CN111206081B

CN111206081B - Nucleic acid detection microsphere, preparation method, kit and high-throughput nucleic acid detection method

Info

Publication number: CN111206081B
Application number: CN201811392278.2A
Authority: CN
Inventors: 盛广济
Original assignee: Sinafo Suzhou Life Technology Co ltd
Current assignee: Sinafo Suzhou Life Technology Co ltd
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2023-06-30
Anticipated expiration: 2038-11-21
Also published as: CN111206081A

Abstract

The application provides a nucleic acid detection microsphere, a preparation method, a kit and a high-throughput nucleic acid detection method. The nucleic acid detection microsphere consists of a nucleosome with fluorescence coding information and a coating layer of a primer or a probe. The nucleic acid detection microspheres are randomly distributed in micro-droplets generated by a nucleic acid reaction solution of a sample to be detected. When the temperature is raised to above 60 ℃, the coating layer is melted, and the primer and the probe are released into a sample nucleic acid reaction liquid to be detected, so that a complete nucleic acid amplification reaction system is formed, and the fluorescence-encoding nucleosome is remained in the nucleic acid amplification liquid and is used as a fluorescence label for marking micro-droplets. And screening out micro-droplets with only one nucleon as effective droplets to enter subsequent analysis by detecting the nucleon with fluorescence coding information in each micro-droplet, thereby obtaining the primer and probe types in the effective micro-droplets. And then reading the report fluorescence signal after the nucleic acid amplification reaction to obtain whether the effective micro-droplets contain corresponding target nucleic acid molecules to be detected.

Description

Nucleic acid detection microsphere, preparation method, kit and high-throughput nucleic acid detection method

Technical Field

The application relates to the field of nucleic acid detection and analysis, in particular to a nucleic acid detection microsphere, a preparation method, a kit and a high-throughput nucleic acid detection method.

Background

In recent years, PCR (Digital PCR) technology has rapidly progressed. Polymerase chain reaction (Polymerase Chain Reaction, PCR) is a molecular biological technique used to amplify specific DNA fragments. PCR includes a droplet PCR detection method and a chip-based detection method.

The microdroplet PCR system performs microdroplet processing on the sample prior to conventional PCR amplification, i.e., the reaction system containing the nucleic acid molecules is divided into thousands of picoliter or nanoliter microdroplets, wherein each microdroplet either does not contain the nucleic acid target molecule to be detected or contains one to several nucleic acid target molecules to be detected. After PCR amplification, the microdroplets are detected, but with conventional PCR detection techniques, only limited target sequences (related to the number of fluorescent channels of the device and the type of probe designed) can be detected. If the PCR detection is to detect more than ten, tens or hundreds of target sequences, the detection needs to be repeated for many times, the workload is increased, a large number of samples are consumed, and the time and the working efficiency are low.

Disclosure of Invention

Based on the above, it is necessary to provide a nucleic acid detection microsphere for high-throughput nucleic acid detection analysis, a preparation method, a kit and a high-throughput nucleic acid detection method, which can realize high-throughput, high-sensitivity and short-detection time, aiming at the problems of repeated detection, large workload and time consumption of the conventional PCR detection technology.

The application provides a nucleic acid detection microsphere for high throughput nucleic acid detection analysis. The nucleic acid detection microsphere includes a core and a coating. The nuclei have fluorescence encoded information. The coating layer wraps the nucleus body, the coating layer comprises a matrix and primers dispersed in the matrix, and the primers uniquely correspond to the nucleus body.

In one embodiment, the coating further comprises probes, and the probes and the primers are dispersed in the matrix and uniquely correspond to the nuclei.

In one embodiment, the matrix is an agarose gel.

In one embodiment, the nucleus is a solid sphere containing a fluorescent dye.

In one embodiment, the diameter of the nucleus is 10 microns to 100 microns.

In one embodiment, the cladding layer has a thickness of 10 microns to 100 microns.

In one embodiment, a method of preparing a nucleic acid detection microsphere comprises:

s110, providing a plurality of nucleosomes and primer solutions;

s120, providing gel powder, adding the gel powder into double distilled water to obtain a gel powder solution, and heating the gel powder solution to be clear to obtain a coating layer preparation solution;

s130, mixing the plurality of nuclear bodies, the primer solution and the coating layer preparation solution at the gel melting temperature to obtain a nucleic acid detection microsphere preparation solution;

s140, at the gel melting temperature, forming a plurality of nucleic acid detection microsphere droplets by forming the nucleic acid detection microsphere preparation solution into droplets;

and S150, cooling the plurality of nucleic acid detection microsphere liquid drops, and obtaining a plurality of nucleic acid detection microspheres through flow separation.

In one embodiment, in step S150, the nucleic acid detection microsphere is a nucleic acid detection microsphere comprising a single of the nuclei.

In one embodiment, in the step S120, the gel powder is agar powder or polyethylene glycol diacrylate.

In one embodiment, the step S140 includes:

s141, providing a liquid-spraying gun head with an outlet end, wherein the liquid-spraying gun head stores the nucleic acid detection microsphere preparation solution and provides an open container storing hydrophobic oil;

S142, inserting the outlet end of the liquid discharge gun head into the position below the liquid surface of the hydrophobic oil at the gel melting temperature;

and S143, the outlet end of the liquid discharge gun head performs instantaneous acceleration movement or speed change period movement under the liquid level of the hydrophobic oil, the nucleic acid detection microsphere preparation solution is discharged from the outlet end of the liquid discharge gun head, and the plurality of nucleic acid detection microsphere liquid drops are formed under the liquid level of the hydrophobic oil.

s210, providing a primer solution, a probe solution and a plurality of nuclei;

s220, providing gel powder, adding the gel powder into double distilled water to obtain a gel powder solution, and heating the gel powder solution until the gel powder solution is clear to obtain a coating preparation solution;

s230, mixing the plurality of nuclear bodies, the primer and the probe solution with the coating preparation solution at the gel melting temperature to obtain a nucleic acid detection microsphere preparation solution;

s240, at the gel melting temperature, forming droplets of the nucleic acid detection microsphere preparation solution into a plurality of droplets of the nucleic acid detection microsphere;

s250, cooling the plurality of nucleic acid detection microsphere droplets, and obtaining a plurality of nucleic acid detection microspheres through flow separation.

In one embodiment, in step S250, the nucleic acid detection microsphere is a nucleic acid detection microsphere comprising a single of the nuclei.

In one embodiment, a kit is used for high throughput nucleic acid detection analysis. The kit comprises the nucleic acid detection microsphere and the nucleic acid reaction solution according to any one of the above embodiments.

