技术领域technical field
本发明涉及生物检测技术领域，具体涉及核酸扩增检测技术及穿戴式医疗设备技术。The invention relates to the technical field of biological detection, in particular to nucleic acid amplification detection technology and wearable medical equipment technology.
背景技术Background technique
可穿戴式设备（wearable device）是直接能穿在身上、或直接整合到用户的衣服或配件中的设备。该设备可以集成多媒体、传感器和无线通信等技术，实现快速的数据获取、超快的数据传输，摆脱传统的手持设备而获得无缝的网络访问体验。具有佩戴舒适甚至无感、使用过程不干扰正常生活、外观适合使用场合和环境等优点。目前全球可穿戴市场规模约为30亿~50亿美元，且未来两到三年有望成长为300亿~500亿美元的巨大市场，特别是随着4G和移动终端的普及，可穿戴设备有巨大的发展潜力。目前应用最广泛的可穿戴设备主要是眼镜、手表、手环和项链等，实现的功能主要是健康监测，如手环监测心率等；安全保障，如蓝牙近距离通信实现钱包防窃等；以及游戏娱乐，如Kinect体感游戏机等。随着支撑技术的不断进步，可穿戴产品朝着更轻便、更隐蔽、更快捷的方向转变，时尚型和功能型将成为可穿戴产品的发展的两条主线。随着硬件技术及互联网技术的发展，可穿戴产品的功能会更加的全面，并且具有巨大的市场潜力以及广阔的发展空间。A wearable device is a device that can be worn directly on the body, or directly integrated into the user's clothing or accessories. The device can integrate multimedia, sensor and wireless communication technologies to achieve fast data acquisition, ultra-fast data transmission, and get rid of traditional handheld devices to obtain a seamless network access experience. It has the advantages of comfortable or even non-sense wearing, no interference with normal life during use, and suitable appearance for the use occasion and environment. At present, the global wearable market size is about 3 billion to 5 billion US dollars, and it is expected to grow into a huge market of 30 billion to 50 billion US dollars in the next two to three years, especially with the popularization of 4G and mobile terminals, wearable devices have a huge potential. development potential. At present, the most widely used wearable devices are mainly glasses, watches, bracelets and necklaces, etc., and the functions realized are mainly health monitoring, such as wristband monitoring heart rate, etc.; security protection, such as Bluetooth short-distance communication to realize wallet anti-theft; and Game entertainment, such as Kinect somatosensory game console, etc. With the continuous advancement of supporting technology, wearable products are changing towards lighter, more concealed, and faster directions. Fashionable and functional models will become the two main lines of development of wearable products. With the development of hardware technology and Internet technology, the functions of wearable products will be more comprehensive, and have huge market potential and broad development space.
医疗管理类可穿戴设备是目前最主要的发展方向。利用集成各种传感器到可穿戴设备上，能够长期实时监测人体中某种参数的指标。由于柔性材料如聚二甲基硅氧烷（PDMS）、聚甲基丙烯酸甲脂（PMMA）等在范德华力的作用下能够和人体皮肤充分接触，并且具有较好的透明性，传热效应和电化学电阻，通过工程处理之后可以做成弯曲的形状，和皮肤更紧密的接触，通常用于集成传感器做成可穿戴设备。将柔性电流传感器集成到隐形眼镜中，用于检测泪液中的葡萄糖含量；将生物传感器集成到护口器中可用于检测唾液中的乳酸含量；近期还研发出了将生物传感器集成到临时纹身中，以非入侵的方式测量人皮肤或间质液体中的汗液成分。将基于聚酯片的传感器集成到柔性材料中，可用于记录人体的脑电图（EEG）、心电图（ECG）和肌电图（EMG），并通过无线数据传输的方式将结果送入电脑终端，实时分析人体的检测状况。虽然目前的可穿戴设备的应用领域非常的广泛，但至今没有可穿戴设备可用于核酸扩增及检测。Medical management wearable devices are currently the most important development direction. By integrating various sensors into wearable devices, it is possible to monitor the indicators of certain parameters in the human body in real time for a long time. Since flexible materials such as polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA) can fully contact with human skin under the action of van der Waals force, and have good transparency, heat transfer effect and Electrochemical resistance can be made into a curved shape after engineering treatment, and it is in closer contact with the skin. It is usually used to integrate sensors into wearable devices. Integrating flexible current sensors into contact lenses to detect glucose levels in tears; integrating biosensors into mouthguards can be used to detect lactic acid levels in saliva; recently also developed biosensors integrated into temporary tattoos , to measure sweat composition in human skin or interstitial fluid in a non-invasive manner. The polyester sheet-based sensor is integrated into a flexible material, which can be used to record the human body's electroencephalogram (EEG), electrocardiogram (ECG) and electromyogram (EMG), and send the results to the computer terminal through wireless data transmission , Real-time analysis of the detection status of the human body. Although the current wearable devices have a wide range of applications, so far no wearable devices can be used for nucleic acid amplification and detection.
