WO2021082951A1 - Digital pcr method, chip, preparation method and circulation system - Google Patents

Abstract

Provided in the present invention are a digital PCR method, a chip, a preparation method and a circulation system, the method comprising: placing a PCR chip which has undergone sample introduction into a reaction chamber; evacuating air from the reaction chamber; performing pressurization of the reaction chamber; putting the pressurized reaction chamber in a reaction device and performing a PCR reaction; performing fluorescence signal analysis of the digital PCR chip.

Description

一种数字PCR方法、芯片、制备方法及循环***A digital PCR method, chip, preparation method and circulation system

本申请要求2019年10月28日递交的申请号为201911030850.5、发明名称为“高压液体浸入式数字PCR方法、数字PCR芯片及其制备方法”以及2019年10月28日递交的申请号为201911031703.X、发明名称为“循环式数字PCR方法、循环***、数字PCR芯片及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application requires that the application number submitted on October 28, 2019 is 201911030850.5, the title of the invention is "High-pressure liquid immersion digital PCR method, digital PCR chip and its preparation method", and the application number submitted on October 28, 2019 is 201911031703. X. The priority of the Chinese patent application titled "Circular Digital PCR Method, Circulation System, Digital PCR Chip and Preparation Method", the entire content of which is incorporated in this application by reference.

技术领域Technical field

本发明涉及核酸检测技术领域，特别涉及一种数字PCR方法、芯片、制备方法及循环***。The invention relates to the technical field of nucleic acid detection, in particular to a digital PCR method, a chip, a preparation method and a circulation system.

背景技术Background technique

聚合酶链式反应(PolymeraseChainReaction，PCR)是一种用于放大扩增特定的DNA片段的分子生物学技术，它可看作是生物体外的特殊DNA复制。现已广泛应用到基因检测、基因扩增、基因工程等分子生物学领域，并且在临床医学、法医学、亲子鉴定以及环境检测等方面发挥着不可替代的作用。然而PCR反应以指数级扩增，在几十分钟内即可扩增百万倍，难于通过PCR产物确定原始PCR模板的含量。为了精确定量分析核酸含量，发明了数字PCR(digitalPCR，dPCR)技术。Polymerase chain reaction (Polymerase Chain Reaction, PCR) is a molecular biology technique used to amplify specific DNA fragments, and it can be regarded as a special DNA replication in vitro. It has been widely used in molecular biology fields such as genetic testing, gene amplification, genetic engineering, and it plays an irreplaceable role in clinical medicine, forensic medicine, paternity testing, and environmental testing. However, the PCR reaction is amplified exponentially, which can be amplified millions of times within tens of minutes, and it is difficult to determine the content of the original PCR template from the PCR product. In order to accurately and quantitatively analyze the nucleic acid content, digital PCR (digitalPCR, dPCR) technology was invented.

数字PCR(dPCR)的基本原理是将PCR样品等量分配到数量众多不同的微反应单元，每个微反应单元包含不同数目的模板分子，在每个反应单元中完成独立的PCR扩增。扩增结束后，包含模板分子的微反应单元会有荧光信号记为阳性，不包含模板分子的微反应单元则 不会有荧光信号，因此记为阴性。不同微反应单元中的模板数量符合泊松分布，因此可以通过计数阳性以及阴性微反应单元的数量，根据公式准确得知初始样品中PCR模板的浓度。微反应单元的反应单元数量、体积精度、均一度以及微反应单元的反应质量决定了整个dPCR的质量。现有dPCR***主要包含阀门式dPCR芯片、油包水微滴式dPCR芯片和开放式阵列dPCR芯片。这些芯片成本高，操作复杂。其中有的芯片还存在热反应慢、体积准确度、均一度差等问题。The basic principle of digital PCR (dPCR) is to distribute PCR samples equally to a large number of different micro-reaction units. Each micro-reaction unit contains a different number of template molecules, and independent PCR amplification is completed in each reaction unit. After the amplification is completed, the micro-reaction unit containing the template molecule will be marked as positive, and the micro-reaction unit that does not contain the template molecule will not have the fluorescent signal, so it is recorded as negative. The number of templates in different microreaction units conforms to the Poisson distribution. Therefore, by counting the number of positive and negative microreaction units, the concentration of PCR template in the initial sample can be accurately known according to the formula. The number of reaction units, volume accuracy, uniformity of the micro-reaction unit, and the reaction quality of the micro-reaction unit determine the quality of the entire dPCR. The existing dPCR system mainly includes a valve type dPCR chip, a water-in-oil droplet type dPCR chip and an open array dPCR chip. These chips are expensive and complicated to operate. Some of the chips also have problems such as slow thermal response, poor volume accuracy, and poor uniformity.

聚二甲基硅氧烷(Polydimethylsiloxane，PDMS)具有透明、生物兼容性好、价值低、易于制作等特性，被广泛应用在dPCR上。PDMS是高分子聚合物，能够透过气体并且能存储一定量的空气，因此在PDMS芯片上容易实现液体微反应单元终端填充。然而，PDMS的气体存储性和透过性带来了一个严重问题，即PDMS芯片受热时容易出现气泡。另外，随着温度增加，会造成PCR过程中各微反应单元的相互污染，还会造成PCR溶液里水分的挥发损失，进而影响PCR反应。并且，液体微反应单元腔体越小，这一现象越明显。目前，采用玻璃、聚对二甲苯基(Parylene)、含油PDMS前聚物等方法来阻止气泡形成以及减少水分蒸发。但是，采用上述方法会导致dPCR芯片的操作难度大，成本高，反应时间长。Polydimethylsiloxane (PDMS) has the characteristics of transparency, good biocompatibility, low value, and easy production, and is widely used in dPCR. PDMS is a high molecular polymer, which can permeate gas and store a certain amount of air, so it is easy to realize the terminal filling of the liquid micro-reaction unit on the PDMS chip. However, the gas storage and permeability of PDMS poses a serious problem, that is, bubbles are prone to appear when the PDMS chip is heated. In addition, as the temperature increases, it will cause mutual contamination of the micro-reaction units during the PCR process, and will also cause the volatilization loss of water in the PCR solution, thereby affecting the PCR reaction. And, the smaller the cavity of the liquid micro-reaction unit, the more obvious this phenomenon. At present, methods such as glass, parylene, and oil-containing PDMS prepolymer are used to prevent bubble formation and reduce water evaporation. However, the use of the above method will cause the dPCR chip to be difficult to operate, high in cost, and long in reaction time.

发明内容Summary of the invention

本发明要解决的技术问题是的问题。The technical problem to be solved by the present invention is the problem.

为解决上述技术问题，第一方面，本申请实施例公开了一种数字PCR方法，所述方法包括：In order to solve the above technical problems, in the first aspect, an embodiment of the present application discloses a digital PCR method, which includes:

S1、将经过进样处理的数字PCR芯片放置于反应室中；S1. Place the sample-injected digital PCR chip in the reaction chamber;

S2、排空所述反应室内的空气；S2, evacuating the air in the reaction chamber;

S3、对所述反应室进行加压处理；S3. Pressurize the reaction chamber;

S4、将加压处理后的所述反应室进行PCR反应；S4. Perform a PCR reaction on the reaction chamber after the pressure treatment;

S5、对所述数字PCR芯片进行荧光信号分析。S5. Perform fluorescence signal analysis on the digital PCR chip.

进一步的，步骤S1中所述进样处理包括以下步骤：Further, the sample injection processing in step S1 includes the following steps:

S11.将进样器与数字PCR芯片组合好，使得所述数字PCR芯片的反应层的进样口、出样口分别与所述进样器的进样池、出样口对准并紧密贴合；S11. Combine the sampler with the digital PCR chip so that the sample inlet and sample outlet of the reaction layer of the digital PCR chip are aligned with and closely adhere to the sample inlet and sample inlet of the sampler respectively Together

S12.将PCR反应液加入到所述进样器的进样池中，在所述进样器的出样口处向外抽气，使所述PCR反应液经过所述数字PCR芯片的反应层的进样口流入所述数字PCR芯片的反应层的主流道，直至部分所述PCR反应液经过所述数字PCR芯片的反应层的出样口从所述进样器的出样口流出；S12. Add the PCR reaction solution to the sampling pool of the sampler, and pump air outward at the sample outlet of the sampler, so that the PCR reaction solution passes through the reaction layer of the digital PCR chip The injection port of the digital PCR chip flows into the main flow channel of the reaction layer of the digital PCR chip, until a part of the PCR reaction solution flows out of the sampling port of the sample injector through the sample outlet of the reaction layer of the digital PCR chip;

S13.自所述进样器的负压腔的抽气口处向外抽气，直至所述PCR反应液充满与所述主流道相连通的各分支流道以及与所述各分支流道相连通的各PCR微反应单元；S13. Pump air from the suction port of the negative pressure chamber of the sampler until the PCR reaction solution is filled with the branch flow channels connected to the main flow channel and communicates with the branch flow channels Each PCR micro-reaction unit;

S14.吸掉所述进样器的进样池处的多余PCR反应液，并滴加隔离油；S14. Aspirate the excess PCR reaction solution at the sample injection pool of the sampler, and drop isolation oil;

S15.在所述进样器的出样口处抽气，使所述隔离油填满所述反应层的主流道，完成对各PCR微反应单元的隔离。S15. Pumping air at the sample outlet of the sample injector, so that the isolation oil fills the main flow channel of the reaction layer, and completes the isolation of each PCR micro-reaction unit.

进一步的，步骤S2中，通过注入中性溶液对所述反应室进行排空气。Further, in step S2, the reaction chamber is evacuated by injecting a neutral solution.

进一步的，步骤S3中，加压处理所述反应室的压强为210-500KPa。Further, in step S3, the pressure of the reaction chamber is 210-500 KPa.

进一步的，加压处理后的所述反应室接入反应设备进行PCR反应，所述反应设备为循环***或PCR仪。Further, the pressure-treated reaction chamber is connected to a reaction device for PCR reaction, and the reaction device is a circulation system or a PCR machine.

进一步的，所述反应室为高压反应室，所述将经过进样处理的数字PCR芯片放置于反应室中，包括：Further, the reaction chamber is a high-pressure reaction chamber, and placing the sample-injected digital PCR chip in the reaction chamber includes:

将经过进样处理的数字PCR芯片浸入高压反应室的液体中。The digital PCR chip that has undergone sample injection processing is immersed in the liquid in the high-pressure reaction chamber.

第二方面，本申请实施例公开了一种数字PCR芯片，所述数字PCR芯片用于执行如权利要求1-8中任一项所述的数字PCR方法；In the second aspect, an embodiment of the present application discloses a digital PCR chip, which is used to execute the digital PCR method according to any one of claims 1-8;

所述数字PCR芯片包括依次设置的基底层和反应层，其中，所述反应层具有主流道、与所述主流道)相连通的各分支流道以及与所述各分支流道相连通的各PCR微反应单元；所述反应层还具有进样口和出样口，所述反应层的进样口与进样器的进样池相连通，所述反 应层的出样口与进样器的出样口相连通。The digital PCR chip includes a base layer and a reaction layer arranged in sequence, wherein the reaction layer has a main flow channel, branch flow channels communicating with the main flow channel, and branch flow channels communicating with the branch flow channels. PCR micro-reaction unit; the reaction layer also has a sample inlet and a sample outlet, the sample inlet of the reaction layer is connected to the sample inlet of the sampler, and the sample outlet of the reaction layer is connected to the sampler The sample outlet is connected.

进一步的，所述反应层采用聚二甲基硅氧烷制成。Further, the reaction layer is made of polydimethylsiloxane.

进一步的，述基底层采用支撑材料制成，用于对所述反应层进行密封及支撑。Further, the base layer is made of a supporting material for sealing and supporting the reaction layer.

进一步的，所述基底层与所述反应层之间还设有空白层，所述空白层采用聚二甲基硅氧烷制成。Further, a blank layer is further provided between the base layer and the reaction layer, and the blank layer is made of polydimethylsiloxane.