In one embodiment, a high throughput nucleic acid detection method comprises:

s310, providing a nucleic acid amplification reaction solution and a plurality of different types of nucleic acid detection microspheres, wherein the nucleic acid detection microspheres comprise a nucleus body and a coating layer, the nucleus body is provided with coding information, the coating layer wraps the nucleus body, the coating layer comprises a matrix and primers dispersed in the matrix, the primers uniquely correspond to the nucleus body, and the nucleus body is a solid sphere containing fluorescent dye;

s320, mixing the nucleic acid detection microspheres with the different types with the nucleic acid amplification reaction solution to obtain a nucleic acid detection solution;

s330, forming a plurality of micro-droplets by micro-droplet the nucleic acid detection solution;

s340, performing nucleic acid amplification on the plurality of micro-droplets to obtain the amplified plurality of micro-droplets;

S350, detecting the nucleosome in each micro-droplet according to the amplified micro-droplets, screening out the micro-droplet containing only one nucleosome, and obtaining a first effective micro-droplet;

s360, detecting fluorescent coding signals of the nucleon in the first effective micro-droplet according to the first effective micro-droplet, obtaining the primer corresponding to the nucleon, reading a report fluorescent signal after nucleic acid amplification reaction, and obtaining whether the corresponding target nucleic acid molecule exists in the first effective micro-droplet.

In one embodiment, in the step S310, the nucleic acid amplification reaction solution is a nucleic acid amplification reaction solution using deoxyribonucleic acid as a template, a reverse transcription nucleic acid amplification reaction solution using ribonucleic acid as a template, or a loop-mediated isothermal amplification reaction solution, and the nucleic acid amplification reaction solution contains a fluorescent dye.

In one embodiment, the step S360 includes:

s361, providing a fluorescence signal detection device, wherein the fluorescence signal detection device comprises a coding fluorescence channel and a fluorescent dye detection channel, and identifying fluorescence coding signals of the nuclei in the effective micro-droplets according to the coding fluorescence channel;

S362, acquiring the primer corresponding to the nucleosome according to the fluorescent coding signal of the nucleosome;

s363, according to the fluorescent dye detection channel, detecting the report fluorescent signal after the nucleic acid amplification reaction in the first effective micro-droplet, obtaining whether the first effective micro-droplet has the corresponding target nucleic acid molecule.

In one embodiment, a high throughput nucleic acid detection method comprises:

s410, providing a nucleic acid amplification reaction solution and a plurality of different types of nucleic acid detection microspheres, wherein the nucleic acid detection microspheres comprise a nucleosome and a coating layer, the nucleosome is provided with coding information, the coating layer wraps the nucleosome, the coating layer comprises a matrix, and a primer and a probe which are dispersed in the matrix, the primer and the probe are uniquely corresponding to the nucleosome, and the nucleosome is a solid sphere with fluorescent coding information;

s420, mixing the nucleic acid detection microspheres with different types with the nucleic acid amplification reaction liquid to obtain a nucleic acid detection liquid;

s430, forming a plurality of micro-droplets by micro-droplet the nucleic acid detection liquid;

s440, performing nucleic acid amplification on the plurality of micro-droplets to obtain the amplified plurality of micro-droplets;

S450, detecting the nucleosome in each micro-droplet according to the amplified micro-droplets, screening out the micro-droplet containing only one nucleosome, and obtaining a second effective micro-droplet;

s460, detecting fluorescent coding signals of the nucleon in the second effective micro-droplet according to the second effective micro-droplet, obtaining the primer and the probe corresponding to the nucleon, and reading report fluorescent signals after nucleic acid amplification reaction to obtain whether the corresponding target nucleic acid molecule exists in the second effective micro-droplet.

Wherein the coating encapsulates the nucleosome to form the nucleic acid detection microsphere. The matrix is an aqueous polymer gel formed in a hydrophobic oil, has no flowability, and is not easily changed in shape and volume. The aqueous polymer gel is in a gel state at room temperature and melts at a temperature higher than room temperature, and does not affect the diffusion and activity of enzymes, reaction solutions, and the like. Meanwhile, the primer dispersed in the matrix can perform qualitative analysis and identification on target detection nucleic acid. The nuclei are high temperature resistant materials, have a special labeling function, and each of the nuclei corresponds to one of the primers and is uniquely corresponding, so that the nucleic acid detection microspheres can be labeled by the nuclei so that tracking detection can be performed.

In the case of performing PCR detection, a plurality of and a plurality of the nucleic acid detecting microspheres are mixed with a nucleic acid amplification reaction solution to be detected, thereby obtaining a nucleic acid detection solution. The nucleic acid detection solution is subjected to microdroplet formation to form a plurality of microdroplets, and the plurality of microdroplets are subjected to PCR reaction. In the PCR reaction process, double-stranded DNA is denatured at 90-95 ℃, then rapidly cooled to 50-60 ℃, the primer is annealed and combined on a target sequence, then rapidly heated to 70-75 ℃, the primer chain is extended along a template under the action of Taq DNA polymerase, and the nucleic acid is amplified in a proper temperature range. In the PCR temperature control process of a plurality of micro-droplets, the coating layer is melted and decomposed, the primer carried in the coating layer is released into the corresponding micro-droplet and reacts with the target nucleic acid molecule contained in the micro-droplet, finally, the nucleosome can be positioned, tracked and identified, and the target nucleic acid molecule is obtained through the primer corresponding to the nucleosome, so that the PCR high-throughput detection is realized.

In the practical application process, a plurality of the nucleic acid detecting microspheres may be prepared in batch. And a plurality of nucleic acid detection microspheres are mixed according to a certain proportion according to the requirement of actually detecting target nucleic acid, and are mixed with nucleic acid amplification reaction liquid to be detected, so that the nucleic acid detection liquid can be obtained, the target nucleic acid can be detected, a plurality of target nucleic acid molecules can be detected at one time, repeated detection for a plurality of times is not required, and the detection method has the advantages of small workload, short detection time and high sensitivity.

Drawings

FIG. 1 is a schematic diagram of the structure of a nucleic acid detecting microsphere provided in the present application;

FIG. 2 is a schematic structural view of a coating layer provided in the present application;

FIG. 3 is a schematic structural view of a core provided herein;

FIG. 4 is a schematic structural view of a coating layer according to one embodiment of the present disclosure;

FIG. 5 is a schematic diagram showing the structure of a nucleic acid detecting microsphere according to one embodiment of the present application;

FIG. 6 is a schematic diagram of a micro-droplet generator provided herein;

fig. 7 is a schematic structural diagram of a different type of microfluidic chip provided in the present application;

FIG. 8 is a schematic structural view of a first effective micro-droplet provided herein;

FIG. 9 is a schematic diagram of the structure of the nucleic acid high-throughput detector provided by the present application;

FIG. 10 is a schematic diagram of a micro-droplet generation device provided herein;

fig. 11 is a schematic structural diagram of a different type of microfluidic chip provided in the present application;

FIG. 12 is a schematic structural view of a second effective micro-droplet provided herein;

fig. 13 is a schematic structural diagram of a fluorescence signal detection device provided in the present application.

Description of the reference numerals

Nucleic acid detecting microsphere 700, coating 710, substrate 711, primer 712, probe 713, nucleus 730, microdroplet 800, first effective microdroplet 810, second effective microdroplet 820, fluorescence detecting device 30, first controller 310, fluorescence detecting assembly 330, camera 331, objective lens 332, second filter 333, excitation light source 340, LED light source 341, collimator 342, first filter 343, dichroic mirror 344, fly eye lens 345, focusing lens 346.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail below by way of examples with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The terms "coupled" and "connected," as used herein, are intended to encompass both direct and indirect coupling (coupling), unless otherwise indicated. In the description of the present application, it should be understood that the terms "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," etc. indicate or refer to an orientation or positional relationship based on that shown in the drawings, merely for convenience of description and to simplify the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present application.