目前核酸扩增技术有很多种，最常见的就是聚合酶链式反应（Polymerase ChainReaction, PCR）。该方发是通过高温下核酸的变性、延伸、复性的方法，但是需要有温度循环的设备，所需条件较高，而一些恒温扩增技术通过酶切割碱基序列，需要一个恒定温度即可实现核酸扩增，为可穿戴设备应用到核酸扩增领域提供了可能。重组酶聚合酶扩增技术（Recombinase Polymerase Amplification, RPA）是一种恒温扩增技术，扩增温度为31~42˚C，接近于人体体温，且能够在十几分钟内将核酸扩增到可检测的水平，是一种快速核酸恒温扩增技术。There are many nucleic acid amplification techniques, the most common of which is the polymerase chain reaction (Polymerase Chain Reaction, PCR). This method is based on the denaturation, extension, and renaturation of nucleic acids at high temperatures, but equipment with temperature cycles is required, and the required conditions are relatively high, while some constant temperature amplification techniques use enzymes to cut base sequences, requiring a constant temperature. Nucleic acid amplification can be realized, which provides the possibility for wearable devices to be applied to the field of nucleic acid amplification. Recombinase Polymerase Amplification (RPA) is a constant temperature amplification technology. The amplification temperature is 31~42˚C, which is close to the body temperature of the human body. The level of detection is a rapid nucleic acid constant temperature amplification technique.
发明内容Contents of the invention
本发明首先要解决的技术问题是提供一种进行核酸扩增检测的装置，可以对核酸进行快速的检测，且所需要的外部设备条件要求低，能够随时随地进行检测。The first technical problem to be solved by the present invention is to provide a device for nucleic acid amplification detection, which can quickly detect nucleic acid, and requires low external equipment conditions, and can perform detection anytime and anywhere.
本发明解决上述技术问题所采用的技术方案是：一种进行核酸扩增检测的装置，包括检测系统和芯片，芯片置于检测系统内，检测系统包括光激发设备和信号采集设备，由光激发设备发出的光，照射到芯片上，在检测区域产生发射光，经过滤得到荧光信号，信号采集设备采集该荧光信号从而进行检测，该芯片能够被集成在穿戴设备上。The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a device for nucleic acid amplification detection, including a detection system and a chip, the chip is placed in the detection system, and the detection system includes light excitation equipment and signal acquisition equipment, which are excited by light. The light emitted by the device is irradiated on the chip, and the emitted light is generated in the detection area. After filtering, the fluorescent signal is obtained. The signal acquisition device collects the fluorescent signal for detection. The chip can be integrated on the wearable device.
在采用上述技术方案的同时，本发明还可以采用一下进一步的技术方案：While adopting above-mentioned technical scheme, the present invention can also adopt following further technical scheme:
检测系统包括壳体，壳体内设有驱动设备、光激发设备、信号采集设备、检测设备和检测腔室，驱动设备包括电源和变压器，驱动设备为光激发设备和信号采集设备供电，光激发设备包括风扇、散热器和led光源，检测设备包括激发片、二向分镜和发射片，信号采集设备为工业相机，激发片置于led光源的前端，发射片置于工业相机的镜头前端，二向分镜和芯片置于检测腔室内，光激发设备位于检测腔室的一侧且led光源朝向检测腔室的方向，工业相机位于检测腔室的上方且镜头朝向检测腔室的方向，芯片置于检测腔室的底部，二向分镜在检测腔室内成45度角放置，其一面朝向光激发设备，另一面朝向工业相机。The detection system includes a housing, in which there are drive equipment, optical excitation equipment, signal acquisition equipment, detection equipment and a detection chamber. The drive equipment includes a power supply and a transformer. The drive equipment supplies power to the optical excitation equipment and signal acquisition equipment. The optical excitation equipment Including fans, heat sinks and LED light sources. The detection equipment includes exciters, dichroic mirrors and emitters. The signal acquisition equipment is an industrial camera. The sub-mirror and the chip are placed in the detection chamber, the optical excitation device is located on one side of the detection chamber and the LED light source is facing the direction of the detection chamber, the industrial camera is located above the detection chamber and the lens is facing the direction of the detection chamber, and the chip is placed At the bottom of the detection chamber, a two-way mirror is placed in the detection chamber at an angle of 45 degrees, with one side facing the light excitation device and the other facing the industrial camera.