第三方面，本申请实施例公开了一种数字PCR芯片制备方法，包括以下步骤：In the third aspect, the embodiment of the application discloses a method for preparing a digital PCR chip, which includes the following steps:

S10、清洗硅片，在所述硅片上制作反应层模具；S10. Cleaning the silicon wafer, and fabricating a reaction layer mold on the silicon wafer;

S20、将聚二甲基硅氧烷以及固化剂混合均匀并脱气后浇注到所述反应层模具上，热固化形成具有主流道、各分支流道以及各PCR微反应单元的聚二甲基硅氧烷膜片；将所述聚二甲基硅氧烷膜片从所述反应层模具上取下并打孔形成进样口和出样口，制成具有进样口、出样口、主流道、各分支流道以及各PCR微反应单元的反应层；S20. Mix the polydimethylsiloxane and the curing agent uniformly and degas and pour it on the reaction layer mold, and heat curing to form a polydimethylsiloxane having a main flow channel, each branch flow channel, and each PCR micro-reaction unit Silicone membrane; the polydimethylsiloxane membrane is removed from the reaction layer mold and punched to form a sample inlet and a sample outlet, so as to have a sample inlet, a sample outlet, Main flow channel, each branch flow channel and the reaction layer of each PCR micro-reaction unit;

S30、将步骤S20获得的聚二甲基硅氧烷反应层结构与基底层经等离子体激活处理后键合在一起，即制成数字PCR芯片。S30, bonding the polydimethylsiloxane reaction layer structure obtained in step S20 and the base layer after plasma activation treatment to form a digital PCR chip.

进一步的，所述将步骤S20获得的聚二甲基硅氧烷反应层结构与基底层经等离子体激活处理后键合在一起，即制成数字PCR芯片，包括：Further, said bonding the polydimethylsiloxane reaction layer structure obtained in step S20 and the base layer after plasma activation treatment to form a digital PCR chip, including:

S301、将聚二甲基硅氧烷以及固化剂混合均匀旋涂在硅片上，固化形成空白层；S301, mixing polydimethylsiloxane and curing agent uniformly and spin-coating on the silicon wafer, and curing to form a blank layer;

S302、将步骤S20获得的反应层与步骤S301获得的空白层一起经等离子体激活处理后键合在一起；S302, bonding the reaction layer obtained in step S20 and the blank layer obtained in step S301 together after plasma activation treatment;

S303、将步骤S302获得的两层聚二甲基硅氧烷键合结构与基底层经等离子体激活处理后键合在一起，即制成数字PCR芯片。S303. The two-layer polydimethylsiloxane bonding structure obtained in step S302 and the base layer are bonded together after plasma activation treatment to form a digital PCR chip.

第四方面，本申请实施例公开了一种循环***，用于执行如权利要求1-5中任一项所述的循环式数字PCR方法，In a fourth aspect, the embodiments of the present application disclose a circulatory system for executing the cyclic digital PCR method according to any one of claims 1-5,

所述循环***包括反应室、液体循环***、温度调节***、压力调节***和控制器，所述反应室与所述液体循环***连接，用于放置 数字PCR芯片；所述控制器分别与所述液体循环***、所述温度调节***和所述压力调节***连接，用于自动控制所述液体循环***为所述反应室输送液体来调节所述反应室的温度，自动控制所述温度调节***调节所述液体循环***中的液体的温度，自动控制所述压力调节***调节所述循环***的压力。The circulation system includes a reaction chamber, a liquid circulation system, a temperature adjustment system, a pressure adjustment system, and a controller. The reaction chamber is connected to the liquid circulation system for placing a digital PCR chip; the controller is connected to the The liquid circulation system, the temperature adjustment system, and the pressure adjustment system are connected to automatically control the liquid circulation system to deliver liquid to the reaction chamber to adjust the temperature of the reaction chamber, and to automatically control the temperature adjustment system to adjust The temperature of the liquid in the liquid circulation system automatically controls the pressure adjustment system to adjust the pressure of the circulation system.

进一步的，所述液体循环***包括恒温储液箱、循环泵、阀门和管路，所述恒温储液箱通过所述管路与所述反应室连接，用于为所述反应室输送恒温的液体来调节所述反应室的温度；所述循环泵和所述阀门均设置在所述管路上，用于驱动恒温的液体在所述管路中循环流动。Further, the liquid circulation system includes a constant temperature liquid storage tank, a circulation pump, a valve, and a pipeline, and the constant temperature liquid storage tank is connected to the reaction chamber through the pipeline, and is used to deliver a constant temperature to the reaction chamber. Liquid is used to adjust the temperature of the reaction chamber; the circulation pump and the valve are both arranged on the pipeline, and are used to drive the constant temperature liquid to circulate in the pipeline.

进一步的，所述温度调节***包括第一温度传感器、第二温度传感器、加热器和冷却器，Further, the temperature adjustment system includes a first temperature sensor, a second temperature sensor, a heater and a cooler,

所述第一温度传感器设置在所述反应室内，用于探测所述反应室内的温度；所述第二温度传感器设置在所述恒温储液箱内，用于探测所述恒温储液箱内的温度；所述加热器和所述冷却器均设置在所述恒温储液箱内，用于对所述恒温储液箱内的液体进行加热或降温。The first temperature sensor is arranged in the reaction chamber and is used to detect the temperature in the reaction chamber; the second temperature sensor is arranged in the constant temperature liquid storage tank and is used to detect the temperature in the constant temperature liquid storage tank. Temperature; the heater and the cooler are both set in the constant temperature liquid storage tank for heating or cooling the liquid in the constant temperature liquid storage tank.

进一步的，所述压力调节***包括压力传感器、增压泵和泄压阀，所述压力传感器设置在所述循环***内，用于探测所述循环***的压力；所述增压泵和所述泄压阀设置在所述循环***内，用于增加或减少所述循环***的压力。Further, the pressure regulating system includes a pressure sensor, a booster pump, and a pressure relief valve. The pressure sensor is arranged in the circulation system for detecting the pressure of the circulation system; the booster pump and the pressure relief valve The pressure relief valve is arranged in the circulation system and is used to increase or decrease the pressure of the circulation system.

进一步的，所述控制器分别与所述循环泵和所述阀门连接，用于根据PCR程序自动控制所述循环泵和所述阀门的工作来为所述反应室输送恒温的液体；所述控制器分别与所述第一温度传感器、所述第二温度传感器、所述加热器和所述冷却器连接，用于根据所述第一温度传感器和所述第二温度传感器探测的温度自动控制所述加热器和所述冷却器的工作对所述恒温储液箱内的液体进行加热或降温；所述控制器分别与所述压力传感器、所述增压泵和所述泄压阀连接，用于根据所述压力传感器探测的压力自动控制所述增压泵和所述泄压阀的工作增加或减少所述循环***的压力。Further, the controller is respectively connected with the circulating pump and the valve, and is used to automatically control the operation of the circulating pump and the valve according to the PCR program to deliver a constant temperature liquid to the reaction chamber; the control The device is respectively connected with the first temperature sensor, the second temperature sensor, the heater and the cooler, and is used to automatically control the plant based on the temperature detected by the first temperature sensor and the second temperature sensor. The heater and the cooler work to heat or cool the liquid in the constant temperature liquid storage tank; the controller is respectively connected with the pressure sensor, the booster pump and the pressure relief valve, The operation of the booster pump and the pressure relief valve is automatically controlled according to the pressure detected by the pressure sensor to increase or decrease the pressure of the circulation system.

进一步的，所述恒温储液箱设置有2-4个。Further, there are 2-4 thermostatic liquid storage tanks.

采用上述技术方案，本申请实施例所述的数字PCR方法、芯片、制备方法及循环***具有如下有益效果：Using the above technical solutions, the digital PCR method, chip, preparation method, and circulation system described in the embodiments of the present application have the following beneficial effects:

1、芯片进样不依赖泵、阀等复杂设备，也不需要使用高粘度热聚合分离油，进样完成后芯片不需要密封，操作简单；1. Chip sampling does not rely on complicated equipment such as pumps and valves, nor does it need to use high-viscosity thermal polymerization separation oil. After sampling is completed, the chip does not need to be sealed, and the operation is simple;

2、利用进样器辅助快速进样，并利用负压进样的方式减少芯片内的气体残留，进样迅速；2. Use the sampler to assist rapid sample injection, and use the negative pressure injection method to reduce the gas residue in the chip, and the sample is quickly injected;

3、芯片在水环境里完成反应，PCR微反应单元内水分损失可以忽略不计，微反应单元体积可以做的更小，单位面积的芯片可以制作更多的微反应单元，芯片整体通量较高；3. The chip completes the reaction in an aqueous environment. The moisture loss in the PCR micro-reaction unit is negligible, the volume of the micro-reaction unit can be made smaller, and the chip per unit area can produce more micro-reaction units, and the overall throughput of the chip is higher. ；

4、芯片各微反应单元体积准确，阵列均匀，易于成像分析；4. The volume of each micro-reaction unit of the chip is accurate, the array is uniform, and it is easy for imaging analysis;

5、高压反应室易与现有的PCR仪器兼容，也易于集成在PCR仪的金属温控模块上；5. The high-pressure reaction chamber is easily compatible with the existing PCR instrument, and is also easy to integrate into the metal temperature control module of the PCR instrument;

6、与反应室相比，恒温储液箱的体积更大，热交换迅速，能够快速加热或者冷却芯片；6. Compared with the reaction chamber, the constant temperature liquid storage tank has a larger volume, rapid heat exchange, and can quickly heat or cool the chip;

7、恒温储液箱的温度可以独立控制，温控效果极好，没有金属浴***不可避免的温度过冲现象；7. The temperature of the constant temperature storage tank can be independently controlled, and the temperature control effect is excellent, without the inevitable temperature overshoot phenomenon of the metal bath system;

8、反应室易于与荧光检测***集成，能够实现芯片的在线检测，缩短检测时间，适合快速临床检测；8. The reaction chamber is easy to integrate with the fluorescence detection system, which can realize the online detection of the chip, shorten the detection time, and is suitable for rapid clinical detection;

9、芯片厚度小，导热快，反应迅速；9. The chip thickness is small, the heat conduction is fast, and the response is fast;

10、芯片结构简单，容易制作，成本低，自动化程度高；10. The chip structure is simple, easy to manufacture, low cost, and high degree of automation;

11、减少水分蒸发，并可兼容其它dPCR芯片。11. Reduce water evaporation and be compatible with other dPCR chips.

附图说明Description of the drawings

为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly describe the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings that need to be used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained from these drawings without creative work.

图1为PDMS芯片受热时气泡形成机制以及水分蒸发原理图；Figure 1 shows the bubble formation mechanism and the principle diagram of water evaporation when the PDMS chip is heated;

图2为本发明实施例中循环***的结构示意图；Figure 2 is a schematic diagram of the structure of the circulatory system in the embodiment of the present invention;

图3为本发明实施例中循环***的温度曲线图；Fig. 3 is a temperature curve diagram of the circulation system in the embodiment of the present invention;

图4为图3中A区域的放大示意图；Fig. 4 is an enlarged schematic diagram of area A in Fig. 3;

图5为本发明实施例中数字PCR芯片与进样器配合的剖面结构示意图；5 is a schematic cross-sectional structure diagram of the digital PCR chip and the sampler in the embodiment of the present invention;

图6为本发明实施例中进样器的结构示意图；Figure 6 is a schematic diagram of the structure of the sample injector in the embodiment of the present invention;

图7为本发明实施例中数字PCR芯片的反应层的结构示意图；7 is a schematic diagram of the structure of the reaction layer of the digital PCR chip in the embodiment of the present invention;

图8为图7中B区域的放大示意图；Fig. 8 is an enlarged schematic diagram of area B in Fig. 7;

图9为本发明实施例中PCR反应液进入数字PCR芯片的反应层的主流道的示意图；9 is a schematic diagram of the main flow channel of the PCR reaction solution entering the reaction layer of the digital PCR chip in the embodiment of the present invention;

图10为本发明实施例中PCR反应液进入数字PCR芯片的反应层的各分支流道及各PCR微反应单元的示意图；10 is a schematic diagram of each branch flow channel of the PCR reaction solution entering the reaction layer of the digital PCR chip and each PCR micro-reaction unit in an embodiment of the present invention;

图11为本发明实施例中隔离油对各PCR微反应单元进行隔离的示意图；11 is a schematic diagram of the isolation oil for isolating each PCR micro-reaction unit in an embodiment of the present invention;

图12为本发明实施例中数字PCR芯片与进样器配合的剖面结构示意图；12 is a schematic diagram of a cross-sectional structure of the digital PCR chip and the sampler in the embodiment of the present invention;

以下对附图作补充说明：The following is a supplementary explanation to the attached drawings:

1-PDMS芯片，2-PCR溶液，3-气泡，4-微液滴，6-恒温储液箱，7-循环泵，8-阀门，9-管路，10-基底层，101-空白层，11-反应层，111-反应层的进样口，112-反应层的出样口，113-主流道，114-分支流道，115-微反应单元，12-进样池，13-进样器的出样口，14-负压腔，15-抽气口，16-数字PCR芯片。1-PDMS chip, 2-PCR solution, 3-bubbles, 4-micro-droplets, 6-constant temperature storage tank, 7-circulation pump, 8-valve, 9-pipeline, 10-base layer, 101-blank layer ,11-reaction layer, 111-inlet of reaction layer, 112-outlet of reaction layer, 113-main flow channel, 114- branch flow channel, 115-micro reaction unit, 12-injection cell, 13-inlet The sample outlet of the sampler, 14-negative pressure cavity, 15-exhaust port, 16-digital PCR chip.

