WO2022036860A1 - 基于CRISPR和Cas的数字化核酸扩增检测方法和集成化检测系统 - Google Patents

基于CRISPR和Cas的数字化核酸扩增检测方法和集成化检测系统 Download PDF

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WO2022036860A1
WO2022036860A1 PCT/CN2020/124875 CN2020124875W WO2022036860A1 WO 2022036860 A1 WO2022036860 A1 WO 2022036860A1 CN 2020124875 W CN2020124875 W CN 2020124875W WO 2022036860 A1 WO2022036860 A1 WO 2022036860A1
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nucleic acid
droplet
acid amplification
detection
crispr
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French (fr)
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叶尊忠
吴翠
应义斌
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浙江大学
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Definitions

  • the invention relates to the technical field of nucleic acid detection microfluidics, in particular to a CRISPR/Cas-based digital nucleic acid amplification detection method and an integrated detection system.
  • dPCR digital polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • RPA Recombinase Polymerase Amplification
  • MDA Multiple Displacement Amplification
  • the fluorescent substances in the fluorescence analysis method based on nucleic acid amplification mainly include fluorescent dyes and fluorescent probes: (1) fluorescent dyes such as SYBR series dyes, EvaGreen, FAM, etc.; (2) fluorescent probes such as Taqman probes.
  • fluorescent dyes are not specific and cannot distinguish the products of specific amplification and non-specific amplification.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • Cas Cas protein
  • CRISPR-associated protein, Cas cooperate to form a CRISPR/Cas system with nucleic acid cutting ability.
  • CRISPR/Cas can be used as a nucleic acid detection tool.
  • the CRISPR/Cas12a system can identify and capture the target strand according to the designed gRNA (guide Ribonucleic Acid), and its DNA (Deoxyribonucleic Acid) enzyme cleavage activity is activated, thereby efficiently cleaving the single-stranded fluorescent probe in the system, To achieve specific detection of target DNA.
  • gRNA guide Ribonucleic Acid
  • DNA Deoxyribonucleic Acid
  • CRISPR/Cas has begun to be used in nucleic acid detection, it has not been used in conjunction with digital nucleic acid amplification.
  • detection systems based on digital nucleic acid amplification technology often require multiple instruments such as droplet generators, nucleic acid amplifiers, and droplet signal detectors, which are poorly integrated, and the operation steps are cumbersome, which is still in the stage of improvement;
  • the working temperature of nucleic acid amplification technology for example, PCR technology needs to be denatured into a single strand at a high temperature of 95 °C
  • the tolerance temperature of the reagents in the CRISPR/Cas system generally 37 °C. Opening the cap and adding the CRISPR/Cas system after the increase will cause aerosol contamination, which is likely to cause false positives in subsequent test results.
  • the purpose of the present invention is to provide a CRISPR/Cas-based digital nucleic acid amplification detection method and an integrated detection system in order to solve the problems of poor integration of the digital detection system in the background technology and the use of the CRISPR/Cas system to easily cause aerosol pollution.
  • This method combines CRISPR/Cas with digital nucleic acid amplification technology to achieve absolute quantitative and highly specific detection of target nucleic acid molecules.
  • An integrated detection system for CRISPR/Cas is an integrated detection system for CRISPR/Cas.
  • the technical scheme adopted in the present invention mainly includes:
  • It includes an integrated reaction chip, a temperature control module, a light source, and an optical signal detector; the integrated reaction chip is distributed with an amplification system droplet generation area, a nucleic acid amplification area, a detection system droplet generation area, and a droplet fusion area. area and optical detection area; between the droplet generation area and the nucleic acid amplification area of the amplification system, between the nucleic acid amplification area and the droplet fusion area, between the detection system droplet generation area and the droplet fusion area, and between the droplet fusion area Microchannels are used to communicate between the area and the optical detection area;
  • the light source and the optical signal detector are respectively located on the upper and lower sides of the optical detection area, and the temperature control module is placed below or above the nucleic acid amplification area, and heats the nucleic acid amplification area.
  • the integrated reaction chip is provided with a quick connection structure, and the temperature control module is connected to the nucleic acid amplification region on the integrated reaction chip through the quick connection structure.
  • a cooling device is placed in the droplet generation area of the detection system, or a cooling channel is arranged around, and cooling liquid is added to the cooling channel.
  • the width of the microchannel is the same as the size of a single droplet or smaller than the diameter of the droplet, so that only a single droplet can pass through the microchannel in sequence.
  • a CRISPR/Cas-based digital nucleic acid amplification detection method :
  • the solution of the nucleic acid amplification system enters the droplet generation area of the amplification system and is divided into tens of thousands of amplified microdroplets, and the nucleic acid amplification area is heated by the temperature control module; and then the amplified microdroplets are driven to pass through the microdroplets.
  • the channel enters the nucleic acid amplification area according to the fixed flow rate to realize digital nucleic acid amplification;
  • the droplet generation area of the amplification system controls the generation of droplets by adjusting the structure and the two-phase flow rate ratio through the micro-pipe structure based on the T-channel method, the flow focusing method or the coaxial flow focusing method.
  • Two-phase refers to a two-phase liquid of a solution/droplet and its surrounding oil.
  • the diameters of the amplified microdroplets range from nanometers to micrometers, and the number can range from tens of thousands to millions, but the diameters of the droplets produced each time are the same, and the size of the droplets prepared each time is uniform. Sex is better.
