所需的PCR循环数过多，从本质上无法满足快速检测的要求。因此，提高荧光探测灵敏度是实现快速PCR的重要手段，通过实现弱光检测能力，来达到大幅减少PCR扩增循环数的需求。实现弱光探测能力，一方面可从高信噪比、高纯度、高亮度荧光探针标记的样品制备方法出发，另一方面，优化光学系统，提高荧光弱信号的探测灵敏度是更本质的解决方法。
目前提高探测灵敏度的方法主要有以下两种：一：采用共聚焦式和光纤式两种光学结构，其优点是结构简单、荧光收集效率高，可具有更高检测精度，但由于采用点扫描方式，导致成像时间长，对于多通道检测，更是大大降低检测时间，另外，该方法对于光源和系统稳定性要求高，要避免由于发光强度的波动以及系统不稳定影响测量结果的准确性。二：设计大视场高通量的物镜来提高荧光收集效率，但这往往导致设备庞大且昂贵。
因此，由于传统光学检测技术的限制，使得信息获取效率低，从而制约探测灵敏度和通量，导致检测效率较低。
发明内容
本发明的目的是提供一种高效的核酸检测和基因测序方法及其装置，从而解决现有技术中的核酸检测和基因测序方法探测灵敏度低、通量低、导致检测效率低的问题。
为了解决上述技术问题，本发明采用以下技术方案：
根据本发明的第一方面，提供一种高效的核酸检测和基因测序方法， 包括以下步骤：S1：构建空间和光谱定标矩阵A作为先验信息；S2：采用荧光探针标记目标核酸序列，制备具有空间分布的核酸芯片，光源激发核酸芯片发出多色荧光信号，采用成像模块和面阵探测器依次对所述多色荧光信号进行调制、编码和采集，获得荧光二维强度测量矩阵Y；以及S3：通过关联重构算法来将所述定标矩阵A与所述测量矩阵Y进行关联计算，求解Y＝AX，重构出目标信号X，即所述标记目标核酸序列的荧光分子空间、光谱和强度分布信息，实现高效的核酸检测和基因测序。
根据本发明提供的上述方法的工作原理在于：采用不同荧光探针标记目标核酸序列，光源激发并对多色荧光信号进行处理并采集，对采集的信号利用光学关联成像方法重构出标记的荧光探针的空间、光谱和强度分布信息，实现高通量、高灵敏、快速、多重核酸检测和基因测序。
在所述步骤S1中，所述空间和光谱定标矩阵A采用实验标定或光线追迹和波动光学计算，或深度学习训练获得，通过标定面上不同空间位置、不同波长的点光源被成像模块成像到面阵探测器上的光强分布，从而构建出所述空间和光谱定标矩阵A。
根据本发明的一个优选方案，在所述步骤S2中，所述成像模块包括投影透镜组、多通道滤光片组，面阵探测器探测的是基于点扩散函数、高斯光斑或艾里斑的多色荧光二维强度测量矩阵。
根据本发明的另一优选方案，在所述步骤S2中，所述成像模块包括投影透镜组、多通道滤光片组和空间调制模块，其中空间调制模块采用空间随机相位调制器实现光场的随机调制，从而获得荧光信号的散斑图像，面阵探测器探测的是基于散斑图案的多色荧光二维强度测量矩阵。
根据本发明的又一优选方案，在所述步骤S2中，所述成像模块包括投影透镜组、多通道滤光片组和空间编码模块，其中空间编码模块采用液晶空间光调制器或DMD构建特定二维编码矩阵，面阵探测器探测的是经过编码的多色荧光二维强度测量矩阵。
根据本发明的再一优选方案，在所述步骤S2中，所述成像模块包括投影透镜组、多通道滤光片组和色散元件，色散元件对多色荧光信号的光谱色散分光，面阵探测器探测的是基于光谱信号的多色荧光二维强度测量矩阵。
在所述步骤S3中，针对探测到多色荧光二维强度信息，包括散斑信号、高斯光斑或艾里斑信号、编码信息和光谱信息，所述关联重构算法选自以下方法中的任意一种：
1)压缩感知算法，结合矩阵映射理论和光学关联成像算法，通过寻找信号的最优解，可快速恢复目标信号空间、光谱强度信息；
2)深度学习算法，通过构建神经网络模型，利用不同光子数水平下的弱荧光信号不断训练和优化网络，从而实现荧光弱信号图像的恢复；
3)极大似然估计算法，通过弱信号与强信号之间的统计概率关系，建立弱信号与需要恢复的信号之间的似然函数，利用似然函数与弱信号的外部先验信息结合构建目标函数，通过最优化方法优化似然函数，以此完成荧光弱信号的恢复，或是与压缩感知算法结合，实现基于稀疏泊松压缩感知算法；