具体实施方式Detailed ways

下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例，都属于本申请保护的范围。The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this application.

此处所称的“一个实施例”或“实施例”是指可包含于本申请至少一个实现方式中的特定特征、结构或特性。在本申请的描述中，需要理解的是，术语“上”、“下”、“顶”、“底”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含的包括一个或者更多个该特征。而且，术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本申请的实施例能够以除了在这里图示或描述的那些以外的顺序实施。The "one embodiment" or "embodiment" referred to herein refers to a specific feature, structure, or characteristic that can be included in at least one implementation of the present application. In the description of this application, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "top", "bottom", etc. is based on the orientation or positional relationship shown in the drawings, and is only for It is convenient to describe the application and simplify the description, instead of indicating or implying that the device or element referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore cannot be understood as a limitation of the application. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of these features. Moreover, the terms "first", "second", etc. are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in a sequence other than those illustrated or described herein.

PDMS芯片1由于气体存储性和透过性带来了一个严重问题，即PDMS芯片1受热中容易出现气泡3。本发明的发明人经过长期研究发现了PDMS芯片1受热时的气泡3形成机制以及水分蒸发原理。图1为PDMS芯片1受热时气泡3形成机制以及水分蒸发原理图。如图1所示，从图1中可以看出，当PDMS芯片1暴露于空气中时，PCR溶液2里的水分蒸发程度受温度影响显著。对于PDMS芯片1，升温过程中，水蒸发形成水蒸气进入PDMS芯片1的PDMS层的微孔内，再加上空气本身热膨胀作用，引起微孔内气体体积和压力的明显增加，大部分气体会溢出PDMS层外。然而，由于微孔的流阻大，部分气体来不及透过PDMS层释放到空气中，气体就会克服液体压力在微反应单元115内形成小的气泡3，气泡3周围的液体中的水分子更容易蒸发进入气泡3，使得气泡3体积迅速增大。气泡3扩展到相邻的PCR微反应单元115，引起相邻反应单元内的PCR溶液2气化，这一连锁反应使得气化在整条微通道的微反应单元115内发生，进而扩展到整个芯片。降温过程中，微孔内的水蒸气凝结形成微液滴4，气体分子减少，形成负压，PDMS层外部空气被吸入到微孔内， 在PCR反应的每个温度循环中，PDMS层就像“泵”一样不停的吸入空气形成气泡3，排出水分，造成PCR溶液2失水，各微反应单元115交叉污染，进而造成PCR反应失败。The PDMS chip 1 has a serious problem due to the gas storage and permeability, that is, bubbles 3 are prone to appear when the PDMS chip 1 is heated. The inventor of the present invention has discovered through long-term research that the bubble 3 formation mechanism and the principle of water evaporation when the PDMS chip 1 is heated. Fig. 1 is a diagram showing the formation mechanism of bubbles 3 and the principle of water evaporation when the PDMS chip 1 is heated. As shown in Figure 1, it can be seen from Figure 1 that when the PDMS chip 1 is exposed to the air, the degree of evaporation of water in the PCR solution 2 is significantly affected by the temperature. For PDMS chip 1, during the heating process, water evaporates to form water vapor into the micropores of the PDMS layer of PDMS chip 1. Coupled with the thermal expansion of the air itself, the gas volume and pressure in the micropores increase significantly. Most of the gas will Overflow outside the PDMS layer. However, due to the large flow resistance of the micropores, part of the gas is too late to pass through the PDMS layer to be released into the air. The gas will overcome the liquid pressure to form small bubbles 3 in the micro-reaction unit 115, and the water molecules in the liquid around the bubbles 3 are even greater. It is easy to evaporate into the bubble 3, so that the volume of the bubble 3 increases rapidly. The bubble 3 expands to the adjacent PCR micro-reaction unit 115, causing the PCR solution 2 in the adjacent reaction unit to vaporize. This chain reaction causes vaporization to occur in the entire micro-channel micro-reaction unit 115, and then expands to the entire micro-channel. chip. During the cooling process, the water vapor in the micropores condenses to form microdroplets 4, the gas molecules are reduced, and a negative pressure is formed. The air outside the PDMS layer is sucked into the micropores. In each temperature cycle of the PCR reaction, the PDMS layer is like Like the “pump”, air is continuously sucked in to form bubbles 3, which discharges water, causing the PCR solution 2 to lose water, and the micro-reaction units 115 are cross-contaminated, thereby causing the PCR reaction to fail.

目前，通常采用玻璃、聚对二甲苯基Parylene、含油PDMS前聚物等方法来阻止气泡3形成以及减少水分蒸发。但是，采用上述方法会导致dPCR芯片的操作难度大，成本高，反应时间长。因此，需要提出一种新的数字PCR方案。At present, methods such as glass, parylene, and oil-containing PDMS precursor are usually used to prevent the formation of bubbles 3 and reduce water evaporation. However, the use of the above method will cause the dPCR chip to be difficult to operate, high in cost, and long in reaction time. Therefore, a new digital PCR scheme needs to be proposed.

实施例1：Example 1:

针对上述问题，本发明实施例提供了一种循环***，该循环***用于实施数字PCR方法。图2为本发明实施例中循环***的结构示意图，如图2所示，所述循环***包括反应室、液体循环***、温度调节***图中未示出、压力调节***图中未示出和控制器图中未示出。In view of the above-mentioned problems, an embodiment of the present invention provides a circulatory system, which is used to implement a digital PCR method. Figure 2 is a schematic structural diagram of the circulation system in the embodiment of the present invention. As shown in Figure 2, the circulation system includes a reaction chamber, a liquid circulation system, a temperature adjustment system not shown in the figure, and a pressure adjustment system not shown in the figure. The controller is not shown in the figure.

所述反应室与所述液体循环***连接，用于放置数字PCR芯片16，以便于进行PCR反应。The reaction chamber is connected to the liquid circulation system, and is used to place a digital PCR chip 16 to facilitate PCR reaction.

所述控制器与所述液体循环***连接，用于自动控制所述液体循环***为所述反应室输送液体来调节所述反应室的温度；所述控制器还与所述温度调节***连接，用于自动控制所述温度调节***调节所述液体循环***中的液体的温度；所述控制器还与所述压力调节***连接，用于自动控制所述压力调节***调节所述循环***的压力。The controller is connected to the liquid circulation system, and is used to automatically control the liquid circulation system to deliver liquid to the reaction chamber to adjust the temperature of the reaction chamber; the controller is also connected to the temperature adjustment system, Used to automatically control the temperature adjustment system to adjust the temperature of the liquid in the liquid circulation system; the controller is also connected to the pressure adjustment system, and is used to automatically control the pressure adjustment system to adjust the pressure of the circulation system .

在一个具体的实施例中，所述控制器可以为可编程逻辑控制器PLC。In a specific embodiment, the controller may be a programmable logic controller PLC.

具体的，如图2所示，所述液体循环***包括恒温储液箱6、循环泵7、阀门8和管路9。所述恒温储液箱6通过所述管路9与所述反应室连接，用于为所述反应室输送恒温的液体来调节所述反应室的温度。所述恒温储液箱6内的液体优选为水溶液，当然，也可以为其他溶液，本发明实施例对此不作限制。本发明实施例中，所述恒温储液箱6的数量可以根据实际需要进行设定。优选的，所述恒温储液箱6设置有2-4个。每个所述恒温储液箱6可以根据PCR反应需要设置相应的温度，例如94℃、72℃、55℃、0℃等。所述循环泵7和所述 阀门8均设置在所述管路9上，用于驱动所述恒温储液箱6中恒温的液体在所述管路9中循环流动。本发明实施例中，所述循环泵7和所述阀门8的数量也可以根据实际需要进行设定。Specifically, as shown in FIG. 2, the liquid circulation system includes a constant temperature liquid storage tank 6, a circulation pump 7, a valve 8 and a pipeline 9. The constant temperature liquid storage tank 6 is connected to the reaction chamber through the pipeline 9 and is used to deliver a constant temperature liquid to the reaction chamber to adjust the temperature of the reaction chamber. The liquid in the constant temperature liquid storage tank 6 is preferably an aqueous solution, of course, it can also be other solutions, which is not limited in the embodiment of the present invention. In the embodiment of the present invention, the number of the constant temperature liquid storage tank 6 can be set according to actual needs. Preferably, the constant temperature liquid storage tank 6 is provided with 2-4. Each of the constant temperature liquid storage tanks 6 can be set to a corresponding temperature according to the needs of the PCR reaction, for example, 94°C, 72°C, 55°C, 0°C, and so on. The circulation pump 7 and the valve 8 are both arranged on the pipeline 9 for driving the constant temperature liquid in the thermostatic storage tank 6 to circulate in the pipeline 9. In the embodiment of the present invention, the number of the circulating pump 7 and the valve 8 can also be set according to actual needs.

具体的，所述温度调节***包括第一温度传感器、第二温度传感器、加热器和冷却器。所述第一温度传感器设置在所述反应室内，用于探测所述反应室内的温度。所述第二温度传感器设置在所述恒温储液箱6内，用于探测所述恒温储液箱6内的温度。所述加热器和所述冷却器均设置在所述恒温储液箱6内，用于对所述恒温储液箱6内的液体进行加热或降温。Specifically, the temperature adjustment system includes a first temperature sensor, a second temperature sensor, a heater, and a cooler. The first temperature sensor is arranged in the reaction chamber and is used to detect the temperature in the reaction chamber. The second temperature sensor is arranged in the constant temperature liquid storage tank 6 for detecting the temperature in the constant temperature liquid storage tank 6. The heater and the cooler are both arranged in the constant temperature liquid storage tank 6 for heating or cooling the liquid in the constant temperature liquid storage tank 6.

具体的，所述压力调节***包括压力传感器、增压泵和泄压阀。所述压力传感器设置在所述循环***内，用于探测所述循环***的压力。所述增压泵和所述泄压阀设置在所述循环***内，用于根据需要增加或减少所述循环***的压力。Specifically, the pressure regulating system includes a pressure sensor, a booster pump and a pressure relief valve. The pressure sensor is arranged in the circulatory system for detecting the pressure of the circulatory system. The booster pump and the pressure relief valve are arranged in the circulation system for increasing or decreasing the pressure of the circulation system as required.

具体的，所述循环泵7和所述阀门8分别与所述控制器连接，所述控制器用于根据PCR程序自动控制所述循环泵7和所述阀门8开启或关闭来为所述反应室输送恒温的液体。所述第一温度传感器、所述第二温度传感器、所述加热器和所述冷却器分别与所述控制器连接，所述控制器用于根据所述第一温度传感器和所述第二温度传感器探测的温度自动控制所述加热器和所述冷却器开启或关闭对所述恒温储液箱6内的液体进行加热或降温。所述压力传感器、所述增压泵和所述泄压阀分别与所述控制器连接，所述控制器用于根据所述压力传感器探测的压力自动控制所述增压泵和所述泄压阀开启或关闭来增加或减少所述循环***的压力。Specifically, the circulation pump 7 and the valve 8 are respectively connected to the controller, and the controller is used to automatically control the circulation pump 7 and the valve 8 to open or close according to the PCR program to provide the reaction chamber Convey constant temperature liquid. The first temperature sensor, the second temperature sensor, the heater, and the cooler are respectively connected to the controller, and the controller is configured to respond according to the first temperature sensor and the second temperature sensor The detected temperature automatically controls the opening or closing of the heater and the cooler to heat or cool the liquid in the constant temperature liquid storage tank 6. The pressure sensor, the booster pump, and the pressure relief valve are respectively connected to the controller, and the controller is used to automatically control the booster pump and the pressure relief valve according to the pressure detected by the pressure sensor Turn on or off to increase or decrease the pressure of the circulatory system.

