WO2023186084A1 - Chip device and instrument for nucleic acid detection, and application thereof - Google Patents
Chip device and instrument for nucleic acid detection, and application thereof Download PDFInfo
- Publication number
- WO2023186084A1 WO2023186084A1 PCT/CN2023/085437 CN2023085437W WO2023186084A1 WO 2023186084 A1 WO2023186084 A1 WO 2023186084A1 CN 2023085437 W CN2023085437 W CN 2023085437W WO 2023186084 A1 WO2023186084 A1 WO 2023186084A1
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- chamber
- sample
- nucleic acid
- amplification reaction
- chip device
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Definitions
- the pressurized chamber increases the air pressure in the chamber through thermal expansion.
- thermal expansion generally refers to the phenomenon that the length or volume of a solid or liquid material changes due to a change in temperature when the pressure remains constant.
- the thermal expansion may be in the form of physical thermal expansion pressurization or chemical thermal expansion pressurization.
- the nucleic acid amplification reaction of the method is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
- LAMP loop-mediated DNA isothermal amplification
- FIG. 2 is a schematic diagram of an exemplary chip device for detecting nucleic acids in samples provided by the present invention, configuring reagents and working after adding samples.
- FIG. 2A is a schematic cross-sectional view of an exemplary chip device for nucleic acid detection in samples provided by the present invention after reagents are configured therein.
- Figure 2B is a schematic diagram of the operation of the exemplary chip device after adding a sample.
- the invention provides a chip device for detecting nucleic acids in samples.
- the chip device is usually used to detect the presence and amount of nucleic acid in a sample after extracting and amplifying it.
- the chip device for nucleic acid detection in samples provided by the present invention includes a substrate 1, a sample chamber 2 arranged vertically on the substrate, and a pressurized chamber 3 arranged parallel to the sample chamber 2, and An amplification reaction chamber 4 is provided in the chip device substrate.
- the sample chamber 2 and the pressurizing chamber 3 are cylindrical chambers surrounded by walls perpendicular to the bottom plate.
- the sample chamber 2 and the pressurizing chamber 3 are connected through an air flow channel 5 .
- the sample chamber 2 is connected to the amplification reaction chamber 4 through the liquid flow channel 6 provided in the substrate below it.
- the bottom of the sample chamber 2 has an opening communicating with the liquid flow channel 6 provided in the bottom plate.
- the sample chamber 2 is used to accommodate the sample to be detected and to separate nucleic acids in the sample.
- the top of the sample chamber has an openable or closable cover, such as a nut 21 as shown in FIG. 1 , which has internal threads and is matched with external threads outside the sample chamber.
- the cover of the sample chamber is a gland, which has a handle fixedly connected to the sample chamber.
- the chip device for nucleic acid detection in this embodiment is particularly suitable for nucleic acid extraction from samples using the direct extraction method, that is, the solution obtained after the sample contacts and reacts with the nucleic acid extraction reagent contains nucleic acids that can be used for subsequent amplification reactions.
- Samples suitable for nucleic acid extraction and amplification using the direct extraction method include synthetic clones, in vitro transcribed RNA, plasmids, serum, plasma, urine, cotton swab eluates, sputum, alveolar lavage fluid and other samples .
- Nucleic acid extraction reagents used to extract the above samples usually contain lysis reagents, including various surfactants such as SDS, Triton, NP-40, etc., as well as other chemical reagents such as buffers, protease inhibitors, reducing agents, etc., as well as various lysis agents.
- lysis reagents including various surfactants such as SDS, Triton, NP-40, etc., as well as other chemical reagents such as buffers, protease inhibitors, reducing agents, etc., as well as various lysis agents.
- the main functions of the nucleic acid extraction reagent are: (1) using detergents to destroy lipid bilayers and rupture cells; (2) dissolve proteins; (3) promote protein denaturation; (4) inhibit proteases and nucleases. active.