4)基于稀疏约束的图像重构算法，通过标记荧光信号具有稀疏特性，结合噪声不能进行稀疏表达特点，对需要恢复的信号施加稀疏约束并结合噪声方差分布构建优化问题，进而利用最优化算法恢复原始荧光弱信号，或是与压缩感知算法结合，实现基于稀疏约束压缩感知算法。
根据本发明的第二方面，提供一种高效的核酸检测和基因测序装置，用于实现上述高效的核酸检测和基因测序方法。该装置包括：激发光源模块，根据核酸样品单重或多重靶点荧光标记的需求采用单路激发光源或多路激发光源激发；成像模块，包括：投影透镜组和多通道滤光片组；以及面阵探测器；其中，待检测的核酸样品经单路激发光源或多路激发光源激发样品后发出一色或多色荧光信号，经过成像模块调制、编码，再利用面阵探测器采样，最后采用关联重构算法恢复出样品中一色或多色荧光分子的空间、光谱和强度分布信息，实现对核酸样品的高通量、高灵敏、快速、多重核酸检测和测序。
所述待检测的核酸样品可以是用于核酸检测的实时荧光定量PCR(qPCR)样品，或数字PCR(dPCR)芯片，或用于基因测序的基因芯片等。核酸样品可根据检测需求标记的多种荧光基团。
所述激发光源模块根据荧光检测需求采用多路或单路激发光源。所述的激发光源是高功率、窄带的LED光源或激光。该激发光源模块无机械装置，结构简单，体积小，波长利用率高。
上述的成像模块分为以下4种：
1)由投影透镜组、多通道滤光片组组成，其中所述的投影透镜组可以采用大孔径短焦的复合透镜，或者高数值孔径物镜，或投影物镜，再或者微透镜阵列，来实现大视场，高效率的荧光信号收集。所述的多通道滤光片组包括二向色镜和探测模块前的滤光片，二向色镜用于将光源反射进所述透镜组， 并透过所述透镜组收集的反射的荧光信号；所述滤波片为多通道滤光片，抑制激发光源的干扰，获取高信噪比的多色荧光信号。经过该成像模块，面阵探测器探测的是荧光信号高斯光斑或艾里斑信号。
2)由投影透镜组、多通道滤光片组和空间调制模块组成，所述的投影透镜组、多通道滤光片组与上述1相同。空间调制模块则采用空间随机相位调制器对荧光信号实现光场随机调制，获得荧光信号的散斑图像，面阵探测器探测的是整个成像面上散斑图案。所述的空间随机相位调制器为具有一定高宽比范围、颗粒随机分布的毛玻璃，或电脑编程控制产生随机相位的相位调制器。
3)由投影透镜组、多通道滤光片组和空间编码模块组成，其中空间编码模块采用液晶空间光调制器或数字微镜器件(DMD)构建特定二维编码矩阵，对荧光信号进行强度编码，面阵探测器探测的是荧光信号编码的强度信息。
4)由投影透镜组、多通道滤光片组和色散元件组成，所述的荧光收集透镜组、多通道滤光片组与上述第一种成像模块相同。所述色散元件采用光栅或棱镜，实现光谱色散分光，面阵探测器探测的是荧光信号空间和光谱信息。
所述面阵探测器采用由像增强器和高速CMOS相机结合构成的单光子相机，或采用光电倍增管(PMT)/雪崩二极管(APD)的二维阵列，具有纳秒级高速电子快门及皮秒级高精度时序控制，可实现单光子灵敏度的高速探测，同时有效的抑制背景光的干扰。亦可采用其它高灵敏的CMOS或CCD探测器。
本发明提供一种高效的核酸检测和基因测序方法，采用荧光探针标记目标核酸序列，光源激发并对荧光信号进行处理并采集，对采集的信号利用光学关联成像方法重构出标记的荧光探针的空间、光谱和强度分布信息，实现高通量、高灵敏、快速、多重核酸、基因检测和测序。基于该方法提供了一种高效的核酸检测和基因测序装置。本发明将基于光联成像的图像重构方法和算法应用到核酸、基因检测和测序中，利用先验信息，大大提高信息获取效率，以及图像恢复的信噪比、重构精度和速度，从而缩短核酸、基因检测和测序时间，提高了核酸检测效率、探测灵敏度、通量和准确度。
本发明所述的高效的核酸检测和基因测序方法和装置与现有方法和装置相比具有以下优点：