本发明实施例将所述数字PCR芯片16放置于上述循环***的反应室的液体中，与所述反应室相比，所述恒温储液箱6的体积更大，热交换迅速，能够快速加热或者冷却所述数字PCR芯片16。此外，本发明实施例两步法dPCR，变性温度设为94度，引物结合及延伸温度设为60度可以独立控制所述恒温储液箱6例如高温储液箱和低温储液箱的温度。图3为本发明实施例中循环***的温度曲线图；图4 为图3中A区域的放大示意图；从图3和图4中可以看出，采用本发明实施例的循环***实施数字PCR方法，由于水比热大，且与芯片无死角接触，热交换迅速，能够快速加热或者冷却芯片；此外，与反应室相比储水器体积很大，各储水器独分布，能够准确控制各储水器温度以及反应室内水流交换速度，温控效果极好，没有金属浴***不可避免的温度过冲现象。In the embodiment of the present invention, the digital PCR chip 16 is placed in the liquid in the reaction chamber of the above-mentioned circulation system. Compared with the reaction chamber, the constant temperature liquid storage tank 6 has a larger volume, rapid heat exchange, and can be heated quickly. Alternatively, the digital PCR chip 16 is cooled. In addition, in the two-step method dPCR in the embodiment of the present invention, the denaturation temperature is set to 94 degrees, and the primer binding and extension temperature is set to 60 degrees to independently control the temperature of the constant temperature liquid storage tank 6, such as the high temperature liquid storage tank and the low temperature liquid storage tank. Fig. 3 is a temperature curve diagram of the circulatory system in an embodiment of the present invention; Fig. 4 is an enlarged schematic diagram of area A in Fig. 3; as can be seen from Figs. 3 and 4, the circulatory system according to an embodiment of the present invention is used to implement the digital PCR method , Because the specific heat of water is large and there is no dead angle contact with the chip, the heat exchange is rapid, and the chip can be heated or cooled quickly; in addition, compared with the reaction chamber, the volume of the water reservoir is large, and each water reservoir is distributed independently, which can accurately control each The temperature of the water reservoir and the exchange rate of the water flow in the reaction chamber have excellent temperature control effects, and there is no temperature overshoot that is inevitable in the metal bath system.

相应的，基于上述循环***，本发明实施例提供了一种数字PCR方法，该数字PCR方法为循环式数字PCR方法，该方法包括以下步骤：Correspondingly, based on the above-mentioned circulatory system, an embodiment of the present invention provides a digital PCR method. The digital PCR method is a cyclic digital PCR method. The method includes the following steps:

S1、将经过进样处理的数字PCR芯片16放置于循环***的反应室中。S1. Place the digital PCR chip 16 that has undergone sample injection processing in the reaction chamber of the circulatory system.

本发明实施例中，如图5至图7所示，所述数字PCR芯片16在进行进样处理时，需要采用一个进样器辅助进样。所述进样器包括进样池12、出样口和负压腔14，所述负压腔14上设置有抽气口15。In the embodiment of the present invention, as shown in FIG. 5 to FIG. 7, when the digital PCR chip 16 performs sample injection processing, a sample injector is needed to assist the sample injection. The sample injector includes a sample inlet 12, a sample outlet, and a negative pressure cavity 14, and the negative pressure cavity 14 is provided with a suction port 15 thereon.

如图5和图7所示，所述数字PCR芯片16包括从下至上依次设置的基底层10和反应层11，其中，所述反应层11上设置有进样口和出样口，所述反应层的进样口111与所述进样器的进样池12相连通，所述反应层的出样口112与进样器的出样口13相连通。As shown in Figures 5 and 7, the digital PCR chip 16 includes a base layer 10 and a reaction layer 11 arranged in order from bottom to top, wherein the reaction layer 11 is provided with a sample inlet and a sample outlet. The sample inlet 111 of the reaction layer communicates with the sample inlet 12 of the sample injector, and the sample outlet 112 of the reaction layer communicates with the sample outlet 13 of the sample injector.

如图5、图7和图8所示，所述反应层11具有主流道113、与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。As shown in FIG. 5, FIG. 7 and FIG. 8, the reaction layer 11 has a main flow channel 113, each branch flow channel 114 communicating with the main flow channel 113, and each PCR branch communicating with the branch flow channel 114. Micro-reaction unit 115.

具体的，如图5至图8所示，所述进样处理可以包括以下步骤：Specifically, as shown in FIGS. 5 to 8, the sample injection processing may include the following steps:

S11、将进样器与数字PCR芯片16组合好，使得所述数字PCR芯片16的反应层的进样口111、出样口分别与所述进样器的进样池12、出样口对准并紧密贴合。S11. Combine the sampler with the digital PCR chip 16 so that the sample inlet 111 and the sample outlet of the reaction layer of the digital PCR chip 16 are respectively aligned with the sample inlet 12 and the sample outlet of the sampler. Accurate and closely fit.

S12、将PCR反应液加入到所述进样器的进样池12中，在所述进样器的出样口13处向外抽气，使所述PCR反应液经过所述数字PCR芯片16的反应层的进样口111流入所述数字PCR芯片16的反应层11的主流道113，直至部分所述PCR反应液经过所述数字PCR 芯片16的反应层的出样口112从所述进样器的出样口13流出。抽气可以使用注射器或其他可以抽气的设备如抽气泵等。S12. The PCR reaction solution is added to the sampling pool 12 of the sampler, and air is pumped out at the sample outlet 13 of the sampler, so that the PCR reaction solution passes through the digital PCR chip 16 The sample inlet 111 of the reaction layer flows into the main channel 113 of the reaction layer 11 of the digital PCR chip 16, until part of the PCR reaction solution passes through the sample outlet 112 of the reaction layer of the digital PCR chip 16 from the inlet The sample outlet 13 of the sampler flows out. You can use a syringe or other equipment that can pump air, such as an air pump.

其中，所述PCR反应液可以包括上游引物、下游引物、荧光标记DNA探针、DNA模板、dNTP混合液、DNA聚合酶等成分。所述PCR反应液中加入的各成分的种类、浓度以及序列顺序可以根据PCR反应需求进行设置。Wherein, the PCR reaction solution may include upstream primers, downstream primers, fluorescently labeled DNA probes, DNA templates, dNTP mixture, DNA polymerase and other components. The type, concentration, and sequence order of the components added to the PCR reaction solution can be set according to the requirements of the PCR reaction.

在一个具体的实施例中，所述步骤S12可以包括：使用移液器在进样器的进样池12滴加PCR反应液，并使用注射器经软管接头在进样器的出样口13向外抽气，引导所述PCR反应液经过所述数字PCR芯片16的反应层的进样口111流入所述数字PCR芯片16的反应层11的主流道113，如图9所示，直至部分所述PCR反应液经过所述数字PCR芯片16的反应层的出样口112从所述进样器的出样口13流出。In a specific embodiment, the step S12 may include: using a pipette to drop the PCR reaction solution into the sampling pool 12 of the sampler, and using a syringe to connect the sample outlet 13 of the sampler via a hose connector. Exhaust air outwards, and guide the PCR reaction solution to flow into the main flow channel 113 of the reaction layer 11 of the digital PCR chip 16 through the sample inlet 111 of the reaction layer of the digital PCR chip 16, as shown in FIG. The PCR reaction solution passes through the sample outlet 112 of the reaction layer of the digital PCR chip 16 and flows out from the sample outlet 13 of the sampler.

S13、自所述进样器的负压腔14的抽气口15处向外抽气，直至所述PCR反应液充满与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。S13. Pump air from the suction port 15 of the negative pressure cavity 14 of the sampler until the PCR reaction solution is filled with each branch flow channel 114 connected to the main flow channel 113 and is connected to each branch flow channel 114. Each PCR micro-reaction unit 115 communicated with the branch channel 114.

本发明实施例中，所述进样器的负压腔14完全覆盖所述反应层11的主流道113、与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。In the embodiment of the present invention, the negative pressure cavity 14 of the sampler completely covers the main flow channel 113 of the reaction layer 11, the branch flow channels 114 connected to the main flow channel 113, and the branch flow channels 114 connected to the main flow channel 113. 114 connected to each PCR micro-reaction unit 115.

本发明实施例中，如图10所示，先将所述PCR反应液滴加在所述进样器的进样池12，然后在所述进样器的出样口13向外抽气，并在所述进样器的负压腔14的抽气口15向外抽气，能够使经过所述数字PCR芯片16上的所述反应层11迅速地在各PCR微反应单元115产生负压，引导所述PCR反应液充满与所述主流道113相连通的各分支流道114，并进入各PCR微反应单元115。采用负压进样的方式可以减少所述数字PCR芯片16内的气体残留。In the embodiment of the present invention, as shown in FIG. 10, the PCR reaction liquid is first dropped into the sample injection pool 12 of the sampler, and then the gas is pumped out from the sample outlet 13 of the sampler, In addition, air is pumped out from the air extraction port 15 of the negative pressure chamber 14 of the sampler, so that the reaction layer 11 on the digital PCR chip 16 can quickly generate negative pressure in each PCR micro-reaction unit 115, The PCR reaction solution is guided to fill each branch flow channel 114 connected to the main channel 113 and enter each PCR micro-reaction unit 115. The negative pressure sampling method can reduce the residual gas in the digital PCR chip 16.

S14、吸掉所述进样器的进样池12处的多余PCR反应液，并滴加隔离油。S14. Aspirate the excess PCR reaction solution at the sample injection pool 12 of the sampler, and drop the isolation oil.

其中，所述隔离油可以为FC-40。Wherein, the isolation oil can be FC-40.

S15、如图11所示，在所述进样器的出样口13处抽气，使所述隔离油填满所述反应层11的主流道113，完成对各PCR微反应单元115的隔离。S15. As shown in FIG. 11, pump air at the sample outlet 13 of the sampler, so that the isolation oil fills the main flow channel 113 of the reaction layer 11, and completes the isolation of each PCR micro-reaction unit 115 .

S2、排空所述反应室内的空气；S2, evacuating the air in the reaction chamber;

对所述循环***进行加压处理。The circulation system is pressurized.

具体的，所述步骤S2中通过注入中性溶液排空所述反应室内的空气。例如，所述中性溶液可以为水溶液。Specifically, in the step S2, the air in the reaction chamber is evacuated by injecting a neutral solution. For example, the neutral solution may be an aqueous solution.

S3、对所述反应室进行加压处理；S3. Pressurize the reaction chamber;

在排空所述反应室内的空气之后，需要对所述循环***进行加压处理，增加所述循环***的压力。优选的，所述加压处理的压强为0-00KPa。After evacuating the air in the reaction chamber, the circulation system needs to be pressurized to increase the pressure of the circulation system. Preferably, the pressure of the pressure treatment is 0-00KPa.

S4、启动所述循环***进行PCR反应。S4. Start the circulatory system to perform PCR reaction.

具体的，所述步骤S4中的PCR反应的反应参数可以根据实际需要进行设定。Specifically, the reaction parameters of the PCR reaction in the step S4 can be set according to actual needs.

S5、对所述数字PCR芯片16进行荧光信号分析。S5. Perform fluorescence signal analysis on the digital PCR chip 16.

具体的，对PCR反应后的所述循环***进行减压，将所述循环***减至一定的压强，所述压强可以根据实际需要进行设定。然后打开所述反应室，取出所述反应室中的数字PCR芯片16，并对所述数字PCR芯片16进行荧光信号分析。在一些实施方式中，还可以在反应室中设置在线读取信号装置，因此，对于反应后的反应室不经过减压处理取出芯片，而是直接采用在线读取信号分析的方式。所述反应室与荧光检测***集成，能够实现芯片的在线检测。Specifically, the circulatory system after the PCR reaction is decompressed, and the circulatory system is reduced to a certain pressure, and the pressure can be set according to actual needs. Then the reaction chamber is opened, the digital PCR chip 16 in the reaction chamber is taken out, and the fluorescent signal analysis is performed on the digital PCR chip 16. In some embodiments, an online signal reading device may also be provided in the reaction chamber. Therefore, the reaction chamber after the reaction is not subjected to decompression treatment to take out the chip, but the method of online reading signal analysis is directly adopted. The reaction chamber is integrated with the fluorescence detection system, which can realize the online detection of the chip.