芯片包括设置在中部的六边形的反应腔室，该反应腔室的两端分别设有进液口和出液口，反应腔室的上下两面分别设有顶层和底层作为封层，当芯片置于检测腔室内时，其下方设有加热片，加热片由驱动设备供电。The chip includes a hexagonal reaction chamber arranged in the middle. The two ends of the reaction chamber are respectively provided with a liquid inlet and a liquid outlet. The upper and lower sides of the reaction chamber are respectively provided with a top layer and a bottom layer as sealing layers. When it is placed in the detection chamber, there is a heating plate under it, and the heating plate is powered by the driving device.
芯片是具有生物相容性的高分子聚合物芯片，例如PDMS，芯片的顶层包含通道结构，芯片的底层是0.5mm厚的PDMS膜，PDMS通道的厚度为0.5-1.5mm，进液口和出液口的孔径为1-4mm。The chip is a biocompatible polymer chip, such as PDMS. The top layer of the chip contains a channel structure, and the bottom layer of the chip is a 0.5mm thick PDMS membrane. The thickness of the PDMS channel is 0.5-1.5mm. The liquid inlet and outlet The aperture of the liquid port is 1-4mm.
芯片顶层的通道结构为单通道结构或多通道结构，通道的总体积不小于50µL。The channel structure on the top layer of the chip is a single-channel structure or a multi-channel structure, and the total volume of the channel is not less than 50µL.
led光源的功率为5-15W。The power of the led light source is 5-15W.
激发片的带通波长为435-480nm。The bandpass wavelength of the excitation sheet is 435-480nm.
发射片的带通波长为500-560nm。The bandpass wavelength of the emitter is 500-560nm.
该芯片能够被集成在腕表上，佩带在人体手腕处。The chip can be integrated on the watch and worn on the human wrist.
本发明所要解决的另一个技术问题是提供一种进行核酸扩增检测的方法，该方法应用上述的装置，并包括以下步骤：Another technical problem to be solved by the present invention is to provide a method for nucleic acid amplification detection, which uses the above-mentioned device and includes the following steps:
（1）测量人体温度：向芯片中注水，将芯片通过穿戴设备固定在人体皮肤表面，通过热电偶测量芯片中的液体温度，验证用人体体温扩增的可行性；(1) Measure the body temperature: inject water into the chip, fix the chip on the surface of the human skin through a wearable device, measure the temperature of the liquid in the chip through a thermocouple, and verify the feasibility of using human body temperature to amplify;
（2）构建核酸检测的标准曲线：(2) Construct a standard curve for nucleic acid detection:
（2.1）从原始浓度的核酸模板开始按比例稀释，得到梯度稀释的模板浓度；(2.1) Proportionally dilute from the original concentration of the nucleic acid template to obtain the template concentration of the gradient dilution;
（2.2）按照RPA试剂盒说明书上的方法，配置好反应所需混合物；(2.2) According to the method on the RPA kit manual, prepare the mixture required for the reaction;
（2.3）在混合物中加入不同浓度的模板和醋酸镁充分混匀；(2.3) Add different concentrations of templates and magnesium acetate to the mixture and mix thoroughly;
（2.4）将混匀后的反应物从进液口打入芯片，放入检测系统，拍照记录荧光强度；(2.4) Put the mixed reactant into the chip from the liquid inlet, put it into the detection system, take pictures and record the fluorescence intensity;
（2.5）将芯片佩戴在人体皮肤表面，通过人体体温进行扩增，反应一定时间后，取出芯片放入检测系统中拍照记录；(2.5) Wear the chip on the surface of the human skin, amplify it through body temperature, and after a certain period of time, take out the chip and put it into the detection system to take pictures and record it;
重复步骤（2.1）-（2.5）至少三次，Repeat steps (2.1)-(2.5) at least three times,
（2.6）ImageJ分析检测区域的绿色荧光强度，根据反应结束后的荧光强度建立标准曲线；(2.6) ImageJ analyzes the green fluorescence intensity of the detection area, and establishes a standard curve based on the fluorescence intensity after the reaction;
（3）样本浓度检测：按照步骤（2.1）-（2.5）检测样本并将检测结果带入标准曲线中，得到样本中的核酸浓度。(3) Sample concentration test: Test the sample according to steps (2.1)-(2.5) and bring the test results into the standard curve to obtain the nucleic acid concentration in the sample.