  • the amplification technique may be polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-dependent amplification (NASBA), rolling circle amplification (RCA), helicase-dependent amplification technology (HDA), recombinase polymerase amplification (RPA), multiple strand displacement amplification (MDA), etc.
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence-dependent amplification
  • RCA rolling circle amplification
  • HDA helicase-dependent amplification technology
  • RPA recombinase polymerase amplification
  • MDA multiple strand displacement amplification
  • the solution of the CRISPR/Cas system is evenly divided into tens of thousands of detection droplets in the droplet generation area of the detection system, wherein the detection droplets contain single-stranded oligonucleotides labeled with fluorescent groups as fluorescent probes acid probe;
  • the droplet generation area of the detection system controls the generation of droplets by adjusting the structure and the two-phase flow rate ratio through the micro-pipe structure based on the T-channel method, the flow focusing method or the coaxial flow focusing method.
  • the diameter of the detection microdroplets ranges from nanometers to micrometers, and the number thereof ranges from tens of thousands to millions, but the diameters of the droplets produced each time are the same, and the size of the droplets prepared each time is the same. Uniformity is good.
  • the amplification droplets and the detection droplets enter the droplet fusion zone at a fixed flow rate after the amplification of the nucleic acid amplification area is completed, and the collision and aggregation between droplets are used to carry out an amplification droplet and an amplification droplet respectively.
  • the mixed droplets enter the optical detection area, and the target strand in the mixed droplets is identified and captured by gRNA, and its DNA enzyme cleavage activity is activated;
  • the fluorescent probe is cleaved, so that the fluorescent group and the quenching group are separated, and a fluorescent signal is emitted under the excitation of the light source, and the fluorescence signal of a single mixed droplet is analyzed by an optical signal detector.
  • a portion of the sample to be tested is added to the nucleic acid amplification system.
  • the droplet fusion zone is a Y-shaped or T-shaped microchannel, and the three ends of the Y-shaped or T-shaped microchannel are respectively connected with the nucleic acid amplification zone, the detection system droplet generation zone and the optical detection zone through the respective microchannels.
  • the droplets in the microchannel are driven by the oil phase flowing around them, and the flow rate of the oil phase is controlled by the micropump.
  • the mixed droplets are driven to flow through the optical detection area in sequence, and the optical signal conversion is detected by an optical signal detector such as a photomultiplier tube or a photodetector under the excitation of a light source or without a light source (if it is not a fluorescent substance, the excitation light source is not required).
  • an optical signal detector such as a photomultiplier tube or a photodetector under the excitation of a light source or without a light source (if it is not a fluorescent substance, the excitation light source is not required).
  • the optical signal of the mixed droplets is continuously recorded, and the ratio p of negative droplets and positive droplets is obtained by filtering the waveform of the electrical signal, removing the baseline, and dividing the threshold value;
  • Negative droplets refer to mixed droplets with no fluorescent signal, that is, the droplets do not contain nucleic acid molecules; positive droplets refer to mixed droplets with fluorescent signals, that is, the droplets contain at least one nucleic acid molecule.
  • the method finally calculates the average number of nucleic acid molecules ⁇ in each mixed droplet according to the ratio p of the negative and positive droplets according to the following formula, so as to obtain the concentration or copy number of nucleic acid molecules in the sample to be tested:
  • the present invention realizes the combination of digital nucleic acid amplification technology and CRISPR/Cas in the field of nucleic acid detection for the first time, which not only has the characteristics of absolute quantification and high tolerance of digital amplification technology, but also has high sensitivity and high specificity of CRISPR/Cas. Sexual advantages.
  • the integrated detection system based on droplet digital nucleic acid amplification and CRISPR/Cas proposed by the present invention The CRISPR/Cas-based digital nucleic acid amplification integrated detection system integrates droplet generation, nucleic acid amplification, and fluorescence detection. On one chip, the operation steps are simplified, and the cross-contamination caused by the second opening of the cap and adding the detection reagent after nucleic acid amplification is avoided.
  • the present invention only regulates the temperature of the nucleic acid amplification area, and can add cooling equipment or cooling liquid in the droplet generation area of the detection system, which overcomes the influence of high temperature on the activity of the reagents in the CRISPR/Cas system and ensures the accuracy of detection.
  • the temperature control module used in the present invention can be disassembled, and the variable temperature control module or the constant temperature control module can be selected according to the amplification technology used.
  • the optical detection method can also detect two kinds of mixed microdroplets one by one and all mixed microdroplets simultaneously. The choice of the method improves the universality of the system.
  • the present invention has simple structure, small volume, high portability, and is suitable for field use.
  • Fig. 1 is the detection flow chart of the present invention.
  • Fig. 2 is the detection principle diagram of the present invention.
  • FIG. 3 is a top view of the integrated reaction chip in the present invention.
  • FIG. 4 is a top view of the cooling channel in the integrated reaction chip of the present invention.
  • FIG. 5 is a top view of the droplet fusion region (Y-shaped microchannel) in the integrated reaction chip of the present invention.
  • nucleic acid amplification system 1 CRISPR/Cas system 2, integrated reaction chip 3, amplification system droplet generation area 4, microchannel 5, nucleic acid amplification area 6, detection system droplet generation area 7, droplet Fusion zone 8 , optical detection zone 9 , quick connection structure 10 , cooling channel 11 , amplifying droplet 12 , detecting droplet 13 , mixing droplet 14 .