本发明实施例采用上述循环式数字PCR方法，使得进行PCR反应时所述数字PCR芯片16沉浸在所述循环***的反应室的液体中，由于所述数字PCR芯片16周围没有空气，在高温的情况下由于所述数字PCR芯片16外部高压的存在，PDMS微孔内的气体无法溢出PDMS，而在低温的情况下也不会有更多的空气进入PDMS中从而破坏里面的气体平衡，从而阻止了PDMS像“泵”一样的呼吸行为，进而阻止了所述各PCR微反应单元115内气泡3的产生，使所述PCR反 应液得以在稳定的环境下完成热循环反应。另外，水汽可以从所述数字PCR芯片16外表面进入PDMS内，从而减少所述PCR反应液的水损失。The embodiment of the present invention adopts the above-mentioned cyclic digital PCR method, so that the digital PCR chip 16 is immersed in the liquid in the reaction chamber of the circulatory system during the PCR reaction. In this case, due to the presence of high pressure outside the digital PCR chip 16, the gas in the PDMS micropores cannot escape the PDMS, and in the case of low temperature, no more air will enter the PDMS to disrupt the gas balance inside, thereby preventing The PDMS's breathing behavior like a "pump" prevents the generation of bubbles 3 in each PCR micro-reaction unit 115, so that the PCR reaction solution can complete the thermal cycle reaction in a stable environment. In addition, water vapor can enter the PDMS from the outer surface of the digital PCR chip 16, thereby reducing the water loss of the PCR reaction solution.

本发明实施例还提供了一种用于放置于上述循环***的反应室中的数字PCR芯片16。如图5所示，所述数字PCR芯片16包括基底层10和反应层11。The embodiment of the present invention also provides a digital PCR chip 16 for placing in the reaction chamber of the above-mentioned circulation system. As shown in FIG. 5, the digital PCR chip 16 includes a base layer 10 and a reaction layer 11.

优选的，所述反应层11采用聚二甲基硅氧烷PDMS制成。如图7和图8所示，所述反应层11具有主流道113、与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。Preferably, the reaction layer 11 is made of polydimethylsiloxane PDMS. As shown in FIGS. 7 and 8, the reaction layer 11 has a main flow channel 113, branch flow channels 114 communicating with the main flow channel 113, and PCR micro-reaction units communicating with the branch flow channels 114 115.

如图5所示，所述反应层11还具有进样口和出样口，所述反应层的进样口111与所述进样器的进样池12相连通，所述反应层的出样口112与进样器的出样口13相连通。As shown in Figure 5, the reaction layer 11 also has a sample inlet and a sample outlet. The sample inlet 111 of the reaction layer communicates with the sample pool 12 of the sampler, and the outlet of the reaction layer The sample port 112 is communicated with the sample outlet 13 of the sampler.

优选的，所述基底层10采用支撑材料如玻璃等材料制成。所述基底层10位于所述反应层11的下方，用于支撑所述反应层11。Preferably, the base layer 10 is made of a supporting material such as glass. The base layer 10 is located below the reaction layer 11 and is used to support the reaction layer 11.

相应的，本发明实施例还提供了一种制备如图5和图8所示的上述数字PCR芯片16的方法，该方法包括以下步骤：Correspondingly, the embodiment of the present invention also provides a method for preparing the above-mentioned digital PCR chip 16 as shown in FIG. 5 and FIG. 8. The method includes the following steps:

S10、清洗硅片，在所述硅片上制作反应层11模具。S10. The silicon wafer is cleaned, and a reaction layer 11 mold is fabricated on the silicon wafer.

在一个具体的实施例中，所述步骤可以包括：使用Phiranha溶液清洗硅片，再用去离子水冲洗干净，氮气吹干，并在170-180℃热板上烘烤20-30min；等离子体处理1-2min，然后旋涂SU8300510μm，进行光刻、显影，制作出反应层11的与主流道113相连通的各分支流道114；在160-170℃热板上烘烤20-30min，再等离子体处理1-2min，然后旋涂SU8302525μm，进行光刻、显影，制作出反应层11的主流道113以及与各分支流道114相连通的各PCR微反应单元115，从而制成反应层11模具。In a specific embodiment, the steps may include: cleaning the silicon wafer with Phiranha solution, rinsing with deionized water, drying with nitrogen, and baking on a hot plate at 170-180°C for 20-30 minutes; plasma Treat for 1-2min, then spin-coating SU8300510μm, perform photolithography and development, and produce the branch runners 114 of the reaction layer 11 that communicate with the main runner 113; bake on a hot plate at 160-170℃ for 20-30min, and then Plasma treatment for 1-2 min, then spin-coating SU8302525μm, photolithography and development, the main channel 113 of the reaction layer 11 and the PCR micro-reaction units 115 connected to each branch channel 114 are produced, thereby forming the reaction layer 11 Mold.

S20、将聚二甲基硅氧烷以及固化剂混合均匀并脱气后浇注到所述反应层11模具上，热固化形成具有主流道113、各分支流道114以及各PCR微反应单元115的聚二甲基硅氧烷膜片；将所述聚二甲 基硅氧烷膜片从所述反应层11模具上取下并打孔形成进样口和出样口，制成具有进样口、出样口、主流道113、各分支流道114以及各PCR微反应单元115的反应层11。S20. The polydimethylsiloxane and curing agent are mixed uniformly and degassed, and then poured onto the mold of the reaction layer 11, and thermally cured to form a main flow channel 113, each branch flow channel 114, and each PCR micro-reaction unit 115 Polydimethylsiloxane membrane; the polydimethylsiloxane membrane is removed from the mold of the reaction layer 11 and punched to form a sample inlet and a sample outlet, making it have a sample inlet , The sample outlet, the main flow channel 113, each branch flow channel 114, and the reaction layer 11 of each PCR micro-reaction unit 115.

在一个具体的实施例中，所述步骤可以包括：采用道康宁Sylgard184PDM，将前聚体和固化剂按照质量比5:1-10:1的比例混合均匀，并用真空脱气法除去混合物中的气泡3，然后将预聚物浇注在具有图形结构的反应层11模具上，在60-80℃热板上烘烤20-30min后形成具有主流道113、各分支流道114以及各PCR微反应单元115的聚二甲基硅氧烷膜片，将该聚二甲基硅氧烷膜片从反应层11模具上揭下后切割并打孔形成进样口和出样口，从而制成具有进样口、出样口、主流道113、各分支流道114以及各PCR微反应单元115的反应层11。In a specific embodiment, the step may include: using Dow Corning Sylgard 184PDM, mixing the precursor and curing agent uniformly in a mass ratio of 5:1-10:1, and removing bubbles in the mixture by vacuum degassing 3. Then pour the prepolymer on the reaction layer 11 mold with a pattern structure, and bake it on a hot plate at 60-80°C for 20-30 minutes to form a main flow channel 113, each branch flow channel 114 and each PCR micro-reaction unit 115 polydimethylsiloxane film, the polydimethylsiloxane film is removed from the mold of the reaction layer 11, and then cut and punched to form a sample inlet and a sample outlet, thereby making it have an inlet The sample port, the sample outlet, the main flow channel 113, each branch flow channel 114, and the reaction layer 11 of each PCR micro-reaction unit 115.

S30、将步骤S20获得的聚二甲基硅氧烷反应层11结构与基底层10经等离子体激活处理后键合在一起，即制成数字PCR芯片16。S30, the structure of the polydimethylsiloxane reaction layer 11 obtained in step S20 and the base layer 10 are bonded together after plasma activation treatment to form a digital PCR chip 16.

在一个具体的实施例中，所述基底层10可以为载玻片。In a specific embodiment, the base layer 10 may be a glass slide.

本发明实施例提供的技术方案具有如下优点：The technical solution provided by the embodiment of the present invention has the following advantages:

1、芯片进样不依赖泵、阀等复杂设备，也不需要使用高粘度热聚合分离油，进样完成后芯片不需要密封，操作简单；1. Chip sampling does not rely on complicated equipment such as pumps and valves, nor does it need to use high-viscosity thermal polymerization separation oil. After sampling is completed, the chip does not need to be sealed, and the operation is simple;

2、利用进样器辅助快速进样，并利用负压进样的方式减少芯片内的气体残留，进样迅速；2. Use the sampler to assist rapid sample injection, and use the negative pressure injection method to reduce the gas residue in the chip, and the sample is quickly injected;

3、与反应室相比，恒温储液箱6的体积更大，热交换迅速，能够快速加热或者冷却芯片；3. Compared with the reaction chamber, the constant temperature liquid storage tank 6 has a larger volume, rapid heat exchange, and can quickly heat or cool the chip;

4、恒温储液箱6的温度可以独立控制，温控效果极好，没有金属浴***不可避免的温度过冲现象；4. The temperature of the constant temperature liquid storage tank 6 can be independently controlled, and the temperature control effect is excellent, without the inevitable temperature overshoot phenomenon of the metal bath system;

5、反应室易于与荧光检测***集成，能够实现芯片的在线检测，缩短检测时间，适合快速临床检测；5. The reaction chamber is easy to integrate with the fluorescence detection system, which can realize the online detection of the chip, shorten the detection time, and is suitable for rapid clinical detection;

6、芯片在水环境里完成反应，PCR微反应单元115内水分损失可以忽略不计，微反应单元115体积可以做的更小，单位面积的芯片可以制作更多的微反应单元115，芯片整体通量较高；6. The chip completes the reaction in an aqueous environment. The moisture loss in the PCR micro-reaction unit 115 can be neglected, the volume of the micro-reaction unit 115 can be made smaller, and more micro-reaction units 115 can be made per unit area of the chip. Higher volume

7、芯片各微反应单元115体积准确，阵列均匀，易于成像分析；7. The volume of each micro-reaction unit 115 of the chip is accurate, the array is uniform, and it is easy for imaging analysis;

8、芯片厚度小，导热快，反应迅速；8. The chip thickness is small, the heat conduction is fast, and the response is fast;

9、芯片结构简单，容易制作，成本低，自动化程度高。9. The chip structure is simple, easy to manufacture, low cost, and high degree of automation.

实施例2：Example 2:

针对上述问题，本发明实施例提供了一种数字PCR方法，该数字PCR方法中的反应室采用高压反应室，数字PCR芯片16浸入高压反应室的液体中进行反应，因此，该数字PCR方法高压液体浸入式数字PCR方法，该方法包括以下步骤：In view of the above-mentioned problems, the embodiment of the present invention provides a digital PCR method. The reaction chamber in the digital PCR method adopts a high-pressure reaction chamber, and the digital PCR chip 16 is immersed in the liquid in the high-pressure reaction chamber for reaction. Therefore, the digital PCR method is high-pressure Liquid immersion digital PCR method, the method includes the following steps:

S1、将经过进样处理的数字PCR芯片16放置于反应室中；S1. Place the digital PCR chip 16 that has undergone sample injection processing in the reaction chamber;

本发明实施例中，反应室采用高压反应室，数字PCR芯片16浸入高压反应室的液体中进行反应。具体的，将经过进样处理的数字PCR芯片16浸入高压反应室的液体中。In the embodiment of the present invention, the reaction chamber adopts a high-pressure reaction chamber, and the digital PCR chip 16 is immersed in the liquid in the high-pressure reaction chamber to perform the reaction. Specifically, the digital PCR chip 16 that has undergone sample injection processing is immersed in the liquid in the high-pressure reaction chamber.

如图6和图12所示，所述数字PCR芯片16在进行进样处理时，需要采用一个进样器辅助进样。所述进样器包括进样池12、出样口和负压腔14，所述负压腔14上设置有抽气口15。As shown in FIG. 6 and FIG. 12, when the digital PCR chip 16 performs sample injection processing, a sample injector needs to be used to assist the sample injection. The sample injector includes a sample inlet 12, a sample outlet, and a negative pressure cavity 14, and the negative pressure cavity 14 is provided with a suction port 15 thereon.