- Commercially available direct-extraction nucleic acid extraction reagents include sample release reagent (model S1014) provided by China Shengxiang Biotechnology Co., Ltd.
- the amount of gas generated in the pressurized chamber 3 is the amount of original gas (i.e., volume) in the sample chamber 2 and the pressurized chamber 3, usually excluding the volume of the nucleic acid processing solution added to the sample chamber. ) about 1-50 times, preferably about 5-20 times.
- the air pressure in the pressurizing chamber 3 can be increased through thermal expansion, including physical thermal expansion pressurization or chemical thermal expansion pressurization.
- a filter element for filtering undesired substances (such as acidic gas or liquid) of the gas is provided in the air flow channel 5 or at its opening with the sample chamber 2 or the pressurized chamber 3 .
- the filter element is a filter membrane 51 disposed at the opening of the air flow channel and the pressurizing chamber 3 .
- the filter element is a filter column disposed in the air flow channel. More preferably, the shapes of the air flow channel and the filter column are set to gradually shrink from one end of the pressurizing chamber to one end of the sample chamber. This prevents the filter column from moving in the air flow path due to gas pressure.
- the filter element is made of filter material that can absorb acidic and/or alkaline substances.
- the workflow of the chip device for nucleic acid detection in samples is: open the cover of the sample chamber, put the sample to be tested (such as a throat swab), and A nucleic acid separation reaction is performed in the sample preservation solution, and the nucleic acid is released into the liquid; the pressurized chamber 3 is heated, and the heating causes the separation medium 81/83 to melt, and the first reactant 83 (for example, sulfuric acid) and the second reactant 84 (for example, sodium bicarbonate) contacts and reacts, and generates gas (such as carbon dioxide).
- the first reactant 83 for example, sulfuric acid
- the second reactant 84 for example, sodium bicarbonate
- the amplified nucleic acid is detected by detecting a fluorescent signal carried by the nucleic acid.
- the primers and oligonucleotides contained in the reaction system of the amplification reaction chamber can be radioactive, fluorescent or non-radioactive detectably labeled by methods well known to those skilled in the art. Commonly used fluorescent dyes and their signal-related wavelengths are shown in Table 1 below.
- a nucleic acid detection chip as shown in Figure 1 was prepared.
- the thickness of substrate 1 is about 2.0mm;
- the cavity volume of sample chamber 2 is about 1.6ml, and the inner diameter is about 7.0mm;
- the cavity volume of booster chamber 3 is about 1.2ml, and the inner diameter is about 7.0mm.
- the size of the cross-section of the liquid flow channel is approximately 0.4mm x 0.5mm.
- sample release agent (Changzhou Jinmag Biotechnology Co., Ltd., Su Chang Mechanical Preparation No. 20200123) into the sample chamber.
- the testing principle of the sample release agent is to use protein denaturants and biochemical reagents to quickly destroy the virus capsid structure in the throat swab sample, thereby causing the virus to cleave and release the nucleic acid; at the same time, the nucleic acid contained is stable
- the agent can effectively prevent the degradation of nucleic acids, thereby achieving the function of extracting and preserving nucleic acids.
- Applicable specimen types throat swab samples or body fluid samples.
- the reagents provided by Beijing Biolab Technology Co., Ltd.'s SARS-CoV-2 novel coronavirus RT-LAMP kit (N gene) were used as test reagents, including SARS-CoV-2N gene RT-LAMP positive control.
- the SARS-CoV-2N gene RT-LAMP primer mixture and 2 ⁇ LAMP MagicMix in the above kit are processed to form a freeze-dried powder and added to the amplification reaction chamber.
- the pressurized chamber is heated to about 70°C, the paraffin melts, HCl and NaHCO react to generate CO gas .
- the sample preservation solution is squeezed into the amplification reaction chamber through the flow channel. Reconstitute the amplification raw materials pre-placed in the amplification reaction chamber.
- a nasopharyngeal swab without SARS nucleic acid adsorption was added to another chip as a control.