1)高灵敏度快速探测，采用关联成像方法，利用先验信息，可大幅度提高图像恢复信噪比和重构精度，结合单光子灵敏度的相机，提高探测灵敏度 与图像信息的获取效率；同时可以在更少的PCR循环数内有效检测荧光信号，缩短检测时间。
2)多重荧光快速探测，采用随机相位调制器、编码和色散元件，并结合关联成像重构方法，可实现单次曝光多色荧光成像。克服传统多重荧光PCR检测和基因测序采用的多通道切换依次曝光方法从而检测速度慢的限制，且消除了荧光信号之间的干扰问题。实现多样本、多基因靶点实时同步快速检测，提高检测效率，降低检测成本。
3)低浓度核酸定量检测，本发明采用高灵敏、高通量的探测方式，突破传统核酸检测和基因测序对低浓度核酸样本的限制，在低浓度核酸检测上具有革命性的优势。
4)仪器的小型化，本发明光学系统结构简单，无机械传动装置，简化了传统PCR仪和基因测序装置多通道荧光切换需要的复杂的光机装置以及为了实现高通量而设计的庞大光学系统，有利于实现小型化。
综上所述，本发明改变传统光学检测方式，开发出了一种用于核酸检测和基因测序的光学关联成像方法，并基于此提出一种小型高效的核酸检测和基因测序装置，实现高通量、高灵敏、快速、多重核酸检测和基因测序。
附图说明
图1为本发明提出的一种高效的核酸检测和基因测序方法原理和流程示意图。
图2为根据本发明的一个实施例的基于空间相位调制器的小型高效的核酸检测和基因测序装置原理示意图。
图3为根据本发明的另一个实施例的基于色散元件的小型高效的核酸检测和基因测序装置原理示意图。
图4为根据本发明的另一个实施例的基于空间编码模块的小型高效的核酸检测和基因测序装置原理示意图。
图5为根据本发明的另一个实施例的小型高效的核酸检测和基因测序装置原理示意图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可 以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。需说明的是，在不冲突的情况下，以下实施例及实施例中的特征可以相互组合。
需要说明的是，以下实施例中所提供的图示仅以示意方式说明本发明的基本构想，遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制，其实际实施时各组件的型态、数量及比例可为一种随意的改变，且其组件布局型态也可能更为复杂。
第一实施例 高效的核酸检测和基因测序方法
如图1所示，根据本发明的第一实施例的一种高效的核酸检测和基因测序方法包括如下步骤：
步骤S1：样品面101上的点光源102发出的单波长λ1的荧光信号经成像模块调制或编码或直接成像到探测器上，探测器探测的二维荧光强度信息103作为定标矩阵A104的一列A(i,1)；接着在样品面101上移动点光源102到下个位置，获得该位置的强度信息作为A(i,2)，继续重复此操作，直到获得样品面101上每个位置点光源的强度信息103，作为定标矩阵A(i,1)～A(i,n)；选择另一个波长λ2的点光源，重复上述操作，获得λ1～λm不同波长下、不同空间位置下点光源在探测面上的强度信息，构建空间和光谱定标矩阵Ai×j，其中i是二维探测器的像素数，j是m×n。该过程可以通过实验标定或光线追迹和波动光学计算，或深度学习训练获得。
步骤S2：采用不同荧光探针标记目标核酸序列106，制备具有空间分布的核酸芯片105，激发核酸芯片105发出多色荧光信号，采用成像模块和面阵探测器依次对其进行调制、编码和采集，获得多色荧光强度信息107，构建二维强度测量矩阵Y108；
步骤S3：通过关联重构算法来将所述定标矩阵104与所述测量矩阵108进行关联计算，求解Y＝AX，重构出目标信号X109，即所述核酸芯片105中标记目标核酸序列的荧光分子空间、光谱和强度分布信息。
需要说明的是，在所述步骤S1和S2中，成像模块可以分为4种：
1.包括投影透镜组、多通道滤光片组，面阵探测器探测的是基于点扩散函数、高斯光斑或艾里斑的多色荧光二维强度测量矩阵。