如图12所示，所述数字PCR芯片16包括从下至上依次设置的基底层10、空白层101和反应层11，其中，所述反应层11上设置有进样口和出样口，所述反应层的进样口111与所述进样器的进样池12相连通，所述反应层的出样口112与进样器的出样口13相连通。As shown in FIG. 12, the digital PCR chip 16 includes a base layer 10, a blank layer 101, and a reaction layer 11 sequentially arranged from bottom to top, wherein the reaction layer 11 is provided with a sample inlet and a sample outlet, so The sample inlet 111 of the reaction layer communicates with the sample inlet 12 of the sample injector, and the sample outlet 112 of the reaction layer communicates with the sample outlet 13 of the sample injector.

参考图8所示，所述反应层11具有主流道113、与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。Referring to FIG. 8, the reaction layer 11 has a main flow channel 113, branch flow channels 114 communicating with the main flow channel 113, and PCR micro-reaction units 115 communicating with the branch flow channels 114.

具体的，所述进样处理可以包括以下步骤：Specifically, the sample injection processing may include the following steps:

S11、将进样器与数字PCR芯片16组合好，使得所述数字PCR芯片16的反应层的进样口1117、出样口7分别与所述进样器的进样池128、出样口9对准并紧密贴合。S11. Combine the sampler with the digital PCR chip 16 so that the sample inlet 1117 and the sample outlet 7 of the reaction layer of the digital PCR chip 16 are respectively connected to the sample inlet 128 and the sample outlet of the sampler. 9 Align and fit tightly.

S12、将PCR反应液加入到所述进样器的进样池12中，在所述进样器的出样口13处向外抽气，使所述PCR反应液经过所述数字PCR芯片16的反应层的进样口111流入所述数字PCR芯片16的反 应层11的主流道113，直至部分所述PCR反应液经过所述数字PCR芯片16的反应层的出样口112从所述进样器的出样口13流出。抽气可以使用注射器或其他可以抽气的设备如抽气泵等。S12. The PCR reaction solution is added to the sampling pool 12 of the sampler, and air is pumped out at the sample outlet 13 of the sampler, so that the PCR reaction solution passes through the digital PCR chip 16 The sample inlet 111 of the reaction layer flows into the main channel 113 of the reaction layer 11 of the digital PCR chip 16, until part of the PCR reaction solution passes through the sample outlet 112 of the reaction layer of the digital PCR chip 16 from the inlet The sample outlet 13 of the sampler flows out. You can use a syringe or other equipment that can pump air, such as an air pump.

其中，所述PCR反应液可以包括上游引物、下游引物、荧光标记DNA探针、DNA模板、dNTP混合液、DNA聚合酶等成分。所述PCR反应液中加入的各成分的种类、浓度以及序列顺序可以根据PCR反应需求进行设置。Wherein, the PCR reaction solution may include upstream primers, downstream primers, fluorescently labeled DNA probes, DNA templates, dNTP mixture, DNA polymerase and other components. The type, concentration, and sequence order of the components added to the PCR reaction solution can be set according to the requirements of the PCR reaction.

在一个具体的实施例中，所述步骤S11可以包括：使用移液器在进样器的进样池12滴加PCR反应液，并使用注射器经软管接头在进样器的出样口13向外抽气，引导所述PCR反应液经过所述数字PCR芯片16的反应层的进样口111流入所述数字PCR芯片16的反应层11的主流道113，参考图9所示，直至部分所述PCR反应液经过所述数字PCR芯片16的反应层的出样口112从所述进样器的出样口13流出。In a specific embodiment, the step S11 may include: using a pipette to drop the PCR reaction solution into the sampling pool 12 of the sampler, and using a syringe to connect to the sample outlet 13 of the sampler via a hose connector. Exhaust air outward, and guide the PCR reaction solution to flow into the main flow channel 113 of the reaction layer 11 of the digital PCR chip 16 through the injection port 111 of the reaction layer of the digital PCR chip 16, as shown in FIG. The PCR reaction solution passes through the sample outlet 112 of the reaction layer of the digital PCR chip 16 and flows out from the sample outlet 13 of the sampler.

S13、自所述进样器的负压腔14的抽气口15处向外抽气，直至所述PCR反应液充满与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。S13. Pump air from the suction port 15 of the negative pressure cavity 14 of the sampler until the PCR reaction solution is filled with each branch flow channel 114 connected to the main flow channel 113 and is connected to each branch flow channel 114. Each PCR micro-reaction unit 115 communicated with the branch channel 114.

本发明实施例中，所述进样器的负压腔14完全覆盖所述反应层11的主流道113、与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。In the embodiment of the present invention, the negative pressure cavity 14 of the sampler completely covers the main flow channel 113 of the reaction layer 11, the branch flow channels 114 connected to the main flow channel 113, and the branch flow channels 114 connected to the main flow channel 113. 114 connected to each PCR micro-reaction unit 115.

本发明实施例中，参考图10所示，先将所述PCR反应液滴加在所述进样器的进样池12，然后在所述进样器的出样口13向外抽气，并在所述进样器的负压腔14的抽气口15向外抽气，能够使经过所述数字PCR芯片16上的所述反应层11迅速地在各PCR微反应单元115产生负压，引导所述PCR反应液充满与所述主流道113相连通的各分支流道114，并进入各PCR微反应单元115。采用负压进样的方式可以减少所述数字PCR芯片16内的气体残留。In the embodiment of the present invention, referring to FIG. 10, the PCR reaction solution is first dropped into the sample inlet 12 of the sampler, and then the gas is pumped outward from the sample outlet 13 of the sampler. In addition, air is pumped out from the air extraction port 15 of the negative pressure chamber 14 of the sampler, so that the reaction layer 11 on the digital PCR chip 16 can quickly generate negative pressure in each PCR micro-reaction unit 115, The PCR reaction solution is guided to fill each branch flow channel 114 connected to the main channel 113 and enter each PCR micro-reaction unit 115. The negative pressure sampling method can reduce the residual gas in the digital PCR chip 16.

S14、吸掉所述进样器的进样池12处的多余PCR反应液，并滴加隔离油。S14. Aspirate the excess PCR reaction solution at the sample injection pool 12 of the sampler, and drop the isolation oil.

其中，所述隔离油可以为FC-40。Wherein, the isolation oil can be FC-40.

S15、参考图11所示，在所述进样器的出样口13处抽气，使所述隔离油填满所述反应层11的主流道113，完成对各PCR微反应单元115的隔离。S15. As shown in FIG. 11, air is pumped at the sample outlet 13 of the sampler, so that the isolation oil fills the main channel 113 of the reaction layer 11, and the isolation of each PCR micro-reaction unit 115 is completed .

本发明实施例中，所述高压反应室的压强为210-500KPa。所述高压反应室的液体可以为中性溶液，例如水溶液。In the embodiment of the present invention, the pressure of the high-pressure reaction chamber is 210-500KPa. The liquid in the high-pressure reaction chamber may be a neutral solution, such as an aqueous solution.

S2、排空所述反应室内的空气。S2. Evacuate the air in the reaction chamber.

具体的，所述步骤S2中通过注中性溶液对高压反应室进行排空气。Specifically, in the step S2, the high-pressure reaction chamber is evacuated by injecting a neutral solution.

S3、对所述反应室进行加压处理。S3, pressurize the reaction chamber.

在排空所述高压反应室内的空气之后，需要增加所述高压反应室的压力，再执行步骤S4。After evacuating the air in the high-pressure reaction chamber, the pressure of the high-pressure reaction chamber needs to be increased, and then step S4 is performed.

S4、将加压处理后的所述反应室接入反应设备进行PCR反应。S4. Connect the pressurized reaction chamber to the reaction device to perform PCR reaction.

本发明实施例中，反应设备采用PCR仪，将增压后的所述高压反应室放到PCR仪上进行PCR反应。具体的，所述步骤S4中的PCR反应的反应参数可以根据实际需要进行设定，将PCR反应后的所述高压反应室进行降温。本发明实施例中，高压反应室并非必须与PCR仪可拆卸连接，高压反应室还可以为集成在PCR仪上的反应室。In the embodiment of the present invention, a PCR machine is used as the reaction equipment, and the pressurized high-pressure reaction chamber is placed on the PCR machine for PCR reaction. Specifically, the reaction parameters of the PCR reaction in the step S4 can be set according to actual needs, and the temperature of the high-pressure reaction chamber after the PCR reaction is lowered. In the embodiment of the present invention, the high-pressure reaction chamber does not have to be detachably connected to the PCR machine, and the high-pressure reaction chamber may also be a reaction chamber integrated on the PCR machine.

S5、对所述数字PCR芯片16进行荧光信号分析。S5. Perform fluorescence signal analysis on the digital PCR chip 16.

具体的，将降温后的高压反应室降压，然后打开所述高压反应室，取出所述高压反应室中的数字PCR芯片16，并对所述数字PCR芯片16进行荧光信号分析。在一些实施方式中，还可以在高压反应室中设置在线读取信号装置，因此，对于反应后的高压反应室不经过减压处理取出芯片，而是直接采用在线读取信号分析的方式。Specifically, the pressure of the reduced high-pressure reaction chamber is reduced, and then the high-pressure reaction chamber is opened, the digital PCR chip 16 in the high-pressure reaction chamber is taken out, and the fluorescence signal analysis of the digital PCR chip 16 is performed. In some embodiments, an online signal reading device may also be provided in the high-pressure reaction chamber. Therefore, the high-pressure reaction chamber after the reaction does not undergo decompression processing to take out the chip, but directly adopts the method of online reading signal analysis.

本发明实施例采用上述高压液体浸入式数字PCR方法，使得进行PCR反应时所述数字PCR芯片16沉浸在所述高压反应室的液体内，由于所述数字PCR芯片16周围没有空气，在高温的情况下由于所述数字PCR芯片16外部高压的存在，PDMS微孔内的气体无法溢出PDMS，而在低温的情况下也不会有更多的空气进入PDMS中从 而破坏里面的气体平衡，从而阻止了PDMS像“泵”一样的呼吸行为，进而阻止了所述各PCR微反应单元115内气泡3的产生，使所述PCR反应液得以在稳定的环境下完成热循环反应。另外，水汽可以从所述数字PCR芯片16外表面进入PDMS内，从而减少所述PCR反应液的水损失。The embodiment of the present invention adopts the above-mentioned high-pressure liquid immersion digital PCR method, so that the digital PCR chip 16 is immersed in the liquid in the high-pressure reaction chamber when the PCR reaction is performed. In this case, due to the presence of high pressure outside the digital PCR chip 16, the gas in the PDMS micropores cannot escape the PDMS, and in the case of low temperature, no more air will enter the PDMS to disrupt the gas balance inside, thereby preventing The PDMS's breathing behavior like a "pump" prevents the generation of bubbles 3 in each PCR micro-reaction unit 115, so that the PCR reaction solution can complete the thermal cycle reaction in a stable environment. In addition, water vapor can enter the PDMS from the outer surface of the digital PCR chip 16, thereby reducing the water loss of the PCR reaction solution.

本发明实施例还提供了一种用于执行上述高压液体浸入式数字PCR方法的数字PCR芯片16。如图12所示，所述数字PCR芯片16包括基底层10、空白层101和反应层11。The embodiment of the present invention also provides a digital PCR chip 16 for executing the above-mentioned high-pressure liquid immersion digital PCR method. As shown in FIG. 12, the digital PCR chip 16 includes a base layer 10, a blank layer 101 and a reaction layer 11.

优选的，所述反应层11采用聚二甲基硅氧烷PDMS制成。所述反应层11设置于所述空白层101的上方。在一些实施例中，所述反应层11和所述空白层101可以通过不可逆的等离子体键合贴合在一起。Preferably, the reaction layer 11 is made of polydimethylsiloxane PDMS. The reaction layer 11 is disposed above the blank layer 101. In some embodiments, the reaction layer 11 and the blank layer 101 may be bonded together by irreversible plasma bonding.

参考图7和图8所示，所述反应层11具有主流道113、与所述主流道113相连通的各分支流道114以及与所述各分支流道114相连通的各PCR微反应单元115。Referring to FIGS. 7 and 8, the reaction layer 11 has a main flow channel 113, branch flow channels 114 connected to the main flow channel 113, and PCR micro-reaction units connected to the branch flow channels 114. 115.