In this application, unless expressly stated or limited otherwise, a first feature "up" or "down" a second feature may be the first and second features in direct contact, or the first and second features in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.

Referring to FIGS. 1-3, the present application provides a nucleic acid detection microsphere 700 for high throughput nucleic acid detection analysis. The nucleic acid detecting microsphere 700 includes a core 730 and a coating 710. The nucleus 730 has fluorescence encoded information. The coating 710 encapsulates the core 730, the coating 710 includes a matrix 711 and primers 712 dispersed in the matrix 711, and the primers 712 uniquely correspond to the core 730.

Wherein the coating 710 encapsulates the core 730 to form the nucleic acid detection microsphere 700. The matrix 711 is an aqueous polymer gel formed in a hydrophobic oil, has no fluidity, and is not easily changed in shape and volume. The aqueous polymer gel is in a gel state at room temperature and melts at a temperature higher than room temperature, and does not affect the diffusion and activity of enzymes, reaction solutions, and the like. Meanwhile, the primer 712 dispersed in the matrix 711 can perform qualitative analysis and identification of the target nucleic acid. The nucleus 730 is a high temperature resistant material with fluorescent coding information. The fluorescence encoded information is displayed by the fluorescence encoded signal of the nucleus 730, thereby achieving a specific labeling function by the fluorescence encoded signal. And each of the cores 730 corresponds to one of the primers 712 and is uniquely corresponding, so that the nucleic acid detecting microsphere 700 can be labeled by the core 730 so that tracking detection can be performed.

In the case of performing PCR detection, a plurality of and a plurality of kinds of the nucleic acid detecting microspheres 700 are mixed with a nucleic acid amplification reaction solution to be detected, thereby obtaining a nucleic acid detection solution. The nucleic acid detection solution is subjected to microdroplet formation to form a plurality of microdroplets, and the plurality of microdroplets are subjected to PCR reaction. In the PCR reaction process, double-stranded DNA is denatured at 90-95 ℃, then rapidly cooled to 50-60 ℃, the primer is annealed and combined on a target sequence, then rapidly heated to 70-75 ℃, the primer chain is extended along a template under the action of Taq DNA polymerase, and the nucleic acid is amplified in a proper temperature range. In the process of performing PCR temperature control on the plurality of microdroplets, the coating 710 is melted and decomposed, the primer 712 carried in the coating 710 is released into the corresponding microdroplet and reacts with the target nucleic acid molecule contained in the microdroplet, and finally the nucleosome 730 can be positioned, tracked and identified, and the target nucleic acid molecule is obtained through the primer 712 corresponding to the nucleosome 730, so as to realize PCR high-throughput detection.

In the practical application process, a plurality of the nucleic acid detecting microspheres 700 may be prepared in batch. And mixing a plurality of nucleic acid detection microspheres 700 according to a certain proportion according to the requirement of actually detecting target nucleic acid, and mixing with a nucleic acid amplification reaction solution to be detected to obtain a nucleic acid detection solution, so that the target nucleic acid can be detected, a plurality of target nucleic acid molecules can be detected at one time, repeated detection for a plurality of times is not required, and the detection method has the advantages of small workload, short detection time and high sensitivity.

Referring to fig. 4-5, in one embodiment, the coating 710 further includes probes 713, and the probes 713 and the primers 712 are dispersed in the matrix 711 and correspond to the core 730 only.

The probe 713 may be a fluorescent probe capable of indicating nucleic acid amplification, or may be an oligonucleotide probe containing both a fluorescent group and a quenching group, such as a TaqMan fluorescent probe. The probes 713 are dispersed in the matrix 711 to perform qualitative analysis and identification of the target nucleic acid molecules. In the PCR temperature control process of the droplets, the coating 710 is melted and decomposed, the primer 712 and the probe 713 carried in the coating 710 are released into the corresponding droplets, react with the target detection nucleic acid molecules contained in the droplets, and finally the nucleosome 730 can be positioned, tracked and identified, and the target nucleic acid molecules are obtained through the primer 712 and the probe 713 corresponding to the nucleosome 730, so as to realize PCR high-throughput detection.

In one embodiment, the primers 712 may be configured as different types of primers, and when performing a batch test, a plurality of the primers 712 may be configured as different types of primers that may be used to detect different types of target nucleic acid molecules. Meanwhile, one of the primers 712 corresponds to one of the nuclei 730, that is, each of the primers 712 has its corresponding representative number, that is, the nuclei 730, so that identification can be performed by detection of the nuclei 730.

In one embodiment, 100 nucleic acid detecting microspheres 700 are taken as an example, 100 nucleic acid detecting microspheres 700 correspond to 100 different primers 712, that is, 100 nucleic acid detecting microspheres 700 correspond to 100 different nuclei 730. That is, 100 different ones of the primers 712 correspond to 100 different ones of the nuclei 730. And in the process of carrying out PCR heating on a plurality of micro-droplets containing 100 nucleic acid detection microspheres 700, the coating 710 in each nucleic acid detection microsphere 700 is melted and decomposed, and the primers 712 carried in the coating 710 are released into the corresponding micro-droplets for PCR amplification. In this case, 1 nucleus 730 is contained in a part of the microdroplet, the primer 712 contained in the microdroplet can be obtained from the nucleus 730, and then the target nucleic acid molecule in the microdroplet can be obtained from the primer 712, thereby realizing PCR detection.

In one embodiment, the matrix is an agarose gel. Agarose gel is a gel prepared by taking agarose as a supporting medium, the melting point of the agarose is between 62 ℃ and 65 ℃, the agarose gel can maintain the liquid state for several hours at 37 ℃ after being melted, and the agarose gel is solidified into gel at 30 ℃. In the process of performing PCR temperature control on a plurality of microdroplets, the coating 710 is melted and decomposed, and the primer 712 and the probe 713 carried in the coating 710 are released into the corresponding microdroplet, so that a PCR reaction between the primer 712 and the target nucleic acid molecules contained in the microdroplet is performed, and whether the nucleic acid is amplified is indicated by a fluorescent dye or the probe 713.

In one embodiment, the nucleus 730 is a solid sphere containing a fluorescent dye.

The material of the core body 730 may be a high temperature resistant material, such as polyimide, polytetrafluoroethylene, polyphenylene sulfide, or polyamide. Meanwhile, the core body 730 contains fluorescent dye, and can emit fluorescent signals. The nuclei 730 may be encoded by different types of fluorescent dyes and intensities of fluorescence, resulting in a plurality of different types of the nuclei 730 such that the nuclei 730 have fluorescent encoding signals, thereby enabling encoding of a plurality of the nucleic acid detection microspheres 700.