2.包括投影透镜组、多通道滤光片组和空间调制模块，其中空间调制模块采用空间随机相位调制器实现光场的随机调制，从而获得荧光信号的散斑图 像，面阵探测器探测的是基于散斑图案的多色荧光二维强度测量矩阵。
3.包括投影透镜组、多通道滤光片组和空间编码模块，其中空间编码模块采用液晶空间光调制器或DMD构建特定二维编码矩阵，面阵探测器探测的是经过编码的多色荧光二维强度测量矩阵。
4.包括投影透镜组、多通道滤光片组和色散元件，色散元件对多色荧光信号的光谱色散分光，面阵探测器探测的是基于光谱信号的多色荧光二维强度测量矩阵。
因此，在所述步骤S1和S2中，探测到的二维荧光强度信息103和多色荧光强度信息107可以是基于点扩散函数、高斯光斑或艾里斑的多色荧光二维强度信息，或基于散斑图案的多色荧光二维强度信息，或编码的多色荧光二维强度信息，或基于光谱信号的多色荧光二维强度信息。
需要说明的是，在所述步骤S3中关联成像算法具体包括以下4种：
1.压缩感知算法，充分利用发展的压缩感知理论和算法，结合矩阵映射理论和算法，可快速恢复测量的荧光分子空间、光谱强度信息。
2.深度学习算法，通过构建神经网络模型，包括卷积神经网络、全连接网络、生成对抗网络及其组合，通过利用不同光子数水平下的弱荧光信号去不断训练和优化网络，从而实现荧光弱信号图像的恢复。
3.极大似然估计算法，通过弱信号与强信号之间的统计概率关系，建立弱信号与需要恢复的信号之间的似然函数，利用似然函数与弱信号的外部先验信息结合构建目标函数，通过最优化方法实现似然函数最大，再结合压缩感知算法结合，以此完成原始信号的恢复。
4.基于稀疏约束的图像重构算法，通过标记荧光信号具有稀疏特性，结合噪声不能进行稀疏表达特点，对需要恢复的信号施加稀疏约束并结合噪声方差分布构建优化问题，进而利用最优化算法恢复原始荧光弱信号，并与压缩感知算法结合，实现基于稀疏约束压缩感知算法。
第二实施例 基于空间相位调制器的小型高效的核酸检测和基因测序装置
如图2所示为根据本发明的第一实施例的一种小型高效的核酸检测和基因测序装置，包括激发光源模块201、成像模块202～206、面阵探测器207。多路或单路的激发光源201经过二向色镜202反射，通过投影透镜组203照射到待检测的核酸样品204上，激发样品产生的荧光信号通过投影透镜组203，再经过二向色镜202透射，多通道滤光片组205进一步滤去激发光源的干扰， 以及空间相位调制器206对荧光信号进行光场随机调制，获得荧光信号的散斑图像。再利用面阵探测器207对整个成像面上的散斑图像采样，最后采用关联成像重构算法恢复出样品中荧光分子的空间、光谱和强度分布信息，实现高通量、快速的多重核酸检测和基因测序。
在本实例中，激发光源模块201采用多路或单路的LED光源或激光。
在本实例中，待检测的核酸样品204可以是用于核酸检测的实时荧光定量PCR(qPCR)样品，或数字PCR(dPCR)芯片，或用于基因测序的基因芯片等。核酸样品可根据检测需求标记的多种荧光基团。
在本实例中，投影透镜组203可以采用大孔径短焦的复合透镜，或者高数值孔径物镜或投影物镜，再或者微透镜阵列，来实现大视场，高效率的荧光信号收集。
空间随机相位调制器206为具有一定高宽比范围、颗粒随机分布的毛玻璃，或电脑编程控制产生随机相位的相位调制器。
在本实例中，面阵探测器207可采用由像增强器和高速CMOS相机结合构成的单光子相机，或采用光电倍增管(PMT)/雪崩二极管(APD)的二维阵列，具有纳秒级高速电子快门及皮秒级高精度时序控制。
在本实施例中，针对面阵探测器探测到荧光散斑信号，荧光弱信号图像重构算法主要基于散斑场的关联成像重构算法，具体算法参照第一实施例中步骤S3。
由此，在本实施例中，基于散斑场随机测量的光学关联成像算法的好处在于，所述随机测量方法通过对荧光信号进行光场随机调制，提升了信号的随机特性，更能满足压缩感知随机测量的要求，大幅度提高了信号重构的定位精度和密度，并且具有光谱分辨能力，实现单次曝光多色成像，该方法极大地提升了信息获取效率，能实现快速、高通量、高灵敏的核酸检测和基因测序。