  • the specific implementation includes an integrated reaction chip 3, a temperature control module, a light source, and an optical signal detector; as shown in FIG. 3, the integrated reaction chip 3 is distributed with an amplification system droplet generation area 4, a nucleic acid amplification area 6, Detection system droplet generation area 7, droplet fusion area 8 and optical detection area 9; between the droplet generation area 4 of the amplification system and the nucleic acid amplification area 6, between the nucleic acid amplification area 6 and the droplet fusion area 8, In the detection system, the microchannel 5 is used for communication between the droplet generation area 7 and the droplet fusion area 8 and between the droplet fusion area 8 and the optical detection area 9 .
  • the light source and the optical signal detector are located on the upper and lower sides of the optical detection area 9 respectively, and the temperature control module is placed above or below the nucleic acid amplification area 6 to heat the nucleic acid amplification area 6 . After the digital nucleic acid amplification is completed, the temperature control module can be turned off or removed.
  • the temperature control module is suitable for the variable temperature environment of PCR, and can also realize the constant temperature environment suitable for constant temperature amplification technologies such as LAMP, RPA, and NASBA.
  • the integrated reaction chip 3 is provided with a quick connection structure 10, and the temperature control module is connected to the nucleic acid amplification area 6 on the integrated reaction chip (3) through the quick connection structure 10, so that the temperature control module and the nucleic acid amplification area 6 pass through the rapid connection.
  • the connection structure 10 is positioned and fixed.
  • the quick connection structure 10 may be a connection structure based on magnet adsorption, a snap structure based on simple rotation, a snap structure based on push-type self-locking, and the like.
  • a cooling device is placed in the droplet generation area 7 of the detection system during the amplification process, or a cooling channel 11 is arranged around it, as shown in FIG. 4 , a cooling liquid is added to the cooling channel 11 . In this way, the temperature of the droplet generating area 7 of the detection system is lowered during the amplification process.
  • the width of the microchannel 5 is the same as the size of a single droplet, so that only a single droplet can pass through the microchannel 5 in sequence.
  • the light source may be a light emitting diode LED, a laser diode, or the like.
  • the optical signal detector can be Photomultiplier, PMT, photodiode, CCDCharge Coupled Device, mobile phone with camera function, optical microscope, etc.
  • Cooling equipment which can cool fans, heat sinks, liquids, etc.
  • the droplet fusion area 8 is a Y-shaped microchannel, and the three ends of the Y-shaped microchannel are respectively connected to the nucleic acid amplification area 6 , the detection system droplet generation area 7 and the optical detection area 9 through the respective microchannels 5 . Connected.
  • the droplet generation area 4 of the amplification system and the droplet generation area 7 of the detection system are both droplet generation areas, and the micro-channel structure designed based on the T-channel method, flow focusing method or coaxial flow focusing method can be used. structure and two-phase flow rate ratio to control droplet formation and its size.
  • the nucleic acid amplification region 6 and the temperature control module are positioned and fixed through a quick connection structure, for example, a connection structure based on magnet adsorption.
  • the temperature control module can be selected according to the amplification technology used. If the polymerase chain reaction (PCR) is used, the temperature control module can choose the variable temperature control module or the multi-temperature zone control module; if the constant temperature amplification technology such as LAMP is used, the temperature control module can be selected. Select a single zone control module.
  • the temperature control module In order to prevent the temperature of the nucleic acid amplification zone 6 from affecting the activity of the reagents in the CRISPR/Cas system, the temperature control module only regulates the temperature of the nucleic acid amplification zone on the integrated reaction chip. After the digital nucleic acid amplification is completed, it can be turned off or removed. the temperature control module.
  • the amplification microdroplet 12 completes the amplification and the detection microdroplet 13 enters the droplet fusion zone 8 at a certain flow rate, through a suitable flow channel design, the two microdroplets are contacted and fused together, and then the CRISPR reaction is performed, and the Detection of optical signals to achieve target detection.
  • the CRISPR/Cas system includes fluorescently labeled single-stranded oligonucleotide probes. After the CRISPR reaction is complete, the target strand can be identified and captured by gRNA, and its DNA enzyme cleavage activity is activated. If the mixed droplet 14 contains the target chain, the fluorescent probe is cleaved, so that the reporter fluorescent group and the quenching fluorescent group are separated, and a fluorescent signal is emitted under the excitation of a light source of a certain wavelength.
  • optical signal detection it can be implemented in two different ways:
  • the mixed microdroplets 14 are detected one by one: In this way, the mixed microdroplets 14 flow through the optical detection area one by one at a certain rate, and are excited by a light source of a certain wavelength (if it is not a fluorescent substance, no The excitation light source), the optical signal can be converted into an electrical signal by an optical detector such as a PMT or a photodetector, and the optical signal of the mixed droplet 14 can be continuously recorded. step, the ratio of negative and positive droplets can be obtained.
  • the mixed microdroplets 14 are gathered in the optical detection area, and can be excited by a light source of a certain wavelength (if it is not a fluorescent substance, the excitation light source is not required), the camera or A mobile phone with a camera function takes pictures of the optical detection area to obtain an optical image.
  • image processing steps such as region of interest acquisition, filtering, threshold segmentation, and counting, the ratio of negative and positive droplets can be obtained.