In one embodiment, the present application employs two different types of fluorescent dyes, each fluorescent dye employing 10 different levels of fluorescent signal intensity, such that the differently labeled nuclei 730 in 10×10=100 can be obtained, such that the nuclei 730 have fluorescence encoded information, thereby obtaining the differently labeled nucleic acid detection microspheres 700 in 10×10=100. Wherein each of the nucleic acid detecting microspheres 700 corresponds to one of the nuclei 730, one of the nuclei 730 corresponds to one of the primers 712, and one of the nuclei 730 corresponds to one of the probes 713. The nucleic acid detection microspheres 700 can be used for carrying out PCR detection on a plurality of different kinds of target nucleic acid molecules at one time, repeated detection for a plurality of times is not needed, and the method has the characteristics of small workload, short detection time, high flux and high sensitivity.

In one embodiment, the diameter of the nucleus 730 is 10 microns to 100 microns. The thickness of the coating layer is 10-100 micrometers.

The nucleic acid detection microsphere 700 typically has a diameter of 20 microns to 150 microns, allowing for the collection of a sufficient number of microdroplets at the time of image acquisition. Wherein the diameter of the core body 730 may be 10 micrometers to 100 micrometers, and the thickness of the coating layer may be 10 micrometers to 100 micrometers. The nucleic acid detecting microsphere 700 is preferably not too large or too small, and if too small, it is not easily recognized, and if too large, it is easy to block the outlet end of the droplet generator when generating a plurality of droplets, and the generation of a plurality of droplets is hindered. By setting the diameter of the nucleic acid detecting microsphere 700, the droplets can be recognized by the fluorescence detecting device when being regenerated, and more droplets can be covered as much as possible, so that image acquisition is facilitated, and the droplets are not easily blocked from being generated at the outlet end of the droplet generating device.

s110, providing a plurality of nuclei 730 and primer solutions;

S130, mixing the plurality of nuclei 730, the primer solution and the coating preparation solution at a gel melting temperature to obtain a nucleic acid detection microsphere preparation solution;

and S150, cooling the plurality of nucleic acid detection microsphere droplets, and obtaining a plurality of nucleic acid detection microspheres 700 through flow separation.

In the step S110, a plurality of the nucleosomes 730 are solid spheres of the same kind containing fluorescent dye, so that the nucleic acid detection microsphere 700 is prepared such that one of the nucleic acid detection microsphere 700 uniquely corresponds to one of the nucleosomes 730 and one of the primers 712 uniquely corresponds to one of the nucleosomes 730.

And, the primer solution contains the primer 712. When the dry powder primer 712 was diluted with sterilized ultrapure water, the concentration of the primer was diluted to 100. Mu.M, that is, 100. Mu. Mol/L. Then, 100. Mu.L of the primer solution having a concentration of 100. Mu.M was put into 900. Mu.L of the coating preparation solution to prepare a primer having a concentration of 10. Mu.M (. Mu. Mol/L).

In the step S120, the gel powder may be an agar powder, ethylene glycol diacrylate, or the like, which can be used to prepare a gel. The coating preparation liquid can be agar powder solution, agar powder with the mass ratio of 1.5% -4.5% and 10ml double distilled water are provided, and the agar powder is added into the double distilled water to be dissolved at high temperature until the agar powder is clear, so that the coating preparation liquid is obtained. Wherein the coating preparation liquid is agar powder solution.

In the step S130, the plurality of primers 712 contained in the primer solution are the same type of primers, and the plurality of nuclei 730 are the same type of fluorescence nuclei. The gel melting temperature is the temperature at which the gel converts to a liquid solution. Wherein the melting point of the agarose is between 62 ℃ and 65 ℃ and the agarose is solidified into gel at 30 ℃. Therefore, the plurality of primers 712 and the plurality of cores 730 are added to the coating preparation solution, that is, the agar powder solution, at a high temperature of 62 to 65℃to obtain the nucleic acid detecting microsphere preparation solution.

In one embodiment, in the step S130, when the primer 712 and the nucleosome 730 are added to the agarose solution, the concentration of the nucleosome 730 is generally selected according to the size of the nucleic acid detection microsphere 700 to be generated.

Referring to fig. 6 to 7, in the step S140, a plurality of nucleic acid detection microsphere droplets are formed in hydrophobic oil by a microfluidic chip, a microfluidic generator, or a microfluidic droplet generation device in a high temperature environment.

Referring to fig. 6, in one embodiment, the step S140 includes:

The application provides a micro-droplet generation device, the micro-droplet generation device includes a liquid discharge gun head, a fluid driving mechanism and a motion control mechanism. The liquid-spraying gun head is provided with an outlet end and an inlet end, the liquid-spraying gun head is driven by the fluid driving mechanism to suck nucleic acid detection microsphere preparation solution into the liquid-spraying gun head through the inlet end, and the outlet end of the liquid-spraying gun head is inserted into a container storing oily liquid, so that the outlet end of the liquid-spraying gun head enters the liquid level of the oily liquid. Simultaneously, the motion control mechanism performs instantaneous acceleration motion or speed change period motion under the liquid level of the oily liquid, so that the nucleic acid detection microsphere preparation solution is discharged from the outlet end of the liquid discharge gun head, and a plurality of nucleic acid detection microsphere liquid drops are formed under the liquid level of the oily liquid. The oily liquid and the nucleic acid detection microsphere preparation solution are two liquids which are mutually insoluble or have interface reaction, and the oily liquid can be mineral oil (including n-tetradecane and the like), vegetable oil, silicone oil, perfluoroalkyl oil and the like.

In step S150, the plurality of nucleic acid detecting microsphere droplets are cooled to about 30 ℃ at room temperature, and at this time, the plurality of nucleic acid detecting microspheres 700 are obtained by flow sorting. The flow sorting irradiates a plurality of droplets of the nucleic acid detection microsphere in a high-speed flow state with a high-energy laser. Since the plurality of nucleic acid detecting microsphere droplets contains 0, 1 or more nuclei 730, and the nuclei 730 are solid spheres containing fluorescent dye, the intensities of the generated scattered light and the emitted fluorescence can be measured, and thus the screening can be performed to obtain the nucleic acid detecting microsphere 700 containing only a single nucleus 730.

At this time, the plurality of cooled nucleic acid detecting microspheres 700 are in a gel state, which can be conveniently stored and transported in a normal temperature environment, and is advantageous for mass transportation for PCR detection.

s210, providing a primer solution, a probe solution and a plurality of nuclei 730;

S230, mixing the plurality of nuclei 730, the primer solution and the probe solution with the coating preparation solution at a gel melting temperature to obtain a nucleic acid detection microsphere preparation solution;

In the step S210, the probe solution includes the probe 713 for detecting whether amplification of nucleic acid occurs, and may be an oligonucleotide probe including both a fluorescent group and a quenching group, such as a TaqMan fluorescent probe. In the PCR temperature control process of the droplets, the coating 710 is melted and decomposed, the primer 712 and the probe 713 carried in the coating 710 are released into the corresponding droplets, react with the target detection nucleic acid molecules contained in the droplets, and finally the nucleosome 730 can be positioned, tracked and identified, and the target nucleic acid molecules are obtained through the primer 712 and the probe 713 corresponding to the nucleosome 730, so as to realize PCR high-throughput detection.