本发明的有益效果是：本发明设计了与人体皮肤直接接触的可穿戴设备，将含有RPA试剂的芯片插入手表中，利用手腕处的体温提供RPA扩增所需的温度，通过自带的光学系统检测反应后产生的荧光信号。利用本发明的装置可以对核酸进行快速的检测，甚至可以定量核酸的含量，且所需要的外部设备条件要求低，在贫困地区也可以广泛使用，对贫困地区易发生的重大传染病如HIV、HBV、Ebola和Zika等检测具有重大的意义，对一些突发性疾病病原检测如Mers、流感等也有重要的意义。由于检测所需要的时间短，只要20分钟左右，也适用于出入境的人员做传染病检测。该方法在快速核酸检测、传染病防治等方面拥有广阔的商业潜力。The beneficial effects of the present invention are: the present invention designs a wearable device that is in direct contact with the human skin, inserts a chip containing the RPA reagent into the watch, uses the body temperature at the wrist to provide the temperature required for RPA amplification, and uses the built-in optical The system detects the fluorescent signal generated after the reaction. The device of the present invention can quickly detect nucleic acid, and even quantify the content of nucleic acid, and the required external equipment conditions are low, and can be widely used in poverty-stricken areas. The detection of HBV, Ebola and Zika is of great significance, and it is also of great significance for the detection of some sudden disease pathogens such as Mers and influenza. Since the time required for the test is short, only about 20 minutes, it is also suitable for people entering and leaving the country for infectious disease testing. This method has broad commercial potential in rapid nucleic acid detection, prevention and treatment of infectious diseases, etc.
附图说明Description of drawings
图1是芯片的结构图。Figure 1 is a structural diagram of the chip.
图2是芯片的分解状态示意图。FIG. 2 is a schematic diagram of a disassembled state of the chip.
图3是检测装置的结构图。Fig. 3 is a structural diagram of the detection device.
图4是实时荧光扩增结果图。Fig. 4 is a diagram of real-time fluorescence amplification results.
图5是核酸浓度检测标准曲线。Figure 5 is a nucleic acid concentration detection standard curve.
图中标号：1-芯片，2-顶层，2-1-进液口，2-2-出液口，2-3-反应腔室，3-底层，4-加热片，5-光激发设备，5-1-风扇，5-2-散热器，5-3-led光源，6-电源，7-驱动设备，8-工业相机，9-检测系统，9-1-发射片，9-2-二巷分镜，9-3-激发片，10-二号挡板，11-检测腔室。Labels in the figure: 1-chip, 2-top layer, 2-1-liquid inlet, 2-2-liquid outlet, 2-3-reaction chamber, 3-bottom layer, 4-heating plate, 5-light excitation device , 5-1-fan, 5-2-radiator, 5-3-led light source, 6-power supply, 7-drive device, 8-industrial camera, 9-detection system, 9-1-emitter, 9-2 -Second-lane mirror, 9-3-exciting sheet, 10-No. 2 baffle, 11-detection chamber.
具体实施方式detailed description
以下结合实施例对本发明的技术方案作进一步详细说明，实施例仅用于解释本发明的实施方式或过程，不是对本发明保护范围的限定。The technical solutions of the present invention will be described in further detail below in conjunction with the examples. The examples are only used to explain the implementation or process of the present invention, and are not intended to limit the protection scope of the present invention.
实施例1，一种进行核酸扩增检测的装置，参照附图1-3。Example 1, a device for performing nucleic acid amplification detection, see Figures 1-3.