  • the proportion p of positive droplets can be obtained.
  • the average number of nucleic acid molecules ⁇ in each mixed droplet 14 can be calculated according to the following formula, so that Obtain the concentration or copy number of nucleic acid molecules in the sample to be tested.
  • the temperature control module is turned off, and the amplification droplet 12 and the detection droplet 13 flow into the droplet fusion zone 8 at a certain flow rate, as shown in FIG.
  • the droplets are fused one by one, and then the CRISPR reaction is performed in the mixed droplets 14;
  • the mixed microdroplets 14 are detected one by one: In this way, the mixed microdroplets 14 flow through the optical detection area one by one at a certain rate. The signal is converted into an electrical signal to record the optical signal of the mixed droplet 14;
  • the mixed microdroplets 14 converge in the optical detection area.
  • the optical detection area can be photographed by a camera or a mobile phone with a camera function to obtain optical image;
  • step 6) correspond to two different optical signal processing and result analysis methods respectively:
  • the ratio of negative and positive droplets can be obtained by filtering the signal waveform, removing the baseline, dividing the threshold value, etc.;
  • the ratio of negative and positive droplets can be obtained through image processing steps such as region of interest acquisition, filtering, threshold segmentation, and counting;
  • the proportion of positive droplets can be obtained, and the concentration or copy number of nucleic acid molecules in the sample to be tested can be calculated according to the Poisson distribution principle, and the test result can be obtained;

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Abstract

本发明公开了一种基于CRISPR/Cas的数字化核酸扩增检测方法和集成化检测系统。集成化检测系统包括一个集成化反应芯片、温度控制模块、光源、光学信号检测器；将核酸扩增体系均分成扩增微液滴，而后将数字化核酸扩增后的扩增微液滴和包含有CRISPR/Cas体系的检测微液滴混合进行CRISPR反应，反应结束后，通过检测光学信号实现目标物的高特异性检测，并得到待测样品中核酸分子的浓度或者拷贝数，实现目标物的高灵敏绝对定量检测。本发明既具有数字化扩增技术绝对定量的特点，又具有CRISPR/Cas高灵敏、高特异性检测的优点，将检测过程集成在一个芯片上，简化了操作步骤，同时避免了交叉污染等问题。

Description

基于CRISPR和Cas的数字化核酸扩增检测方法和集成化检测系统 技术领域
本发明涉及核酸检测微流控技术领域，具体涉及一种基于CRISPR/Cas的数字化核酸扩增检测方法和集成化检测系统。
背景技术