Wherein the plurality of nuclei 730 are the same kind of solid spheres containing fluorescent dye, so that the nucleic acid detection microsphere 700 is prepared such that one nucleic acid detection microsphere 700 corresponds uniquely to one of the nuclei 730, and one primer 712 corresponds uniquely to one of the nuclei 730, and one probe 713 corresponds uniquely to one of the primers 712.

In the step S220, the coating preparation liquid may be the same as the preparation method in the step S120.

In the step S240, a preparation method for forming a plurality of the nucleic acid detecting microsphere droplets may be the same as the step S140 method.

In the step S250, a method of obtaining a plurality of the plurality of nucleic acid detecting microspheres 700 may be the same as the step 150 method.

In one embodiment, in the step S220, the gel powder is agar powder or polyethylene glycol diacrylate or the like.

Wherein the nucleic acid reaction solution contains enzymes, dNTPs, fluorescent dyes, ions and the like required for PCR amplification. If the probe 713 is contained in the nucleic acid detecting microsphere 700, the fluorescent dye may not be contained in the nucleic acid reaction solution.

The kit may be used to store, carry a plurality of different ones of the nucleic acid detecting microspheres 700. Wherein, the nucleic acid detecting microsphere 700 may be stored in glycerol.

In one embodiment, kits and solutions are prepared for use exclusively in digital PCR to reduce or avoid potential contamination of a template DNA sample with exogenous DNA. All instruments and consumables used should be sterilized at high temperature and dried at high temperature.

Referring to FIG. 8, in one embodiment, a high throughput nucleic acid detection method comprises:

s310, providing a nucleic acid amplification reaction solution and a plurality of different types of nucleic acid detection microspheres 700, wherein the nucleic acid detection microspheres 700 comprise a nucleus body 730 and a coating layer 710, the nucleus body 730 is provided with coding information, the coating layer 710 wraps the nucleus body 730, the coating layer 710 comprises a matrix 711 and primers 712 dispersed in the matrix 711, the primers 712 are uniquely corresponding to the nucleus body 730, and the nucleus body 730 is a solid sphere containing fluorescent dye;

S320, mixing the nucleic acid detection microspheres 700 with the plurality of different types with the nucleic acid amplification reaction solution to obtain a nucleic acid detection solution;

s330, forming a plurality of micro-droplets 800 by micro-droplet the nucleic acid detection solution;

s340, performing nucleic acid amplification on the plurality of micro-droplets 800 to obtain the plurality of micro-droplets 800 after amplification is completed;

s350, detecting the nucleous 730 in each micro-droplet 800 according to the amplified plurality of micro-droplets 800, and screening out the micro-droplets 800 only containing one nucleous 730 to obtain a first effective micro-droplet 810;

s360, detecting fluorescent signals of the nucleon 730 in the first effective micro-droplet 810 according to the first effective micro-droplet 810, obtaining the primer 712 corresponding to the nucleon 730, and reading the reported fluorescent signals after the nucleic acid amplification reaction to obtain whether the corresponding target nucleic acid molecules exist in the first effective micro-droplet 810.

In the step S310, the nucleic acid amplification reaction solution is a nucleic acid amplification reaction solution using deoxyribonucleic acid as a template, a reverse transcription nucleic acid amplification reaction solution using ribonucleic acid as a template, or a loop-mediated isothermal amplification reaction solution, and the nucleic acid amplification reaction solution contains a fluorescent dye. The nucleic acid amplification reaction solution includes a nucleic acid template, a reaction buffer, deoxyribonucleoside triphosphates, a polymerase, a divalent metal cation, and the like, and if the probe 713 is not present in the coating 710, the reaction buffer contains a fluorescent dye.

The nucleic acid amplification reaction solution may be a nucleic acid amplification reaction solution (may be referred to as a DNA amplification reaction solution) using deoxyribonucleic acid (DNA) as a template, a reverse transcription nucleic acid amplification reaction solution (may be referred to as an RNA reverse transcription reaction solution) using ribonucleic acid (RNA) as a template, or other nucleic acid amplification reaction solutions such as a loop-mediated isothermal amplification (LAMP) reaction solution. The DNA amplification reaction solution is characterized by containing dNTPs required by DNA amplification, a reaction buffer solution, inorganic salt ions, polymerase, a DNA template to be detected and fluorescent dye. The fluorescent dye may be a fluorescent dye bound to DNA such as SYBR Green.

In the PCR reaction system, SYBR Green fluorescent dye is added in a free state to emit weak fluorescence, but once the SYBR Green fluorescent dye is combined with double-stranded DNA, the fluorescence is greatly enhanced, and a fluorescence signal is emitted, so that the increase of the fluorescence signal and the increase of a PCR product can be completely synchronous. At this time, the reporter fluorescent signal after the nucleic acid amplification reaction can be obtained by detecting the fluorescent signal emitted from the SYBR Green fluorescent dye, thereby obtaining whether the corresponding target nucleic acid molecule is present in the first-effect micro-droplet 810.

Wherein the size, shape, and the contained primer 712 of the nucleic acid detecting microsphere 700 among the plurality of different types of the nucleic acid detecting microsphere 700 may be the same or different. The plurality of nucleic acid detecting microspheres 700 may contain different types of the primers 712 to detect different types of the target nucleic acid molecules.

In the step S320, when the plurality of different types of nucleic acid detecting microspheres 700 are mixed with the nucleic acid amplification reaction solution to form the nucleic acid detecting solution, the concentration of the nucleic acid detecting microspheres 700 in the nucleic acid detecting solution may be adjusted so that the number of single packages is the largest when the plurality of micro droplets 800 are generated, and the distribution of the micro spheres conforms to the poisson distribution theoretical model. At this time, the probability of having one of the nuclei 730 in each of the micro-droplets 800 is calculated as p (x=1) =λe ^-λ ，p’(x＝1)＝e ^-λ -λe ^-λ =0, where λ=1, i.e. when 1 core body 730 is contained in each micro-droplet 800 on average, the probability that the core body 730 is individually wrapped is the largest. At this time, the probability that 1 of the nuclei 730 are contained in each of the microdroplets 800 is p (x=1) =e ^-1 ＝0.368。

Referring to fig. 9, in the step S330, in one embodiment, the present application provides a nucleic acid high-throughput detector including a micro-droplet generation device, a temperature control device, a fluorescent signal detection device, an analysis device, and a controller. The micro-droplet generator is configured to micro-droplet the nucleic acid detection liquid to form the plurality of micro-droplets 800. The temperature control device is connected with the micro-droplet generation device through a track, and is used for transferring the micro-droplets to the temperature control device, and performing temperature circulation through the temperature control device to realize nucleic acid amplification. And after the amplification of the plurality of micro-droplets is finished, performing fluorescence detection on the plurality of micro-droplets after the nucleic acid amplification is finished through the fluorescence signal detection device. The controller is respectively connected with the micro-droplet generation device, the temperature control device and the fluorescent signal detection device and used for controlling the micro-droplet generation device, the temperature control device and the fluorescent signal detection device.