本发明提供一种进行核酸扩增检测的装置，该装置能够利用穿戴设备佩带在人体皮肤表面，从而感受人体温度，进行核酸扩增检测，具体来说，本发明的装置包括检测系统9和芯片1，芯片1可以被置于穿戴设备，例如腕带或腕表中，从而与人体皮肤直接接触。The present invention provides a device for nucleic acid amplification detection. The device can be worn on the surface of human skin by using a wearable device, so as to sense the temperature of the human body and perform nucleic acid amplification detection. Specifically, the device of the present invention includes a detection system 9 and a chip 1. The chip 1 can be placed in a wearable device, such as a wristband or a watch, so as to be in direct contact with human skin.
芯片1的具体结构是：芯片1包括设置在中部的六边形的反应腔室2-3，该反应腔室2-3的两端分别设有进液口2-1和出液口2-2，反应腔室2-3的上下两面分别设有顶层2和底层3作为封层，当芯片1置于检测系统9的检测腔室内时，其下方设有加热片4，加热片4由检测系统9内的驱动设备7供电。The specific structure of the chip 1 is: the chip 1 includes a hexagonal reaction chamber 2-3 arranged in the middle, and the two ends of the reaction chamber 2-3 are respectively provided with a liquid inlet 2-1 and a liquid outlet 2- 2. The upper and lower sides of the reaction chamber 2-3 are respectively provided with a top layer 2 and a bottom layer 3 as a sealing layer. When the chip 1 is placed in the detection chamber of the detection system 9, a heating plate 4 is provided below it, and the heating plate 4 is detected by the chip 1. The drives 7 within the system 9 are powered.
本发明中的芯片1是具有生物相容性的高分子聚合物芯片，例如PDMS，芯片1的顶层2中包含通道结构，通道结构可以是单通道结构或多通道结构，只需要保证通道的总体积不小于50µL。当芯片1位PDMS时，芯片1的底层3是0.5mm厚的PDMS膜，PDMS通道的厚度为0.5-1.5mm，进液口2-1和出液口2-2的孔径为1-4mm。The chip 1 in the present invention is a biocompatible polymer chip, such as PDMS. The top layer 2 of the chip 1 contains a channel structure. The channel structure can be a single-channel structure or a multi-channel structure. The volume is not less than 50µL. When the chip is equipped with PDMS, the bottom layer 3 of the chip 1 is a 0.5mm thick PDMS membrane, the thickness of the PDMS channel is 0.5-1.5mm, and the apertures of the liquid inlet 2-1 and the liquid outlet 2-2 are 1-4mm.
检测系统9包括光激发设备5和信号采集设备，由光激发设备5发出的光，照射到芯片1上，在检测区域产生发射光，经过滤得到荧光信号，信号采集设备采集该荧光信号从而进行检测。The detection system 9 includes a light excitation device 5 and a signal acquisition device. The light emitted by the light excitation device 5 is irradiated onto the chip 1, and emitted light is generated in the detection area, which is filtered to obtain a fluorescent signal. The signal acquisition device collects the fluorescent signal to perform detection.
检测系统9包括壳体，壳体内设有驱动设备7、光激发设备5、信号采集设备、检测设备和检测腔室11，驱动设备7为光激发设备和信号采集设备供电，驱动设备7包括电源6和变压器，变压器将电源6的电源转换为 不同的电压，以适应检测系统中不同用电装置的需求。The detection system 9 includes a housing, and the housing is provided with a drive device 7, an optical excitation device 5, a signal acquisition device, a detection device and a detection chamber 11. The drive device 7 supplies power for the optical excitation device and the signal acquisition device, and the drive device 7 includes a power supply 6 and a transformer, the transformer converts the power of the power supply 6 into different voltages to meet the needs of different electrical devices in the detection system.
光激发设备5包括风扇5-1、散热器5-2和led光源5-3，风扇5-1的额定电压为12V，led光源5-3的功率为11W，额定电压为11V，所需电压由电源6提供，且由驱动模块7中的变压器转换为对应的电压。Light excitation device 5 comprises fan 5-1, radiator 5-2 and led light source 5-3, and the rated voltage of fan 5-1 is 12V, and the power of led light source 5-3 is 11W, and rated voltage is 11V, and required voltage Provided by the power supply 6, and converted to a corresponding voltage by the transformer in the drive module 7.