如图12所示，所述反应层11还具有进样口和出样口，所述反应层的进样口1117所述进样器的进样池12相连通，所述反应层的出样口112与进样器的出样口13相连通。As shown in FIG. 12, the reaction layer 11 also has a sample inlet and a sample outlet. The sample inlet 1117 of the reaction layer is connected to the sample inlet 12 of the sampler, and the sample outlet of the reaction layer The port 112 is communicated with the sample outlet 13 of the sampler.

优选的，所述空白层101采用聚二甲基硅氧烷PDMS制成。所述空白层101设置于所述基底层10的上方。在一些实施例中，所述空白层101可以旋涂于硅片上。Preferably, the blank layer 101 is made of polydimethylsiloxane PDMS. The blank layer 101 is disposed above the base layer 10. In some embodiments, the blank layer 101 may be spin-coated on a silicon wafer.

优选的，所述基底层10采用支撑材料如玻璃等材料制成。所述基底层10位于所述空白层101和所述反应层11的下方，用于支撑所述空白层101和所述反应层11。Preferably, the base layer 10 is made of a supporting material such as glass. The base layer 10 is located below the blank layer 101 and the reaction layer 11 and is used to support the blank layer 101 and the reaction layer 11.

相应的，本发明实施例还提供了一种制备如上所述的数字PCR芯片16的方法，该方法包括以下步骤：Correspondingly, the embodiment of the present invention also provides a method for preparing the digital PCR chip 16 as described above, and the method includes the following steps:

S10、清洗硅片，在所述硅片上制作反应层11模具。S10. The silicon wafer is cleaned, and a reaction layer 11 mold is fabricated on the silicon wafer.

在一个具体的实施例中，所述步骤可以包括：使用Phiranha溶液清洗硅片，再用去离子水冲洗干净，氮气吹干，并在170-180℃热板 上烘烤0-0min；等离子体处理-min，然后旋涂SU8300510μm，进行光刻、显影，制作出反应层11的与主流道113相连通的各分支流道114；在160-170℃热板上烘烤20-30min，再等离子体处理1-2min，然后旋涂SU8302525μm，进行光刻、显影，制作出反应层11的主流道113以及与各分支流道114相连通的各PCR微反应单元115，从而制成反应层11模具。In a specific embodiment, the steps may include: cleaning the silicon wafer with Phiranha solution, rinsing it with deionized water, drying with nitrogen, and baking it on a hot plate at 170-180°C for 0-0 min; plasma Process for -min, then spin-coating SU8300510μm, perform photolithography and development, to produce the branch runners 114 of the reaction layer 11 communicating with the main channel 113; bake on a hot plate at 160-170℃ for 20-30min, and then plasma Body treatment for 1-2min, then spin-coating SU8302525μm, photolithography and development, to produce the main channel 113 of the reaction layer 11 and each PCR micro-reaction unit 115 connected to each branch channel 114, thereby making the reaction layer 11 mold .

S20、将聚二甲基硅氧烷以及固化剂混合均匀并脱气后浇注到所述反应层11模具上，热固化形成具有主流道113、各分支流道114以及各PCR微反应单元115的聚二甲基硅氧烷膜片；将所述聚二甲基硅氧烷膜片从所述反应层11模具上取下并打孔形成进样口和出样口，制成具有进样口、出样口、主流道113、各分支流道114以及各PCR微反应单元115的反应层11。S20. The polydimethylsiloxane and curing agent are mixed uniformly and degassed, and then poured onto the mold of the reaction layer 11, and thermally cured to form a main flow channel 113, each branch flow channel 114, and each PCR micro-reaction unit 115 Polydimethylsiloxane membrane; the polydimethylsiloxane membrane is removed from the mold of the reaction layer 11 and punched to form a sample inlet and a sample outlet, making it have a sample inlet , The sample outlet, the main flow channel 113, each branch flow channel 114, and the reaction layer 11 of each PCR micro-reaction unit 115.

在一个具体的实施例中，所述步骤S20可以包括：采用道康宁Sylgard184PDM，将前聚体和固化剂按照质量比5:1-10:1的比例混合均匀，并用真空脱气法除去混合物中的气泡3，然后将预聚物浇注在具有图形结构的反应层11模具上，在60-80℃热板上烘烤20-30min后形成具有主流道113、各分支流道114以及各PCR微反应单元115的聚二甲基硅氧烷膜片，将该聚二甲基硅氧烷膜片从反应层11模具上揭下后切割并打孔形成进样口和出样口，从而制成具有进样口、出样口、主流道113、各分支流道114以及各PCR微反应单元115的反应层11。In a specific embodiment, the step S20 may include: using Dow Corning Sylgard 184PDM, mixing the precursor and curing agent uniformly in a mass ratio of 5:1-10:1, and removing the mixture from the mixture by vacuum degassing. Bubble 3, then pour the prepolymer on the patterned reaction layer 11 mold, and bake it on a hot plate at 60-80°C for 20-30 minutes to form a main flow channel 113, branch flow channels 114, and PCR micro-reactions The polydimethylsiloxane film of unit 115 is removed from the mold of the reaction layer 11, and then cut and punched to form a sample inlet and a sample outlet, thereby making The sample inlet, the sample outlet, the main flow channel 113, each branch flow channel 114, and the reaction layer 11 of each PCR micro-reaction unit 115.

S301、将聚二甲基硅氧烷以及固化剂混合均匀旋涂在硅片上，固化形成空白层101。S301, mixing polydimethylsiloxane and curing agent uniformly and spin-coating on the silicon wafer, and curing to form a blank layer 101.

在一个具体的实施例中，所述步骤S301可以包括：将Sylgard184PDMS预聚物在3000-5000rpm下旋涂在硅片上，60-80℃热板上烘烤10-20min后形成空白层101。In a specific embodiment, the step S301 may include: spin-coating the Sylgard 184 PDMS prepolymer on a silicon wafer at 3000-5000 rpm, and baking on a hot plate at 60-80° C. for 10-20 minutes to form the blank layer 101.

S302、将步骤S20获得的反应层11与步骤S301获得的空白层101一起经等离子体激活处理后键合在一起。S302, bonding the reaction layer 11 obtained in step S20 and the blank layer 101 obtained in step S301 together after plasma activation treatment.

在一个具体的实施例中，所述步骤S302可以包括：In a specific embodiment, the step S302 may include:

将步骤S20获得的反应层11与步骤S301获得的空白层101的表面先用等离子体处理，然后键合在一起，80℃热板上烘烤对准了的芯片1-2h，实现反应层11与空白层101之间的永久键合。The surface of the reaction layer 11 obtained in step S20 and the surface of the blank layer 101 obtained in step S301 are first treated with plasma, and then bonded together, and the aligned chips are baked on a hot plate at 80°C for 1-2 hours to realize the reaction layer 11 Permanent bond with the blank layer 101.

S303、将步骤S302获得两层聚二甲基硅氧烷键合结构与载玻片经等离子体激活处理后键合在一起即制成数字PCR芯片16。S303, the two-layer polydimethylsiloxane bonding structure obtained in step S302 and the glass slide are bonded together after plasma activation treatment to form the digital PCR chip 16.

本发明实施例提供的技术方案具有如下优点：The technical solution provided by the embodiment of the present invention has the following advantages:

1、芯片进样不依赖泵、阀等复杂设备，也不需要使用高粘度热聚合分离油，进样完成后芯片不需要密封，操作简单；1. Chip sampling does not rely on complicated equipment such as pumps and valves, nor does it need to use high-viscosity thermal polymerization separation oil. After sampling is completed, the chip does not need to be sealed, and the operation is simple;

2、利用进样器辅助快速进样，并利用负压进样的方式减少芯片内的气体残留，进样迅速；2. Use the sampler to assist rapid sample injection, and use the negative pressure injection method to reduce the gas residue in the chip, and the sample is quickly injected;

3、芯片在水环境里完成反应，PCR微反应单元115内水分损失可以忽略不计，微反应单元115体积可以做的更小，单位面积的芯片可以制作更多的微反应单元115，芯片整体通量较高；3. The chip completes the reaction in an aqueous environment. The moisture loss in the PCR micro-reaction unit 115 can be neglected, the volume of the micro-reaction unit 115 can be made smaller, and more micro-reaction units 115 can be made per unit area of the chip. Higher volume

4、芯片各微反应单元115体积准确，阵列均匀，易于成像分析；4. The volume of each micro-reaction unit 115 of the chip is accurate, the array is uniform, and it is easy for imaging analysis;

5、高压反应室易与现有的PCR仪器兼容，也易于集成在PCR仪的金属温控模块上；5. The high-pressure reaction chamber is easily compatible with the existing PCR instrument, and is also easy to integrate into the metal temperature control module of the PCR instrument;

6、芯片厚度小，导热快，反应迅速；6. The chip thickness is small, the heat conduction is fast, and the response is fast;

7、芯片结构简单，容易制作，成本低，自动化程度高；7. The chip structure is simple, easy to manufacture, low cost, and high degree of automation;

8、减少水分蒸发，并可兼容其它dPCR芯片。8. Reduce water evaporation and be compatible with other dPCR chips.

以上所述仅为本申请的较佳实施例，并不用以限制本申请，凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。The above descriptions are only preferred embodiments of this application, and are not intended to limit this application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included in the protection of this application. Within range.

Claims (18)

1. 一种数字PCR方法，其特征在于，所述方法包括：A digital PCR method, characterized in that the method includes:

   S1、将经过进样处理的数字PCR芯片(16)放置于反应室中；S1. Place the digital PCR chip (16) that has undergone sample injection processing in the reaction chamber;

   S2、排空所述反应室内的空气；S2, evacuating the air in the reaction chamber;

   S3、对所述反应室进行加压处理；S3. Pressurize the reaction chamber;

   S4、将加压处理后的所述反应室进行PCR反应；S4. Perform a PCR reaction on the reaction chamber after the pressure treatment;

   S5、对所述数字PCR芯片(16)进行荧光信号分析。S5. Perform fluorescence signal analysis on the digital PCR chip (16).
2. 根据权利要求1所述的数字PCR方法，其特征在于，步骤S1中所述进样处理包括以下步骤：The digital PCR method according to claim 1, wherein the sample injection processing in step S1 comprises the following steps:

   S11.将进样器与数字PCR芯片(16)组合好，使得所述数字PCR芯片(16)的反应层的进样口(111)、出样口分别与所述进样器的进样池(12)、出样口对准并紧密贴合；S11. Combine the sampler with the digital PCR chip (16), so that the sample inlet (111) and the sample outlet of the reaction layer of the digital PCR chip (16) are respectively connected to the sample pool of the sampler (12) Align the sample outlet and fit closely;

   S12.将PCR反应液加入到所述进样器的进样池(12)中，在所述进样器的出样口(13)处向外抽气，使所述PCR反应液经过所述数字PCR芯片(16)的反应层的进样口(111)流入所述数字PCR芯片(16)的反应层(11)的主流道(113)，直至部分所述PCR反应液经过所述数字PCR芯片(16)的反应层的出样口(112)从所述进样器的出样口(13)流出；S12. Add the PCR reaction solution to the sampling pool (12) of the sampler, and pump air outward at the sample outlet (13) of the sampler, so that the PCR reaction solution passes through the The sample inlet (111) of the reaction layer of the digital PCR chip (16) flows into the main channel (113) of the reaction layer (11) of the digital PCR chip (16) until part of the PCR reaction solution passes through the digital PCR The sample outlet (112) of the reaction layer of the chip (16) flows out from the sample outlet (13) of the sampler;

   S13.自所述进样器的负压腔(14)的抽气口(15)处向外抽气，直至所述PCR反应液充满与所述主流道(113)相连通的各分支流道(114)以及与所述各分支流道(114)相连通的各PCR微反应单元(115)；S13. Pump air from the suction port (15) of the negative pressure chamber (14) of the sampler until the PCR reaction solution is filled with the branch flow channels ( 114) and each PCR micro-reaction unit (115) connected to each branch flow channel (114);

   S14.吸掉所述进样器的进样池(12)处的多余PCR反应液，并滴加隔离油；S14. Aspirate the excess PCR reaction solution at the sample injection pool (12) of the sampler, and drop the isolation oil;