The micro-droplet generation device comprises a liquid discharge gun head, a fluid driving mechanism and a motion control mechanism. The liquid-spraying gun head is provided with an outlet end and an inlet end, the liquid-spraying gun head is driven by the fluid driving mechanism to suck the nucleic acid detection liquid into the liquid-spraying gun head through the inlet end, and the outlet end of the liquid-spraying gun head is inserted into a container storing oily liquid, so that the outlet end of the liquid-spraying gun head enters the liquid level of the oily liquid. Meanwhile, the motion control mechanism performs instantaneous acceleration motion or speed change period motion under the liquid surface of the oily liquid, so that the nucleic acid detection liquid is discharged from the outlet end of the liquid discharge gun head, and the plurality of micro-droplets 800 are formed under the liquid surface of the oily liquid. Wherein the oily liquid and the nucleic acid detection liquid are two liquids which are mutually insoluble or have interface reaction, and the oily liquid can be mineral oil (including n-tetradecane and the like), vegetable oil, silicone oil, perfluoroalkyl oil and the like.

Referring to fig. 10-11, in one embodiment, in the step S330, the nucleic acid detecting solution is microdroplet to form a plurality of micro-droplets 800, a microfluidic chip, a microfluidic generator, or a micro-droplet generating device may be used. And the method for preparing the plurality of micro drops 800 is not limited to the above-mentioned device, but may be other devices for preparing the plurality of micro drops 800.

Wherein each micro-droplet 800 may include zero, one or a plurality of the nucleic acid detection microspheres 700, and each micro-droplet 800 contains the nucleic acid amplification reaction solution for performing nucleic acid amplification.

In one embodiment, the droplets 800 formed by the nucleic acid detecting liquid in the step S330 may have the same size or different sizes.

In one embodiment, in the step S330, a microfluidic chip may be used when the nucleic acid detection solution is microdroplet.

When the nucleic acid detection solution is microdroplet, zero, one or a plurality of the nucleic acid detection microspheres 700 may be included in each of the microdroplets 800. When the nucleic acid amplification temperature of the plurality of micro droplets 800 is higher than the melting point of agarose, the coating 710 melts and releases the primer 712, so that the primer 712 and the nucleic acid molecules in the micro droplets 800 are simultaneously amplified by PCR, and at this time, the type of the primer 712 can be obtained by identifying the corresponding nucleosome 730 in the micro droplets 800, thereby obtaining the target nucleic acid molecules.

In one embodiment, in the step S350, the first active micro-droplet 810 includes one of the nuclei 730. The microdroplet 800 is considered an invalid microdroplet for those containing zero or more than 1. Wherein the first effective micro-droplet 810 contains a fluorescent dye and the primer 712. If the fluorescence is greatly enhanced after the fluorescent dye is combined with the double-stranded DNA during the PCR amplification process of the primer 712 and the nucleic acid molecules in the first effective micro-droplet 810, a stronger fluorescent signal can be emitted, so that the first effective micro-droplet 810 has a stronger fluorescent signal, and thus, the type of the corresponding target nucleic acid molecules in the first effective micro-droplet 810 can be obtained according to the core body 730 and the primer 712.

In one embodiment, the step S360 includes:

s361, providing a fluorescence signal detection device, wherein the fluorescence signal detection device comprises a coded fluorescence channel and a fluorescent dye detection channel, and the fluorescence signal of the nucleus 730 in the effective micro-droplet is identified according to the coded fluorescence channel;

s362, acquiring the primer 712 corresponding to the core body 730 according to the fluorescence signal of the core body 730;

s363, according to the fluorescence signal reported after the detection of the nucleic acid amplification reaction in the first effective micro-droplet 810 by the fluorescence dye detection channel, obtaining whether the first effective micro-droplet 810 has the corresponding target nucleic acid molecule.

The nucleus 730 is a solid sphere containing fluorescent dye, and the nucleus 730 can be labeled by using different kinds of fluorescent dye and the intensity of fluorescence. Each fluorescence corresponds to one of the nuclei 730, and each of the nuclei 730 corresponds to one of the primers 712, i.e., each of the primers 712 has its corresponding representation number, i.e., the nucleus 730.

In step S360, the presence or absence of the target nucleic acid molecule is detected based on the presence or absence of the reporter fluorescent signal, thereby realizing qualitative detection. Wherein the first active microdroplets 810 comprising the same type of the nuclei 730 may be grouped together during the screening process. Meanwhile, by detecting the presence or absence of the reporter fluorescence, the proportion of the number of the micro-droplets without the reporter fluorescence signal in the first effective micro-droplet 810 in the core 730 in this category to the total number of the micro-droplets in this category can be obtained, so that the concentration of the target nucleic acid molecules corresponding to the core 730 in the same category can be obtained by calculation according to poisson distribution.

Referring to FIG. 12, in one embodiment, a high throughput nucleic acid detection method comprises:

s410, providing a nucleic acid amplification reaction solution and a plurality of different types of nucleic acid detection microspheres 700, wherein the nucleic acid detection microspheres 700 comprise a nucleus body 730 and a coating layer 710, the nucleus body 730 is provided with coding information, the coating layer 710 wraps the nucleus body 730, the coating layer 710 comprises a matrix 711, and a primer 712 and a probe 713 which are dispersed in the matrix 711, the primer 712 and the probe 713 are uniquely corresponding to the nucleus body 730, and the nucleus body 730 is a solid sphere with fluorescence coding information;

s420, mixing the nucleic acid detection microspheres 700 with the plurality of different types with the nucleic acid amplification reaction solution to obtain a nucleic acid detection solution;

s430, forming a plurality of micro-droplets 800 by micro-droplet the nucleic acid detection solution;

s440, performing nucleic acid amplification on the plurality of micro-droplets 800 to obtain the plurality of micro-droplets 800 after amplification is completed;

s450, detecting the nucleous 730 in each micro-droplet 800 according to the amplified plurality of micro-droplets 800, screening out the micro-droplets 800 containing only one nucleous 730, and obtaining a second effective micro-droplet 820;

S460, detecting fluorescent signals of the nucleon 730 in the second effective micro-droplet 820 according to the second effective micro-droplet 820, obtaining the primer 712 and the probe 713 corresponding to the nucleon 730, and reading the reported fluorescent signals after the nucleic acid amplification reaction to obtain whether the second effective micro-droplet 820 has the corresponding target nucleic acid molecule.

When the nucleic acid detection solution is microdroplet, zero, one or a plurality of the nucleic acid detection microspheres 700 may be included in each of the microdroplets 800. When the nucleic acid amplification temperature of the plurality of microdroplets 800 is higher than the melting point of agarose, the coating 710 melts, releasing the primer 712 and the probe 713. Thus, the primer 712 and the probe 713 are simultaneously amplified by PCR with the nucleic acid molecules in the micro-droplet 800, and the primer 712 and the probe 713, and thus the primer 740, can be obtained by identifying the corresponding nucleotides 730 in the micro-droplet 800, thereby obtaining the target nucleic acid molecules.