自1999年Vogelstein等人提出数字聚合酶链式反应(digital Polymerase Chain Reaction，dPCR)以来，已在食品安全、法医鉴定、精准医疗等研究领域显示出巨大的技术优势和应用前景。dPCR是一种核酸分子绝对定量技术，通过将一个样本分散成几十到几万份至不同的反应单元，每个单元的核酸模板数少于或者等于1个；每个单元分别进行PCR扩增，扩增结束后有核酸模板的反应单元会发出荧光信号，没有模板的反应单元没有荧光信号，因此通过对各个反应单元的荧光信号进行统计学分析得到核酸分子的数量。对于常规PCR和实时荧光PCR，其定量检测都需要已知拷贝数的标准DNA制定标准曲线，然而由于样品测定条件上不会完全一致，会造成PCR扩增效率的差异，从而影响定量结果的准确性。而根据dPCR原理可知，该技术不会受标准曲线和扩增动力学影响，可以实现高灵敏度、高精准度、高耐受性的绝对定量。
由于PCR扩增技术需要对多个反应温度进行精准控制，相关研究人员也提出了基于恒温扩增技术的数字化恒温核酸技术，例如基于环介导等温扩增(Loop-mediated isothermal amplification，LAMP)的dLAMP、基于重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)的dRPA、基于多重链置换扩增(Multiple Displacement Amplification，MDA)的dMDA等。
不论是dPCR还是数字化恒温扩增技术，目前对于阴阳性反应单元的检测大都基于荧光分析方法，通过在样品中加入相应的荧光物质。基于核酸扩增的荧光分析法中的荧光物质主要有荧光染料、荧光探针：(1)荧光染料如SYBR系列染料、EvaGreen、FAM等；(2)荧光探针如Taqman探针。但是其中荧光染料不具有特异性，无法区分特异性扩增和非特异性扩增的产物。
CRISPR(Clustered regularly interspaced short palindromic repeats，规律成簇的具有规律间隔的短回文重复序列)，是大多数细菌及古细菌中一种不断进化适应的免疫防御机制。CRISPR和Cas蛋白(CRISPR-associated protein,Cas)协同作用，组成具有核酸切割能力的CRISPR/Cas系统。基于此，可以将CRISPR/Cas 作为核酸检测工具。例如，CRISPR/Cas12a系统可以根据设计的gRNA(guide Ribonucleic Acid)对目标链进行识别与捕获，其DNA(Deoxyribonucleic Acid)酶切活性被激活，从而对体系中的单链荧光探针进行高效切割，实现目标DNA的特异性检测。
目前，虽然CRISPR/Cas已经开始应用于核酸检测中，但还未将其结合数字化核酸扩增一起使用。一方面是基于数字化核酸扩增技术的检测系统往往需要液滴发生器、核酸扩增仪、液滴信号检测仪等多台仪器，集成度差，操作步骤比较繁琐，仍在待完善阶段；另一方面是由于核酸扩增技术的工作温度(例如PCR技术中需要在95℃高温时变性成单链)远高于CRISPR/Cas体系中试剂的耐受温度(一般为37℃)，若在扩增结束后再开盖加入CRISPR/Cas体系会造成气溶胶污染，对后续的检测结果容易造成假阳性。
因此本领域需要一种可以解决上述问题的方法，将数字核酸扩增技术和CRISPR/Cas一起使用，结合两者绝对定量、高灵敏、高特异性的优点，并开发一种集成化检测系统，更好地应用于核酸检测领域。
发明内容
本发明的目的，为了解决背景技术中数字化检测系统集成度差、采用CRISPR/Cas体系容易造成气溶胶污染等问题，提供一种基于CRISPR/Cas的数字化核酸扩增检测方法和集成化检测系统，利用此方法将CRISPR/Cas与数字核酸扩增技术相结合，实现对目标核酸分子的绝对定量、高特异性的检测，在避免扩增产物污染的同时简化操作步骤，实现基于数字化核酸扩增和CRISPR/Cas的集成化检测系统。
为实现上述目的，本发明采用的技术方案主要包含：
一、一种基于液滴式数字核酸扩增和CRISPR/Cas的集成化检测系统：
包含一个集成化反应芯片、温度控制模块、光源、光学信号检测器；所述集成化反应芯片上分布有扩增体系液滴生成区、核酸扩增区、检测体系液滴生成区、液滴融合区和光学检测区；扩增体系液滴生成区与核酸扩增区之间、核酸扩增区与液滴融合区之间、检测体系液滴生成区与液滴融合区之间以及液滴融合区与光学检测区之间均采用微通道连通；
光源与光学信号检测器分别位于光学检测区的上下两侧，温度控制模块置于所述核酸扩增区下方或上方，并对核酸扩增区实现加热。
所述的集成化反应芯片上设有快速连接结构，温度控制模块通过快速连接结构连接到所述集成化反应芯片上的核酸扩增区。
所述检测体系液滴生成区放置有一个降温设备，或者在周围布置冷却通道， 冷却通道中加入冷却液。
所述的微通道的宽度为单个液滴的大小一致或微小于液滴直径，使得只能单个液滴依次通过微通道。
二、一种基于CRISPR/Cas的数字化核酸扩增检测方法：
将核酸扩增体系的溶液均分成数以万计的扩增微液滴，再选择工作环境实现扩增微液滴内的核酸扩增；同时将CRISPR/Cas体系的溶液均分成数以万计的检测微液滴；将检测微液滴与扩增微液滴分别一一进行融合，之后进行CRISPR反应，进而通过检测光学信号实现目标物的高特异性检测。
所述核酸扩增体系的溶液进入通过扩增体系液滴生成区均分成数以万计的扩增微液滴，通过温度控制模块对核酸扩增区加热；然后驱动扩增微液滴经由微通道按照固定流速进入核酸扩增区，实现数字化核酸扩增；