检测设备包括激发片9-3、二向分镜9-2和发射片9-1，信号采集设备为工业相机8，激发片9-3置于led光源5-3的前端，发射片9-1置于工业相机8的镜头前端且贴紧工业相机8的镜头，二向分镜9-2和芯片1置于检测腔室11内，激发片9-3的中心波长为475 nm，带宽为40 nm，距led光源5-3的距离为2.3 mm；光激发设备5位于检测腔室11的右侧且led光源5-3的发射方向朝向检测腔室的方向，工业相机8位于检测腔室11的上方且镜头朝向检测腔室11的方向，芯片置于检测腔室的底部，检测腔室11是55×55 mm的正方形腔室，二向分镜在检测腔室内成45度角放置，其一面朝向光激发设备，另一面朝向工业相机，二号挡板10是一块黑色的PMMA板，用于遮住干扰光，距激发片9-3的距离为2.3 mm，防止散射光产生背景干扰。The detection equipment includes excitation sheet 9-3, two-way mirror 9-2 and emission sheet 9-1, the signal acquisition device is industrial camera 8, excitation sheet 9-3 is placed at the front end of led light source 5-3, emission sheet 9- 1 is placed on the front end of the lens of the industrial camera 8 and is close to the lens of the industrial camera 8, the dichroic mirror 9-2 and the chip 1 are placed in the detection chamber 11, the central wavelength of the excitation plate 9-3 is 475 nm, and the bandwidth is 40 nm, the distance from the led light source 5-3 is 2.3 mm; the optical excitation device 5 is located on the right side of the detection chamber 11 and the emission direction of the led light source 5-3 faces the direction of the detection chamber, and the industrial camera 8 is located in the detection chamber 11 and the lens is facing the direction of the detection chamber 11, the chip is placed at the bottom of the detection chamber, the detection chamber 11 is a square chamber of 55×55 mm, and the dichroic mirror is placed at an angle of 45 degrees in the detection chamber. One side of it faces the photoexcitation device, and the other side faces the industrial camera. The second baffle 10 is a black PMMA plate, which is used to cover the interfering light. The distance from the excitation plate 9-3 is 2.3 mm to prevent background interference caused by scattered light. .
实施例2，构建标准曲线，参照附图5。Embodiment 2, construct standard curve, refer to accompanying drawing 5.
本实施例使用HIV-1 DNA质粒为检测模板，将混合物注入芯片中，芯片通过手表与人体紧贴，利用人体体温扩增核酸，并检测，建立不同浓度的标准曲线。具体包括如下步骤：In this example, the HIV-1 DNA plasmid is used as the detection template, and the mixture is injected into the chip. The chip is closely attached to the human body through the watch, and the nucleic acid is amplified by human body temperature, and detected, and a standard curve with different concentrations is established. Specifically include the following steps:
（1）从原始浓度109copies/mL的HIV-1 DNA质粒模板开始按1：10的比例稀释，得到梯度稀释的模板浓度。(1) Dilute the HIV-1 DNA plasmid template at a ratio of 1:10 starting from the original concentration of 109 copies/mL to obtain the template concentration of the gradient dilution.
（2）RPA试剂盒购买自twist，按照配套说明书上的方法，29.5 µL Rehydrationbuffer, 2.1 µL Primer-F， 2.1 µL Primer-R，0.6 µL Probe和3.2 µL水，配置好混合物。(2) The RPA kit was purchased from twist, and the mixture was prepared according to the method in the supporting instructions, 29.5 µL Rehydrationbuffer, 2.1 µL Primer-F, 2.1 µL Primer-R, 0.6 µL Probe and 3.2 µL water.
（3）在混合物中加入10 copies/mL HIV-1 DNA模板10 µL，2.5 µL醋酸镁充分混匀，得反应物。(3) Add 10 µL of 10 copies/mL HIV-1 DNA template to the mixture, 2.5 µL of magnesium acetate and mix thoroughly to obtain a reaction product.
（4）在冰上将反应物打入芯片4中，放到检测系统中，CCD工业相机 8拍照记录检测区域初始反应的荧光强度。(4) Put the reactant into the chip 4 on ice and put it into the detection system, and the CCD industrial camera 8 takes pictures and records the fluorescence intensity of the initial reaction in the detection area.