In the step S450, the second effective micro-droplet 820 includes one of the nuclei 730. The microdroplet 800 is considered an invalid microdroplet for the core 730 that contains zero or more than 1. Meanwhile, the second effective micro droplet 820 further includes the probe 713 and the primer 712. When the second effective micro-droplet 820 contains the probe 713, the second effective micro-droplet 820 may not contain fluorescent dye, and the probe 713 plays a role of fluorescent calibration. During PCR amplification of the primer 712 with the nucleic acid molecules in the second effective micro-droplet 820, the probe 713 binds to double-stranded DNA so that the second effective micro-droplet 820 containing the corresponding target nucleic acid molecule can be identified.

Assuming that the probe 713 is bound to double-stranded DNA during PCR amplification of the primer 712 and the nucleic acid molecules in the second effective micro-droplet 820, it is possible to determine whether the second effective micro-droplet 820 contains the corresponding target nucleic acid molecules by recognizing the probe 713. Thus, the type of the corresponding target nucleic acid molecule in the first effective micro-droplet 810 can be obtained from the core 730 and the primer 712.

In one embodiment, the method of microdroplet of the nucleic acid detecting liquid in step S430 is the same as that in step S330.

Referring to fig. 13, in one embodiment, the present application provides a fluorescent signal detection device 30. The fluorescence signal detecting apparatus 30 includes an excitation light source 340, a fluorescence detection assembly 330, and a first controller 310. The excitation light source 340 is disposed above the detection areas of the plurality of micro-droplets 800, and irradiates the detection areas of the plurality of micro-droplets 800 at an oblique angle to form an oblique light path. The fluorescence detection assembly 330 is disposed directly above the detection areas of the droplets 800, and is configured to collect fluorescence images of the droplets 800. The first controller 310 is connected to the excitation light source 340 and the fluorescence detection assembly 330, respectively, and is configured to control the excitation light source 340 and the fluorescence detection assembly 330. The fluorescence signal detection device 30 can perform multiple fluorescence channel imaging and bright field and dark field imaging on the microdroplet. Wherein multiple fluorescent channel imaging is used for detection of microdroplet reaction signals, bright field dark field imaging is used to detect size information of formed microdroplets and to monitor the state of the droplets during the reaction.

The excitation light source 340 includes LED light sources 341 of different colors, a collimator 342, a first filter 343, a dichroic mirror 344, a fly-eye lens 345, and a focusing lens 346. The LED light sources 341 with different colors may generate light with different colors and irradiate the plurality of micro droplets 800. By selecting the different colored LED light sources 341, illumination of different fluorescent colors may be obtained, the different colored LED light sources 341 may be operated in turn. The collimator 342, the first filter 343 and the dichroic mirror 344 are sequentially disposed right in front of the light path emitted by each LED light source. The collimating mirror 342 and the first filter 343 are disposed at a perpendicular angle (90 ° angle) to the optical path. The dichroic mirror 344 is disposed at an angle of 0 ° to 45 ° to the optical path. An optical path formed by the dichroic mirror 344, the optical path being immediately preceded by the fly-eye lens 345 and the focusing lens 346 in order. The fly-eye lens 345 is disposed at a right angle (90 ° angle) to the optical path with the focusing lens 346. The internal fluorescence of the plurality of micro-droplets 800 is excited, collected by the objective lens 332 above through the second filter 333, and enters the camera 331, and the camera 331 collects fluorescence images of the plurality of micro-droplets.

The light path emitted by the excitation light source 340 is obliquely irradiated to the plurality of micro-droplets 800, so that the micro-droplets containing fluorescent substances in the plurality of micro-droplets 800 generate fluorescence. The fluorescence information of the micro-droplets containing fluorescent substances is collected by the fluorescence detection assembly 330, and the fluorescence information of the micro-droplets containing fluorescent substances is transmitted to an analysis device (computer) in the form of a fluorescence image for analysis.

The second controller 310 is configured to switch different filters to form different fluorescence detection channels. The fluorescent signal detection device comprises a coded fluorescent channel, a fluorescent dye detection channel, a fluorescent probe detection channel, a micro-droplet identification channel, a plurality of standby channels and the like.

Wherein, when generating a plurality of the micro droplets 800, a ROX reference dye is added to the nucleic acid detecting liquid. The ROX reference dye does not participate in the PCR reaction and can be used to identify the specific position, profile, number, etc. of the plurality of microdroplets 800. The micro-droplet identification channel is used for identifying the fluorescence of the reference dye in ROX, so as to accurately position each micro-droplet 800. The encoded fluorescence channel is used to identify the fluorescence signal and fluorescence signal intensity of the nuclei 730 for obtaining the first effective micro-droplet 810 containing one of the nuclei 730. The fluorescent dye detection channel or fluorescent probe detection channel is used to identify a reporter fluorescent signal after the nucleic acid amplification reaction in the first effective micro-droplet 810, so as to determine whether the primer 712 or the primer 712 and the probe 713 are subjected to PCR amplification with a target nucleic acid molecule according to the reporter fluorescent signal.

In step S460, the presence or absence of the target nucleic acid molecule is detected based on the presence or absence of the reporter fluorescent signal, thereby achieving qualitative detection. Wherein the second effective microdroplets 820 comprising the same class of the nuclei 730 may be grouped together during the screening process. Meanwhile, by detecting the presence or absence of the reporter fluorescence, the proportion of the number of the micro-droplets without the reporter fluorescence signal in the second effective micro-droplet 820 in the core 730 in this category to the total number of the micro-droplets in this category can be obtained, so that the concentration of the target nucleic acid molecules corresponding to the core 730 in the same category can be obtained by calculation according to poisson distribution.

The first effective micro-droplet 810 can be selected from the plurality of micro-droplets 800 by detecting fluorescent signals of the nuclei 730 in the plurality of micro-droplets 800 through the encoded fluorescent channels. Wherein the first active micro-droplet 810 comprises one of the nuclei 730. Thus, the first effective micro-droplet 810 is detected through the fluorescent dye detection channel, the report fluorescent signal after the nucleic acid amplification reaction is read, and whether the first effective micro-droplet 810 contains the corresponding target nucleic acid molecule is judged. If the first effective micro-droplet 810 contains a corresponding target nucleic acid molecule, the type of the corresponding target nucleic acid molecule can be obtained from the primer 712 corresponding to the core body 730 in the first effective micro-droplet 810.

Similarly, the second effective micro-droplet 810 can be selected from the plurality of micro-droplets 800 by detecting the fluorescent signal of the nuclei 730 in the plurality of micro-droplets 800 through the encoded fluorescent channel. Wherein the second effective micro-droplet 820 contains one of the nuclei 730. Thus, the second effective micro droplet 820 is detected through the fluorescent probe detection channel, the report fluorescent signal after the nucleic acid amplification reaction is read, and whether the second effective micro droplet 820 contains the corresponding target nucleic acid molecule is judged. If the second effective micro-droplet 820 contains a corresponding target nucleic acid molecule, the type of the corresponding target nucleic acid molecule can be obtained from the primer 712 or the probe 713 corresponding to the core body 730 in the second effective micro-droplet 820.