所述扩增体系液滴生成区通过基于T型通道法、流动聚焦法或共轴流聚焦法等方法的微管道结构，调整结构和两相流速比来控制液滴的生成。两相是指溶液/液滴与其周围的油液的两相液体。
所述扩增微液滴直径从纳米级至微米级不等，其数量可从几万颗至几百万颗不等，但是每次产生的液滴直径一致，每次制备的液滴尺寸均一性较好。
所述扩增技术可为聚合酶链式反应(PCR)、环介导等温扩增(LAMP)、依赖核酸序列扩增(NASBA)、滚环扩增技术(RCA)、依赖解旋酶扩增技术(HDA)、重组酶聚合酶扩增(RPA)、多重链置换扩增(MDA)等。
CRISPR/Cas体系的溶液在检测体系液滴生成区中均分成数以万计的检测微液滴，其中检测微液滴包含有作为荧光探针的带有荧光基团标记的单链寡核苷酸探针；
所述检测体系液滴生成区通过基于T型通道法、流动聚焦法或共轴流聚焦法等方法的微管道结构，调整结构和两相流速比来控制液滴的生成。
所述检测微液滴其直径从纳米级至微米级不等，其数量可从几万颗至几百万颗不等，但是每次产生的液滴直径一致，且每次制备的液滴尺寸均一性较好。
所述扩增微液滴在核酸扩增区完成扩增后和所述检测微液滴按固定流速进入液滴融合区，利用液滴间的碰撞和聚合分别进行一个扩增微液滴和一个检测微液滴的一一融合形成混合微液滴，之后混合微液滴进行CRISPR反应；
CRISPR反应完全后，混合微液滴进入光学检测区，通过gRNA对混合微液滴中的目标链进行识别与捕获，其DNA酶切活性被激活；
若混合微液滴中含有目标链，则荧光探针被切割，使得荧光基团和淬灭基团分离，在光源激发下发出荧光信号，通过光学信号检测器分析单个混合微液 滴的荧光信号获得阴性液滴和阳性液滴(有荧光信号)的比例，再计算核酸扩增体系中核酸分子的浓度或者拷贝数，从而获得绝对定量的检测结果。从待测样品中取部分加入核酸扩增体系中。
所述的液滴融合区为Y型或T型微通道，Y型或T型微通道的三端经各自的微通道分别和核酸扩增区、检测体系液滴生成区和光学检测区连通。
微通道中的液滴通过其周围流动的油相驱动，油相的流动速度通过微泵控制。
方法对于阴性液滴和阳性液滴比例的获得，采用以下两种不同的方式之一：
(A)混合微液滴逐个检测：
驱动混合微液滴依次流过光学检测区，在光源激发或者没有光源激发下(若不是荧光物质，则无需该激发光源)，通过光电倍增管或者光电检测器等光学信号检测器检测光信号转化为电信号，连续记录混合微液滴的光学信号，通过对电信号的波形进行滤波处理、去除基线、阈值划分的处理得到阴性液滴和阳性液滴的比例p；
阴性液滴是指不存在荧光信号的混合微液滴，即该液滴中不包含核酸分子；阳性液滴是指存在荧光信号的混合微液滴，即该液滴中至少包含一个核酸分子。
(B)所有微液滴同时检测：所有混合微液滴汇聚在光学检测区并分散排布，在光源激发或者没有光源激发下(若不是荧光物质，则无需该激发光源)，通过相机或者带有拍照功能的手机对光学检测区进行拍照获得光学图像，通过感兴趣区域获取、滤波处理、阈值分割、计数的图像处理得到阴性液滴和阳性液滴的比例p；
方法最后根据阴阳性液滴的比例p，按下述公式计算出每个混合微液滴中的平均核酸分子数λ，从而获得待测样品中核酸分子的浓度或者拷贝数：
λ＝-ln(1-p)。
本发明具有的有益效果是：
1、本发明首次实现了将数字化核酸扩增技术和CRISPR/Cas结合应用于核酸检测领域，既具有数字化扩增技术绝对定量、高耐受性的特点，又具有CRISPR/Cas高灵敏、高特异性的优点。
2、本发明所提出的基于液滴式数字核酸扩增和CRISPR/Cas的集成化检测系统基于CRISPR/Cas的数字化核酸扩增集成化检测系统，将液滴生成、核酸扩增、荧光检测集成在一个芯片上，简化了操作步骤，同时避免了核酸扩增后二次开盖加入检测试剂而造成的交叉污染。
3、本发明仅对核酸扩增区进行温度调控，并且可以在检测体系液滴生成区 增加降温设备或者冷却液，克服了高温对CRISPR/Cas体系中试剂活性的影响，保证检测的准确性。
4、本发明采用的温度控制模块可拆卸，根据所采用的扩增技术选择变温控制模块或者恒温控制模块，光学检测方法亦可在混合微液滴逐个检测和所有混合微液滴同时检测两种方式中选择，提高了系统的使用普适性。
5、本发明结构简单，体积较小，有较高的便携性，适用于野外现场使用。
附图说明
图1是本发明的检测流程图。
图2是本发明的检测原理图。
图3是本发明中的集成化反应芯片俯视图。
图4是本发明中的集成化反应芯片中冷却通道俯视图。
图5是本发明中的集成化反应芯片中液滴融合区(Y型微通道)的俯视图。
图中：核酸扩增体系1，CRISPR/Cas体系2，集成化反应芯片3，扩增体系液滴生成区4，微通道5，核酸扩增区6，检测体系液滴生成区7，液滴融合区8，光学检测区9，快速连接结构10，冷却通道11，扩增微液滴12，检测微液滴13，混合微液滴14。
具体实施方式
下面结合说明书附图，对本发明做进一步说明，但本发明并不局限于以下实施例。
具体实施包含一个集成化反应芯片3、温度控制模块、光源、光学信号检测器；如图3所示，集成化反应芯片3上分布有扩增体系液滴生成区4、核酸扩增区6、检测体系液滴生成区7、液滴融合区8和光学检测区9；扩增体系液滴生成区4与核酸扩增区6之间、核酸扩增区6与液滴融合区8之间、检测体系液滴生成区7与液滴融合区8之间以及液滴融合区8与光学检测区9之间均采用微通道5连通。
光源与光学信号检测器分别位于光学检测区9的上下两侧，温度控制模块置于核酸扩增区6的上方或下方，并对核酸扩增区6实现加热。数字化核酸扩增完成后即可关闭或者移除温度控制模块。