第三实施例 基于色散元件的小型高效的核酸检测和基因测序装置
如图3所示为根据本发明的第三实施例的一种小型高效的核酸检测和基因测序装置，包括激发光源模块301、成像模块302～306、面阵探测器307。多路或单路的激发光源301经过二向色镜302反射，通过投影透镜组303照射到待检测的核酸样品304上，激发样品产生的荧光信号通过投影透镜组303，再经过二向色镜302透射，多通道滤光片组305进一步滤去激发光源的干扰，以及色散元件306对荧光信号进行光谱色散分光，获得荧光信号的光谱信息。 再利用面阵探测器307对整个成像面上的荧光信号空间和光谱信息进行采样，最后采用关联成像重构算法恢复出样品中荧光分子的空间、光谱和强度分布信息，实现高通量、快速的多重核酸检测和基因测序。
在本实例中，激光光源模块301、多通道滤光片组302、投影透镜组303、待测核酸样品304、面阵探测器307同实施例一。
在本实例中，色散元件306可以是光栅或棱镜。
在本实施例中，针对面阵探测器探测到荧光信号空间和光谱信息，光联成像重构算法主要基于空间和光谱信号的关联成像计算，具体算法参照第一实施例中步骤S3。
第四实施例 基于空间编码模块的小型高效的核酸检测和基因测序装置
如图4所示为根据本发明的第四实施例的一种小型高灵敏多重核酸快速检测仪，包括激发光源模块401、成像模块402～406、面阵探测器407。多路或单路的激发光源401经过二向色镜402反射，通过投影透镜组403照射到待检测的核酸样品404上，激发样品产生的荧光信号通过投影透镜组403，再经过二向色镜402透射，多通道滤光片组405进一步滤去激发光源的干扰，以及空间编码模块406对荧光信号进行空间强度编码。再利用面阵探测器407对整个成像面上的编码后的荧光信号空间信息进行采样，最后采用关联成像重构算法恢复出样品中荧光分子的空间、光谱和强度分布信息，实现高通量、快速的多重核酸检测和基因测序。
在本实例中，激光光源模块401、多通道滤光片组402、投影透镜组403、待测核酸样品404、面阵探测器407同实施例一。
在本实例中，空间编码模块406采用液晶空间光调制器或DMD构建特定二维编码矩阵。
在本实施例中，针对面阵探测器探测到荧光信号编码后的空间信息，光联成像重构算法主要基于编码的空间信号的关联成像计算，具体算法参照第一实施例中步骤S3。
第五实施例 小型高效的核酸检测和基因测序装置
如图5所示为根据本发明的第五实施例的一种小型高灵敏多重核酸快速检测仪，包括激发光源模块501、成像模块502～505、面阵探测器506。多路或单路的激发光源501经过二向色镜502反射，通过投影透镜组503照射到待检测的核酸样品504上，激发样品产生的荧光信号通过投影透镜组503， 再经过二向色镜502透射，多通道滤光片组505进一步滤去激发光源的干扰。获得基于点扩散函数(PSF)、高斯光斑或艾里(Airy)斑的荧光信号图像，利用面阵探测器506对整个成像面上的荧光信号直接采样，最后采用关联成像重构算法恢复出样品中荧光分子的空间、光谱和强度分布信息，实现高通量、快速的多重核酸检测和基因测序。
在本实例中，激光光源模块501、投影透镜组503、多通道滤光片组502、待测核酸样品504、面阵探测器506同实施例一。
在本实施例中，针对面阵探测器探测到荧光信号空间分布的点扩散函数(PSF)、高斯光斑或艾里(Airy)斑，关联成像重构算法主要基于采用点扩散函数(PSF)、高斯光斑或艾里(Airy)斑的强度关联计算，具体算法参照第一实施例中步骤S3。
以上所述的，仅为本发明的较佳实施例，并非用以限定本发明的范围，本发明的上述实施例还可以做出各种变化。凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰，皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。