In one embodiment, the fluorescent signal detection means comprises a plurality of the encoded fluorescent channels, which may be used to identify a plurality of different fluorescent-labeled nuclei 730. Specifically, the first encoded fluorescent channel is set to fluorescent a, the second encoded fluorescent channel is set to fluorescent B, and the same 10 gradient concentrations are set, so that 10×10=100 different fluorescent channels and the intensity-labeled nuclei 730 can be identified by the fluorescent signal detection means, that is, 100 different types of the primers 712 or the primers 712 and the probes 713 can be labeled by the 100 fluorescent-labeled nuclei 730. Similarly, a plurality of different types of the nuclei 730 may be obtained, and thus a plurality of different types of the nucleic acid detecting microspheres 700 may be labeled.

The nucleic acid detecting microsphere 700 is composed of the core body 730 having fluorescence coding information, the primer 712, and the coating 710 of the probe 713. The plurality of nucleic acid detecting microspheres 700 are randomly distributed in the nucleic acid amplification reaction solution, and are mixed to obtain the nucleic acid detecting solution, and the nucleic acid detecting solution is subjected to micro-droplet formation to generate the plurality of micro-droplets 800. When the temperature is raised to 60 degrees celsius or higher, the coating 710 melts, releasing the primer 712 and the probe 713 into the micro-droplet 800, thereby forming a complete nucleic acid amplification reaction system, and the nuclei 730 remain in the micro-droplet 800 as fluorescent labels for labeling the micro-droplet 800. After the amplification is completed, if the micro-droplet 800 contains target nucleic acid molecules, the fluorescent dye or the probe 713 will bind to the double-stranded DNA during the amplification process, and the fluorescent signal will be enhanced, so that a report fluorescent signal will be generated.

The microdroplets 800 with only one of the nuclei 730 having fluorescence encoded information are then screened out as valid microdroplets for subsequent analysis by detecting the nuclei 730 having fluorescence encoded information in a plurality of the microdroplets 800. Based on the obtained effective microdroplets (the effective microdroplet is the first effective microdroplet 810 or the second effective microdroplet 820 in the above embodiments), fluorescence signals of the nuclei 730 in the effective microdroplet are detected, and the types of the corresponding primer 712 and probe 713 are obtained. And then obtaining a report fluorescence signal after the nucleic acid amplification reaction in the effective micro-droplet, and judging whether the effective micro-droplet has corresponding target nucleic acid molecules according to the report fluorescence signal. Therefore, by the nucleic acid detecting microsphere 700, the preparation method, the kit and the high-throughput nucleic acid detecting method, it is possible to detect the presence or absence of a plurality of target nucleic acid molecules at a time by adding a plurality of the nucleic acid detecting microspheres 700 at a time, and the concentration of each detection target nucleic acid can be obtained according to poisson distribution.

Therefore, a plurality of target nucleic acids can be detected at one time by mixing a large number of different kinds of the nucleic acid detecting microspheres 700 with the nucleic acid amplification reaction solution to be detected, and repeated detection is not required, so that the workload is small, the time is saved, and the sensitivity is high.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. A nucleic acid detection microsphere for high throughput nucleic acid detection analysis, comprising:

A nucleus, said nucleus having fluorescence encoded information;

the coating layer wraps the nuclear body, the coating layer comprises a matrix and primers dispersed in the matrix, and the primers uniquely correspond to the nuclear body.

2. The nucleic acid detecting microsphere of claim 1, wherein the coating further comprises probes, and wherein the probes and the primers are dispersed in the matrix and correspond uniquely to the core.

3. The nucleic acid detecting microsphere according to claim 1 or 2, wherein the matrix is agarose gel.

4. The nucleic acid detection microsphere of claim 1 or 2, wherein the core is a solid sphere comprising a fluorescent dye.

5. The nucleic acid detecting microsphere of claim 1 or 2, wherein the diameter of the core is 10 microns to 100 microns.

6. The nucleic acid detecting microsphere according to claim 1 or 2, wherein the coating layer has a thickness of 10 to 100 μm.

7. A method for preparing nucleic acid detection microspheres, comprising the steps of:

s110, providing a plurality of nuclear bodies and primer solutions, wherein the nuclear bodies have fluorescence coding information, and the primers uniquely correspond to the nuclear bodies;

8. The method of producing a nucleic acid detecting microsphere according to claim 7, wherein in said step S150, said nucleic acid detecting microsphere is a nucleic acid detecting microsphere comprising a single one of said nuclei.

9. The method of claim 7, wherein in the step S120, the gel powder is agar powder, polyethylene glycol diacrylate, or the like.

10. The method of claim 7, wherein the step S140 comprises:

11. A method for preparing nucleic acid detection microspheres, comprising the steps of:

s210, providing a primer solution, a probe solution and a plurality of nuclei, wherein the nuclei have fluorescence coding information, the primer uniquely corresponds to the nuclei, and the probe uniquely corresponds to the nuclei;

12. The method of producing a nucleic acid detecting microsphere according to claim 11, wherein in said step S250, said nucleic acid detecting microsphere is a nucleic acid detecting microsphere comprising a single one of said nuclei.

13. A kit for high throughput nucleic acid detection analysis, comprising the nucleic acid detection microsphere of any one of claims 1 to 6 and a nucleic acid reaction solution.

14. A high throughput nucleic acid detection method comprising the steps of:

s310, providing a nucleic acid amplification reaction solution and a plurality of different types of nucleic acid detection microspheres, wherein the nucleic acid detection microspheres are the nucleic acid detection microspheres of any one of claims 1 and 3-6;

S360, detecting fluorescence coding information of the nucleon in the first effective micro-droplet according to the first effective micro-droplet, obtaining the primer corresponding to the nucleon, reading a report fluorescence signal after nucleic acid amplification reaction, and obtaining whether the corresponding target nucleic acid molecule exists in the first effective micro-droplet.

15. The method according to claim 14, wherein in the step S310, the nucleic acid amplification reaction solution is a deoxyribonucleic acid-based nucleic acid amplification reaction solution, a ribonucleic acid-based reverse transcription nucleic acid amplification reaction solution or a loop-mediated isothermal amplification reaction solution, and the nucleic acid amplification reaction solution contains a fluorescent dye.

16. The high throughput nucleic acid detection method of claim 14, wherein said step S360 comprises:

s361, providing a fluorescence signal detection device, wherein the fluorescence signal detection device comprises a coded fluorescence channel and a fluorescent dye detection channel, and identifying fluorescence coding information of the nucleon in the first effective micro-droplet according to the coded fluorescence channel;

s362, acquiring the primer corresponding to the nucleosome according to fluorescence coding information of the nucleosome;

17. A high throughput nucleic acid detection method comprising:

s410, providing a nucleic acid amplification reaction solution and a plurality of different types of nucleic acid detection microspheres, wherein the nucleic acid detection microspheres are the nucleic acid detection microspheres of any one of claims 2-6;

s460, detecting fluorescence coding information of the nucleon in the second effective micro-droplet according to the second effective micro-droplet, obtaining the primer and the probe corresponding to the nucleon, reading a report fluorescence signal after nucleic acid amplification reaction, and obtaining whether the corresponding target nucleic acid molecule exists in the second effective micro-droplet.