温度控制模块适用于PCR的变温环境、亦可实现适合于LAMP、RPA、NASBA等恒温扩增技术的恒温环境。
集成化反应芯片3上设有快速连接结构10，温度控制模块通过快速连接结构10连接到集成化反应芯片(3)上的核酸扩增区6，使得温度控制模块与核酸扩增区6通过快速连接结构10进行定位及固定。具体实施中，快速连接结构10 可以为基于磁铁吸附的连接结构、基于简单旋转的卡扣结构、基于按压式自锁的卡扣结构等。
检测体系液滴生成区7在扩增的过程中放置有一个降温设备，或者在周围布置冷却通道11，如图4所示，冷却通道11中加入冷却液。这样在扩增过程中对检测体系液滴生成区7进行降温。
微通道5的宽度为单个液滴的大小一致，使得只能单个液滴依次通过微通道5。
光源可为发光二极管LED、激光二极管等。
光学信号检测器可为光电倍增管Photomultiplier，PMT、光电二极管、CCDCharge Coupled Device、带有拍照功能的手机、光学显微镜等。
降温设备，可为风扇、散热片、液体降温等。
如图5所示，液滴融合区8为Y型微通道，Y型微通道的三端经各自的微通道5分别和核酸扩增区6、检测体系液滴生成区7和光学检测区9连通。
扩增体系液滴生成区4和检测体系液滴生成区7均为液滴生成区，可采用基于T型通道法、流动聚焦法或共轴流聚焦法等方法设计的微管道结构，通过调整结构和两相流速比来控制液滴的生成及其尺寸。
核酸扩增区6与温度控制模块通过快速连接结构进行定位并固定，例如基于磁铁吸附的连接结构。温度控制模块可以根据采用的扩增技术选择，若采用聚合酶链式反应(PCR)，则温度控制模块可以选择变温控制模块或多温区控制模块；若采用恒温扩增技术如LAMP，则可选择单温区控制模块。
为了避免核酸扩增区6的温度影响CRISPR/Cas体系中试剂的活性，温度控制模块仅调控集成化反应芯片上的核酸扩增区的温度，数字化核酸扩增完成后即可关闭或者移除所述温度控制模块。
扩增微液滴12完成扩增后和检测微液滴13按一定流速进入液滴融合区8，通过合适的流道设计，两种微液滴接触并融合在一起，而后进行CRISPR反应，通过检测光学信号实现目标物检测。例如所述CRISPR/Cas体系中，包含有带有荧光标记的单链寡核苷酸探针。CRISPR反应完全后，可通过gRNA对目标链进行识别与捕获，其DNA酶切活性被激活。若混合微液滴14中含有目标链，则荧光探针被切割，使得报告荧光基团和淬灭荧光基团分离，在一定波长的光源激发下发出荧光信号。
对于光学信号检测，可以采用两种不同的方式实现：
(1)混合微液滴14逐个检测：此种方式下，混合微液滴14以一定的速率一个接一个地流过光学检测区，在一定波长的光源激发下(若不是荧光物质， 则无需该激发光源)，可通过PMT或者光电检测器等光学检测器将光信号转化为电信号，连续记录下混合微液滴14的光学信号，通过对信号波形进行滤波处理、去除基线、阈值划分等步骤，可以得到阴阳性液滴比例。
(2)所有微液滴同时检测：此种方式下，混合微液滴14汇聚在光学检测区，在一定波长的光源激发下(若不是荧光物质，则无需该激发光源)，可通过相机或者带有拍照功能的手机对光学检测区进行拍照，获得光学图像，通过感兴趣区域获取、滤波处理、阈值分割、计数等图像处理步骤，可以得到阴阳性液滴比例。
不论方式(1)还是方式(2)，均可得到阳性液滴的比例p，根据泊松分布原理，按下述公式可计算出每个混合微液滴14中的平均核酸分子数λ，从而获得待测样品中核酸分子的浓度或者拷贝数。
λ＝-ln(1-p)
下面结合本发明的方法与集成化检测系统，对本发明的内容及实施过程作进一步阐述：
1)待测液准备：
①根据实际所采用的扩增技术配置所需核酸扩增体系1；
②置合适浓度的CRISPR/Cas体系2；
2)将配置好的核酸扩增体系1加入集成化反应芯片3的扩增体系液滴生成区4，生成进行数字化核酸扩增所需的扩增微液滴12；
3)根据所采用的扩增技术选择合适的温度控制模块，通过快速连接结构10将集成化反应芯片3上的核酸扩增区6固定在温度控制模块上方，打开温度控制模块的电源进行加热，实现核酸扩增；
4)将配置好的CRISPR/Cas体系2加入集成化反应芯片3的检测体系液滴生成区7，生成光学检测所需的检测微液滴13，为了减小扩增加热过程对CRISPR/Cas体系2中试剂活性的影响，可以在冷却通道11中添加冷却液；
5)数字化核酸扩增后，关闭温度控制模块，扩增微液滴12与检测微液滴13以一定的流速流入液滴融合区8，如图5所示，通过Y型微通道后两种液滴分别进行一一融合，而后在混合微液滴14中进行CRISPR反应；
6)CRISPR反应完全后，获取单个混合微液滴14的光学信号，有两种实现方式：
①混合微液滴14逐个检测：此种方式下，混合微液滴14以一定的速率一个接一个地流过光学检测区，在一定波长的光源激发下，可通过PMT或者光电检测器将光信号转化为电信号记录下混合微液滴14的光学信号；
②所有微液滴同时检测：此种方式下，混合微液滴14汇聚在光学检测区，在一定波长的光源激发下，可通过相机或者带有拍照功能的手机对光学检测区进行拍照，获得光学图像；
7)根据步骤6)的两个光学信号采集方式分别对应于两种不同的光学信号处理及结果分析方法：
①得到混合微液滴14中每个液滴的光学信号后，通过对信号波形进行滤波处理、去除基线、阈值划分等步骤，可以得到阴阳性液滴比例；