Claims (9)

  1. 一种高效的核酸检测和基因测序方法，其特征在于，包括以下步骤：
    S0：提供一种高效的核酸检测和基因测序装置，包括：激发光源模块，根据核酸样品单重或多重靶点荧光标记的需求采用单路激发光源或多路激发光源激发；成像模块，包括：投影透镜组和多通道滤光片组，还包括：空间调制模块或空间编码模块或色散元件，其中，所述空间调制模块采用空间随机相位调制器实现光场的随机调制，从而获得荧光信号的散斑图像，所述空间编码模块采用液晶空间光调制器或DMD构建特定二维编码矩阵；以及面阵探测器，所述面阵探测器采用基于微通道板的像增强器和高速CMOS相机结合构成的单光子相机，或采用光电倍增管/雪崩二极管的二维阵列，或高灵敏的面阵CMOS或CCD相机；
    S1：构建空间和光谱定标矩阵A作为先验信息；
    S2：采用荧光探针标记目标核酸序列，制备具有空间分布的核酸芯片，光源激发核酸芯片发出多色荧光信号，采用成像模块和面阵探测器依次对所述多色荧光信号进行调制、编码和采集，获得荧光二维强度测量矩阵Y；以及
    S3：通过关联成像算法来将所述定标矩阵A与所述测量矩阵Y进行关联计算，求解Y＝AX，重构出目标信号X，即所述标记核酸序列的荧光分子空间、光谱和强度分布信息，从而实现高效的核酸检测和基因测序。
  2. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S1中，所述空间和光谱定标矩阵A采用实验标定或光线追迹和波动光学计算或深度学习训练获得，通过标定面上不同空间位置、不同波长的点光源被成像模块成像到面阵探测器上的光强分布，从而构建出所述空间和光谱定标矩阵A。
  3. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S2中，所述成像模块包括投影透镜组、多通道滤光片组，面阵探测器探测的是基于点扩散函数、高斯光斑或艾里斑的多色荧光二维强度测量矩阵。
  4. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S0及S2中，所述成像模块包括投影透镜组、多通道滤光片组和空间调制模块，其中空间调制模块采用空间随机相位调制器实现光场的随机调制，从而获得荧光信号的散斑图像，面阵探测器探测的是基于散斑图案的多色荧光二维强度测量矩阵。
  5. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S0及S2中，所述成像模块包括投影透镜组、多通道滤光片组和空间编码模块，其中空间编码模块采用液晶空间光调制器或DMD构建特定二维编码矩阵，面阵探测器探测的是经过编码的多色荧光二维强度测量矩阵。
  6. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S0及S2中，所述成像模块包括投影透镜组、多通道滤光片组和色散元件，色散元件对多色荧光信号的光谱色散分光，面阵探测器探测的是基于光谱信号的多色荧光二维强度测量矩阵。
  7. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S3中，所述关联成像算法选自以下方法中的任意一种：
    1)压缩感知算法，结合矩阵映射理论和光学关联成像算法，通过
    Figure PCTCN2021114636-appb-100001
    寻找信号的最优解，可快速恢复目标信号空间、光谱强度信息；
    2)深度学习算法，通过构建神经网络模型，利用不同光子数水平下的弱荧光信号不断训练和优化网络，从而实现荧光弱信号图像的恢复；
    3)极大似然估计算法，通过弱信号与强信号之间的统计概率关系，建立弱信号与需要恢复的信号之间的似然函数，利用似然函数与弱信号的外部先验信息结合构建目标函数，通过最优化方法优化似然函数，以此完成荧光弱信号的恢复，或是与压缩感知算法结合，实现基于稀疏泊松压缩感知算法；
    4)基于稀疏约束的图像重构算法，通过标记荧光信号具有稀疏特性，结合噪声不能进行稀疏表达特点，对需要恢复的信号施加稀疏约束并结合噪声方差分布构建优化问题，进而利用最优化算法恢复原始荧光弱信号，或是与压缩感知算法结合，实现基于稀疏约束压缩感知算法。
  8. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S0中激发光源为LED或激光。
  9. 根据权利要求1所述的高效的核酸检测和基因测序方法，其特征在于，在所述步骤S0中的投影透镜组包括：大孔径短焦的复合透镜、高数值孔径物镜、投影物镜、或者微透镜阵列。
PCT/CN2021/114636 2020-09-18 2021-08-26 一种高效的核酸检测和基因测序方法及其装置 WO2022057584A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/026,950 US20230257807A1 (en) 2020-09-18 2021-08-26 Efficient nucleic acid testing and gene sequencing method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010982860.5 2020-09-18
CN202010982860.5A CN111926065B (zh) 2020-09-18 2020-09-18 一种高效的核酸检测和基因测序方法及其装置