（5）将芯片镶嵌在手表中，配戴在人体的手腕处，反应24分钟后，取出芯片置于检测系统中拍照记录。(5) Embed the chip in the watch and wear it on the wrist of the human body. After 24 minutes of reaction, take out the chip and put it in the detection system to take pictures and record it.
（6）将（2）中的模板分别换成模板浓度为105 - 102 copies/m，重复(2)-(5)。(6) Replace the templates in (2) with template concentrations of 105 - 102 copies/m, and repeat (2)-(5).
（7）重复(2) - (6) 三次。(7) Repeat (2) - (6) three times.
（8）ImageJ分析检测区域2-3的绿色荧光强度，根据反应结束后的荧光强度建立标准曲线。(8) ImageJ analyzed the green fluorescence intensity of the detection area 2-3, and established a standard curve based on the fluorescence intensity after the reaction.
（9）阴性对照组使用RNA-free水代替核酸模板，重复(4) - (5)。标准曲线如图4。(9) For the negative control group, use RNA-free water instead of the nucleic acid template, and repeat (4) - (5). The standard curve is shown in Figure 4.
实施例3，核酸扩增检测，参照附图4。Embodiment 3, nucleic acid amplification detection, refer to accompanying drawing 4.
1）在PDMS芯片1中打入50 µL水，放置在加热片上4，插入热电偶测通道中水的温度，调节加热温度，使水的温度达到33~34 ˚C,模仿体温。1) Pour 50 µL of water into the PDMS chip 1, place it on the heating plate 4, insert a thermocouple to measure the temperature of the water in the channel, and adjust the heating temperature so that the temperature of the water reaches 33~34 ˚C, imitating the body temperature.
2）将HIV-1 DNA 质粒浓度从109 copies/mL开始按1:10开始稀释，得到不同的浓度梯度。2) Dilute the HIV-1 DNA plasmid concentration from 109 copies/mL to 1:10 to obtain different concentration gradients.
2）根据RPA试剂说明书的方法，29.5 µL Rehydration buffer, 2.1 µLPrimer-F,2.1 µL Primer-R, 0.6 µL Probe和3.2 µL水，配置好混合物。2) According to the method in the RPA reagent manual, 29.5 µL Rehydration buffer, 2.1 µL Primer-F, 2.1 µL Primer-R, 0.6 µL Probe and 3.2 µL water were used to prepare the mixture.
3）加入10 copies/mL的HIV-1 DNA模板10 µL，醋酸镁2.5 µL，充分混匀，得反应物。3) Add 10 µL of 10 copies/mL HIV-1 DNA template, 2.5 µL of magnesium acetate, and mix thoroughly to obtain a reaction product.
4）在冰上将反应物打入芯片1中，放到检测系统中，CCD工业相机 8拍照记录检测区域初始反应的荧光强度。4) Put the reactant into the chip 1 on ice, put it into the detection system, and take pictures with the CCD industrial camera 8 to record the fluorescence intensity of the initial reaction in the detection area.
5）加热片4开始加热，工业相机 8每隔三分钟记录一次扩增图像，共统计到30分钟。5) The heating plate 4 starts to heat, and the industrial camera 8 records the amplification image every three minutes, and the total counts up to 30 minutes.
6）浓度为105~102 copies/mL HIV-1 DNA作为扩增模板，重复步骤6）~8）。6) Use HIV-1 DNA at a concentration of 105 ~102 copies/mL as the amplification template, and repeat steps 6) to 8).
7）重复5）~9）三次。7) Repeat 5)~9) three times.
8）ImageJ分析检测区域2-3的荧光强度，建立实时荧光扩增曲线。检测结果如图3。8) ImageJ analyzes the fluorescence intensity of the detection area 2-3, and establishes a real-time fluorescence amplification curve. The test results are shown in Figure 3.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710248640.8ACN106979940A (en) | 2017-04-17 | 2017-04-17 | A kind of device and method for carrying out nucleic acid amplification detection |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710248640.8ACN106979940A (en) | 2017-04-17 | 2017-04-17 | A kind of device and method for carrying out nucleic acid amplification detection |
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| CN106979940Atrue CN106979940A (en) | 2017-07-25 |
| Application Number | Title | Priority Date | Filing Date |
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| CN201710248640.8APendingCN106979940A (en) | 2017-04-17 | 2017-04-17 | A kind of device and method for carrying out nucleic acid amplification detection |
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| CN (1) | CN106979940A (en) |
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