   S15.在所述进样器的出样口(13)处抽气，使所述隔离油填满所述反应层(11)的主流道(113)，完成对各PCR微反应单元(115) 的隔离。S15. Exhaust air at the sample outlet (13) of the sampler, so that the isolation oil fills the main flow channel (113) of the reaction layer (11), and completes the PCR micro-reaction unit (115) Isolation.
3. 根据权利要求2所述的数字PCR方法，其特征在于，步骤S2中，通过注入中性溶液对所述反应室进行排空气。The digital PCR method according to claim 2, wherein in step S2, the reaction chamber is evacuated by injecting a neutral solution.
4. 根据权利要求3所述的数字PCR方法，其特征在于，步骤S3中，加压处理所述反应室的压强为210-500KPa。The digital PCR method according to claim 3, wherein in step S3, the pressure of the reaction chamber is 210-500KPa.
5. 根据权利要求1所述的数字PCR方法，其特征在于，加压处理后的所述反应室接入反应设备进行PCR反应，所述反应设备为循环***或PCR仪。The digital PCR method according to claim 1, wherein the pressure-treated reaction chamber is connected to a reaction device for PCR reaction, and the reaction device is a circulation system or a PCR machine.
6. 根据权利要求1所述的数字PCR方法，其特征在于，所述反应室为高压反应室，所述将经过进样处理的数字PCR芯片(16)放置于反应室中，包括：The digital PCR method according to claim 1, wherein the reaction chamber is a high-pressure reaction chamber, and placing the sample-injected digital PCR chip (16) in the reaction chamber comprises:

   将经过进样处理的数字PCR芯片(16)浸入高压反应室的液体中。The digital PCR chip (16) that has undergone sample injection processing is immersed in the liquid in the high-pressure reaction chamber.
7. 一种数字PCR芯片，其特征在于，所述数字PCR芯片(16)用于执行如权利要求1-6中任一项所述的数字PCR方法；A digital PCR chip, characterized in that the digital PCR chip (16) is used to execute the digital PCR method according to any one of claims 1-6;

   所述数字PCR芯片(16)包括依次设置的基底层(10)和反应层(11)，其中，所述反应层(11)具有主流道(113)、与所述主流道(113)相连通的各分支流道(114)以及与所述各分支流道(114)相连通的各PCR微反应单元(115)；所述反应层(11)还具有进样口和出样口，所述反应层的进样口(111)与进样器的进样池(12)相连通，所述反应层的出样口(112)与进样器的出样口(13)相连通。The digital PCR chip (16) includes a base layer (10) and a reaction layer (11) arranged in sequence, wherein the reaction layer (11) has a main flow channel (113) and is connected to the main flow channel (113) Each branch flow channel (114) and each PCR micro-reaction unit (115) connected to each branch flow channel (114); the reaction layer (11) also has a sample inlet and a sample outlet, the The sample inlet (111) of the reaction layer is connected with the sample inlet (12) of the sampler, and the sample outlet (112) of the reaction layer is connected with the sample outlet (13) of the sampler.
8. 根据权利要求7所述的数字PCR芯片，其特征在于，所述反 应层(11)采用聚二甲基硅氧烷制成。The digital PCR chip according to claim 7, wherein the reaction layer (11) is made of polydimethylsiloxane.
9. 根据权利要求8所述的数字PCR芯片，其特征在于，所述基底层(10)采用支撑材料制成，用于对所述反应层(11)进行密封及支撑。The digital PCR chip according to claim 8, characterized in that the base layer (10) is made of a supporting material for sealing and supporting the reaction layer (11).
10. 根据权利要求8所述的数字PCR芯片，其特征在于，所述基底层(10)与所述反应层(11)之间还设有空白层(101)，所述空白层(101)采用聚二甲基硅氧烷制成。The digital PCR chip according to claim 8, wherein a blank layer (101) is provided between the base layer (10) and the reaction layer (11), and the blank layer (101) is made of poly Made of dimethyl siloxane.
11. 一种数字PCR芯片制备方法，其特征在于，包括以下步骤：A method for preparing a digital PCR chip is characterized in that it comprises the following steps:

    S10、清洗硅片，在所述硅片上制作反应层(11)模具；S10, cleaning the silicon wafer, and fabricating a reaction layer (11) mold on the silicon wafer;

    S20、将聚二甲基硅氧烷以及固化剂混合均匀并脱气后浇注到所述反应层(11)模具上，热固化形成具有主流道(113)、各分支流道(114)以及各PCR微反应单元(115)的聚二甲基硅氧烷膜片；将所述聚二甲基硅氧烷膜片从所述反应层(11)模具上取下并打孔形成进样口和出样口，制成具有进样口、出样口、主流道(113)、各分支流道(114)以及各PCR微反应单元(115)的反应层(11)；S20, mixing polydimethylsiloxane and curing agent uniformly and degassing and pouring on the reaction layer (11) mold, thermally curing to form a main flow channel (113), each branch flow channel (114) and each The polydimethylsiloxane membrane of the PCR micro-reaction unit (115); the polydimethylsiloxane membrane is removed from the reaction layer (11) mold and punched to form a sample inlet and The sample outlet is made into a reaction layer (11) having a sample inlet, a sample outlet, a main flow channel (113), each branch flow channel (114), and each PCR micro-reaction unit (115);

    S30、将步骤S20获得的聚二甲基硅氧烷反应层(11)结构与基底层(10)经等离子体激活处理后键合在一起，即制成数字PCR芯片(16)。S30, bonding the polydimethylsiloxane reaction layer (11) structure obtained in step S20 and the base layer (10) after plasma activation treatment to form a digital PCR chip (16).
12. 根据权利要求11所述的数字PCR芯片制备方法，其特征在于，所述将步骤S20获得的聚二甲基硅氧烷反应层(11)结构与基底层(10)经等离子体激活处理后键合在一起，即制成数字PCR芯片(16)，包括：The method for preparing a digital PCR chip according to claim 11, wherein the structure of the polydimethylsiloxane reaction layer (11) obtained in step S20 and the base layer (10) are bonded after plasma activation treatment. Together, a digital PCR chip (16) is made, including:

    S301、将聚二甲基硅氧烷以及固化剂混合均匀旋涂在硅片上，固化形成空白层(101)；S301, mixing polydimethylsiloxane and curing agent uniformly and spin-coating on the silicon wafer, and curing to form a blank layer (101);

    S302、将步骤S20获得的反应层(11)与步骤S301获得的空白 层(101)一起经等离子体激活处理后键合在一起；S302, bonding the reaction layer (11) obtained in step S20 and the blank layer (101) obtained in step S301 together after plasma activation treatment;

    S303、将步骤S302获得的两层聚二甲基硅氧烷键合结构与基底层(10)经等离子体激活处理后键合在一起，即制成数字PCR芯片(16)。S303. The two-layer polydimethylsiloxane bonding structure obtained in step S302 and the base layer (10) are bonded together after plasma activation treatment to form a digital PCR chip (16).
13. 一种循环***，其特征在于，用于执行如权利要求1-6中任一项所述的循环式数字PCR方法，A circulatory system, characterized by being used to execute the cyclic digital PCR method according to any one of claims 1-6,

    所述循环***包括反应室、液体循环***、温度调节***、压力调节***和控制器，所述反应室与所述液体循环***连接，用于放置数字PCR芯片(16)；所述控制器分别与所述液体循环***、所述温度调节***和所述压力调节***连接，用于自动控制所述液体循环***为所述反应室输送液体来调节所述反应室的温度，自动控制所述温度调节***调节所述液体循环***中的液体的温度，自动控制所述压力调节***调节所述循环***的压力。The circulation system includes a reaction chamber, a liquid circulation system, a temperature adjustment system, a pressure adjustment system, and a controller. The reaction chamber is connected to the liquid circulation system for placing a digital PCR chip (16); the controllers are respectively Connected to the liquid circulation system, the temperature adjustment system, and the pressure adjustment system, and is used to automatically control the liquid circulation system to deliver liquid to the reaction chamber to adjust the temperature of the reaction chamber, and to automatically control the temperature The adjustment system adjusts the temperature of the liquid in the liquid circulation system, and automatically controls the pressure adjustment system to adjust the pressure of the circulation system.
14. 根据权利要求13所述的循环***，其特征在于，所述液体循环***包括恒温储液箱(6)、循环泵(7)、阀门(8)和管路(9)，所述恒温储液箱(6)通过所述管路(9)与所述反应室连接，用于为所述反应室输送恒温的液体来调节所述反应室的温度；所述循环泵(7)和所述阀门(8)均设置在所述管路(9)上，用于驱动恒温的液体在所述管路(9)中循环流动。The circulation system according to claim 13, characterized in that the liquid circulation system comprises a constant temperature liquid storage tank (6), a circulation pump (7), a valve (8) and a pipeline (9), and the constant temperature liquid storage The tank (6) is connected to the reaction chamber through the pipeline (9), and is used to deliver a constant temperature liquid to the reaction chamber to adjust the temperature of the reaction chamber; the circulation pump (7) and the valve (8) are all arranged on the pipeline (9), and used for driving the constant temperature liquid to circulate in the pipeline (9).
15. 根据权利要求14所述的循环***，其特征在于，所述温度调节***包括第一温度传感器、第二温度传感器、加热器和冷却器，The circulation system according to claim 14, wherein the temperature adjustment system comprises a first temperature sensor, a second temperature sensor, a heater and a cooler,

    所述第一温度传感器设置在所述反应室内，用于探测所述反应室内的温度；所述第二温度传感器设置在所述恒温储液箱(6)内，用于探测所述恒温储液箱(6)内的温度；所述加热器和所述冷却器均设置在所述恒温储液箱(6)内，用于对所述恒温储液箱(6)内的液体进行加热或降温。The first temperature sensor is arranged in the reaction chamber and is used to detect the temperature in the reaction chamber; the second temperature sensor is arranged in the constant temperature liquid storage tank (6) and is used to detect the constant temperature liquid storage The temperature in the tank (6); the heater and the cooler are both arranged in the constant temperature liquid storage tank (6) for heating or cooling the liquid in the constant temperature liquid storage tank (6) .
16. 根据权利要求15所述的循环***，其特征在于，所述压力调节***包括压力传感器、增压泵和泄压阀，所述压力传感器设置在所述循环***内，用于探测所述循环***的压力；所述增压泵和所述泄压阀设置在所述循环***内，用于增加或减少所述循环***的压力。The circulation system according to claim 15, wherein the pressure adjustment system comprises a pressure sensor, a booster pump, and a pressure relief valve, and the pressure sensor is arranged in the circulation system for detecting the circulation system The pressure; the booster pump and the pressure relief valve are provided in the circulation system for increasing or decreasing the pressure of the circulation system.
17. 根据权利要求16所述的循环***，其特征在于，所述控制器分别与所述循环泵(7)和所述阀门(8)连接，用于根据PCR程序自动控制所述循环泵(7)和所述阀门(8)的工作来为所述反应室输送恒温的液体；所述控制器分别与所述第一温度传感器、所述第二温度传感器、所述加热器和所述冷却器连接，用于根据所述第一温度传感器和所述第二温度传感器探测的温度自动控制所述加热器和所述冷却器的工作对所述恒温储液箱(6)内的液体进行加热或降温；所述控制器分别与所述压力传感器、所述增压泵和所述泄压阀连接，用于根据所述压力传感器探测的压力自动控制所述增压泵和所述泄压阀的工作增加或减少所述循环***的压力。The circulation system according to claim 16, characterized in that the controller is respectively connected with the circulation pump (7) and the valve (8) for automatically controlling the circulation pump (7) according to a PCR program And the valve (8) work to deliver a constant temperature liquid for the reaction chamber; the controller is respectively connected with the first temperature sensor, the second temperature sensor, the heater and the cooler , Used for automatically controlling the work of the heater and the cooler according to the temperature detected by the first temperature sensor and the second temperature sensor to heat or cool the liquid in the constant temperature liquid storage tank (6) The controller is respectively connected with the pressure sensor, the booster pump and the pressure relief valve, and is used to automatically control the work of the booster pump and the pressure relief valve according to the pressure detected by the pressure sensor Increase or decrease the pressure of the circulatory system.
18. 根据权利要求17所述的循环***，其特征在于，所述恒温储液箱(6)设置有2-4个。The circulation system according to claim 17, characterized in that there are 2-4 thermostatic liquid storage tanks (6).

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