Publications (1)

Publication Number Publication Date
WO2022057584A1 true WO2022057584A1 (zh) 2022-03-24

Family

ID=73334557

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/114636 WO2022057584A1 (zh) 2020-09-18 2021-08-26 一种高效的核酸检测和基因测序方法及其装置

Country Status (3)

Country Link
US (1) US20230257807A1 (zh)
CN (1) CN111926065B (zh)
WO (1) WO2022057584A1 (zh)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111926065B (zh) * 2020-09-18 2021-01-29 中国科学院上海高等研究院 一种高效的核酸检测和基因测序方法及其装置
CN113506643A (zh) * 2021-07-08 2021-10-15 沈阳知健健康管理有限公司 一种基因健康管理系统
CN114134025B (zh) * 2021-11-19 2023-08-22 中国科学院长春光学精密机械与物理研究所 基因测序系统及其测序方法
CN114480582B (zh) * 2022-02-07 2024-04-12 华中农业大学 运用于空间组学高密度信号分解稀疏解读的方法及其应用
CN115266662A (zh) * 2022-06-13 2022-11-01 深圳赛陆医疗科技有限公司 一种高光谱测序方法、系统和基因测序仪
CN116033138B (zh) * 2023-03-27 2023-06-02 中国科学院国家空间科学中心 一种单次曝光压缩感知被动三维成像系统及方法
CN117237352B (zh) * 2023-11-14 2024-02-09 之江实验室 基因芯片的图像处理方法、数据处理装置及存储介质