②得到混合微液滴14光学图像后，通过感兴趣区域获取、滤波处理、阈值分割、计数等图像处理步骤，可以得到阴阳性液滴比例；
根据方法①或方法②得到阳性液滴的比例，按泊松分布原理可计算出待测样品中核酸分子的浓度或者拷贝数，得到检测结果；
8)取下集成化反应芯片，更换新的集成化反应芯片或者结束检测。
上述具体实施方式是用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改版，都落入本发明的保护范围。

Claims (8)

  1. 一种基于液滴式数字核酸扩增和CRISPR/Cas的集成化检测系统，其特征在于：包含一个集成化反应芯片(3)、温度控制模块、光源、光学信号检测器；所述集成化反应芯片(3)上分布有扩增体系液滴生成区(4)、核酸扩增区(6)、检测体系液滴生成区(7)、液滴融合区(8)和光学检测区(9)；扩增体系液滴生成区(4)与核酸扩增区(6)之间、核酸扩增区(6)与液滴融合区(8)之间、检测体系液滴生成区(7)与液滴融合区(8)之间以及液滴融合区(8)与光学检测区(9)之间均采用微通道(5)连通；光源与光学信号检测器分别位于光学检测区(9)的上下两侧，温度控制模块置于所述核酸扩增区(6)下方或上方，并对核酸扩增区(6)实现加热。
  2. 根据权利要求1所述的一种基于液滴式数字核酸扩增和CRISPR/Cas的集成化检测系统，其特征在于：所述的集成化反应芯片(3)上设有快速连接结构(10)，温度控制模块通过快速连接结构(10)连接到所述集成化反应芯片(3)上的核酸扩增区(6)。
  3. 根据权利要求1所述的一种基于液滴式数字核酸扩增和CRISPR/Cas的集成化检测系统，其特征在于：所述检测体系液滴生成区(7)放置有一个降温设备，或者在周围布置冷却通道(11)，冷却通道(11)中加入冷却液。
  4. 根据权利要求1所述的一种基于液滴式数字核酸扩增和CRISPR/Cas的集成化检测系统，其特征在于：所述的微通道(5)的宽度为单个液滴的大小一致，使得只能单个液滴依次通过微通道(5)。
  5. 应用于权利要求1-4任一所述集成化检测系统的一种基于CRISPR/Cas的数字化核酸扩增检测方法，其特征在于：将核酸扩增体系(1)的溶液均分成数以万计的扩增微液滴(12)，再选择工作环境实现扩增微液滴(12)内的核酸扩增；同时将CRISPR/Cas体系(2)的溶液均分成数以万计的检测微液滴(13)；将检测微液滴(13)与扩增微液滴(12)分别一一进行融合，之后进行CRISPR反应，进而通过检测光学信号实现目标物的高特异性检测。
  6. 根据权利要求5所述的一种基于CRISPR和Cas的数字化核酸扩增检测方法，其特征在于：所述核酸扩增体系(1)的溶液进入通过扩增体系液滴生成区(4)均分成数以万计的扩增微液滴(12)，通过温度控制模块对核酸扩增区(6)加热；然后驱动扩增微液滴(12)经由微通道(5)按照固定流速进入核酸扩增区(6)，实现数字化核酸扩增；
    CRISPR/Cas体系(2)的溶液在检测体系液滴生成区(7)中均分成数以万 计的检测微液滴(13)，其中检测微液滴(13)包含有作为荧光探针的带有荧光基团标记的单链寡核苷酸探针；
    所述扩增微液滴(12)在核酸扩增区(6)完成扩增后和所述检测微液滴(13)按固定流速进入液滴融合区(8)，利用液滴间的碰撞和聚合分别进行一个扩增微液滴(12)和一个检测微液滴(13)的一一融合形成混合微液滴(14)，之后混合微液滴(14)进行CRISPR反应；
    CRISPR反应完全后，混合微液滴(14)进入光学检测区(9)，通过gRNA对混合微液滴(14)中的目标链进行识别与捕获，DNA酶切活性被激活；若混合微液滴(14)中含有目标链，则荧光探针被切割，使得荧光基团和淬灭基团分离，在光源激发下发出荧光信号，通过光学信号检测器分析单个混合微液滴(14)的荧光信号获得阴性液滴和阳性液滴的比例，再计算核酸扩增体系中核酸分子的浓度或者拷贝数，从而获得检测结果。
  7. 根据权利要求5所述的一种基于CRISPR/Cas的数字化核酸扩增检测方法，其特征在于：所述的液滴融合区(8)为Y型或T型微通道，Y型或T型微通道的三端经各自的微通道(5)分别和核酸扩增区(6)、检测体系液滴生成区(7)和光学检测区(9)连通。
  8. 根据权利要求5所述的一种基于CRISPR/Cas的数字化核酸扩增检测方法，其特征在于：方法对于阴性液滴和阳性液滴比例的获得，采用以下两种方式之一：
    (A)混合微液滴(14)逐个检测：
    驱动混合微液滴(14)依次流过光学检测区(9)，在光源激发或者没有光源激发下，通过光学信号检测器检测光信号转化为电信号，通过对电信号的波形进行滤波处理、去除基线、阈值划分的处理得到阴性液滴和阳性液滴的比例p；
    (B)所有微液滴同时检测：所有混合微液滴(14)汇聚在光学检测区(9)并分散排布，在光源激发或者没有光源激发下，通过相机或者带有拍照功能的手机对光学检测区(9)进行拍照获得光学图像，通过感兴趣区域获取、滤波处理、阈值分割、计数的图像处理得到阴性液滴和阳性液滴的比例p；
    方法最后根据阴阳性液滴的比例p，按下述公式计算出每个混合微液滴(14)中的平均核酸分子数λ，从而获得待测样品中核酸分子的浓度或者拷贝数：
    λ＝-ln(1-p)。
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