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102612652A (zh) * 2009-08-06 2012-07-25 康奈尔大学 用于分子分析的设备和方法
CN103582887A (zh) * 2011-06-07 2014-02-12 皇家飞利浦有限公司 提供核苷酸序列数据
US20180327829A1 (en) * 2015-11-18 2018-11-15 Kalim U. Mir Super-Resolution Sequencing
US20190046985A1 (en) * 2015-09-17 2019-02-14 The Regents Of The University Of California Droplet-trapping devices for bioassays and diagnostics
US20190271645A1 (en) * 2018-03-05 2019-09-05 SmartSens Technology (Cayman) Co., Limited. Dna sequencing system with stacked bsi global shutter image sensor
US20200056229A1 (en) * 2017-11-29 2020-02-20 Xgenomes Corp. Sequencing by emergence
CN111926065A (zh) * 2020-09-18 2020-11-13 中国科学院上海高等研究院 一种高效的核酸检测和基因测序方法及其装置

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102612652A (zh) * 2009-08-06 2012-07-25 康奈尔大学 用于分子分析的设备和方法
CN103582887A (zh) * 2011-06-07 2014-02-12 皇家飞利浦有限公司 提供核苷酸序列数据
US20190046985A1 (en) * 2015-09-17 2019-02-14 The Regents Of The University Of California Droplet-trapping devices for bioassays and diagnostics
US20180327829A1 (en) * 2015-11-18 2018-11-15 Kalim U. Mir Super-Resolution Sequencing
US20200056229A1 (en) * 2017-11-29 2020-02-20 Xgenomes Corp. Sequencing by emergence
US20190271645A1 (en) * 2018-03-05 2019-09-05 SmartSens Technology (Cayman) Co., Limited. Dna sequencing system with stacked bsi global shutter image sensor
CN111926065A (zh) * 2020-09-18 2020-11-13 中国科学院上海高等研究院 一种高效的核酸检测和基因测序方法及其装置

Also Published As

Publication number Publication date
CN111926065A (zh) 2020-11-13
US20230257807A1 (en) 2023-08-17
CN111926065B (zh) 2021-01-29

Similar Documents

Publication Publication Date Title
WO2022057584A1 (zh) 一种高效的核酸检测和基因测序方法及其装置
US11280718B2 (en) Parallel flow cytometer using radiofrequency multiplexing
JP6002232B2 (ja) 時間分解単一光子計数イメージングスペクトルシステム
EP1983331B1 (en) Optical system for reading a biochip
CN201653906U (zh) 生物免疫层析芯片检测仪
WO2023197734A1 (zh) 多通道超分辨基因检测仪及其检测方法
US20020146734A1 (en) Method and apparatus for labeling and analyzing cellular components
CN110132910B (zh) 基于光场多维信息融合显微超分辨成像装置和成像方法
CN110793633B (zh) 基于集束光纤的单像元多光谱计算成像系统及成像方法
CN210215369U (zh) 一种基因测序仪光学系统
US20110017915A1 (en) Drift scanner for rare cell detection
CN110836883A (zh) 基于spad的时间相关拉曼-荧光寿命光谱仪
CN109557070A (zh) 一种基于空间编码光的拉曼成像系统
US10585272B2 (en) Coherent fluorescence super-resolution microscopy
WO2022107557A1 (ja) 生体試料分析システム、情報処理装置、情報処理方法及び生体試料分析方法
CN101107508B (zh) 用于测量粒子动态参数的方法和装置
JP2004184379A (ja) マイクロアレイの読取方法
Liu et al. Parallel scan hyperspectral fluorescence imaging system and biomedical application for microarrays
CN113755311B (zh) 一种基因芯片阅读仪
WO2023223851A1 (ja) 生体試料分析システム、情報処理装置、情報処理方法及び生体試料分析方法
CN1174239C (zh) 光谱像生物芯片检测仪
CN117723515A (zh) 基于单光子雪崩二极管的单像素荧光多维显微成像方法
RU2679605C2 (ru) Флуориметрический анализатор биологических микрочипов
CN105548179A (zh) 一种基于透射光或自发光测定生物芯片的方法及系统
Basiji Multispectral imaging in flow: a technique for advanced cellular studies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21868412

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21868412

Country of ref document: EP

Kind code of ref document: A1