- a nasopharyngeal swab without SARS nucleic acid adsorption was added to another chip as a control, and the remaining steps were the same as in the experimental group.
- the instrument has a chip device receiving and motion control system for receiving the chip device and performing various treatments on the chip, including heating treatment.
- the instrument may also have a signal detection module for detecting nucleic acid amplification products, such as a fluorescence detection system.
- the instrument has a system for temperature controlling the nucleic acid amplification area of the chip.
- the instrument has a nucleic acid amplification result analysis and/or output system.
Abstract
Provided is a chip device for nucleic acid detection of a sample, said device being provided with a substrate, a sample chamber, and a pressurization chamber, as well as an amplification reaction chamber arranged in the substrate. Further provided is an instrument for nucleic acid detection of a sample by utilizing the chip device, and in particular a POCT instrument. Further provided is a method for nucleic acid detection of a sample by utilizing the chip device or instrument.
Description
本申请要求以下中国专利申请的优先权：2022年3月31日提交的、申请号为202210329941.4、发明名称为“检测核酸的芯片装置和仪器以及其应用”，其全部内容通过引用结合在本申请中。This application claims priority to the following Chinese patent application: submitted on March 31, 2022, with application number 202210329941.4, and the invention title is "Chip device and instrument for detecting nucleic acids and applications thereof", the entire content of which is incorporated into this application by reference. middle.
本发明涉及生物技术和设备应用领域，具体涉及一种用于检测样品中核酸的芯片装置和仪器，以及其在生物样品检测中的应用。The present invention relates to the field of biotechnology and equipment applications, and in particular to a chip device and instrument for detecting nucleic acids in samples, and its application in biological sample detection.
核酸的检测是临床试验、病原微生物类型鉴定等诸多领域的核心。通过核酸提取、扩增和检测分析，能够检测诸如癌症、微生物感染和基因标记等各种疾病。The detection of nucleic acids is the core of many fields such as clinical trials and identification of pathogenic microorganism types. Through nucleic acid extraction, amplification and detection analysis, various diseases such as cancer, microbial infections and genetic markers can be detected.
采用利用PCR和实时PCR等方法是对基因进行呈指数地扩增和检测的有效方法。使用PCR/实时PCR装置的基因检测市场在诸如病毒性、性传播疾病和流感等传染性疾病的基因检测迅速展开。基因检测对于癌症治疗的作用已变得明显。然而，采用PCR和RT-PCR的应用的自动化，特别是小型自动化并不容易。Using methods such as PCR and real-time PCR is an effective method to exponentially amplify and detect genes. The genetic testing market using PCR/real-time PCR devices is rapidly expanding in genetic testing for infectious diseases such as viruses, sexually transmitted diseases, and influenza. The role of genetic testing in cancer treatment is becoming clear. However, automation of applications employing PCR and RT-PCR, especially small-scale automation, is not easy.
因此，本领域还需要高速实时、小型和方便操作的核酸扩增和检测装置和仪器，特别是适合用于现场快速检验(point-of-care testing,POCT)的芯片装置和仪器。Therefore, this field also needs high-speed, real-time, small and easy-to-operate nucleic acid amplification and detection devices and instruments, especially chip devices and instruments suitable for rapid on-site testing (point-of-care testing, POCT).
发明内容Contents of the invention
本发明提供了一种用于样品中核酸检测的芯片装置，其具有基板，样品腔和增压腔，以及设置在所述基板内的扩增反应腔，其中，所述样品腔和增压腔之间通过空气流道连通，以及样品腔通过设置在基板内的液体流
道与所述扩增反应腔连通，The invention provides a chip device for nucleic acid detection in samples, which has a substrate, a sample chamber and a pressurizing chamber, and an amplification reaction chamber arranged in the substrate, wherein the sample chamber and the pressurizing chamber are connected through an air flow channel, and the sample chamber is connected through a liquid flow set in the substrate. The channel is connected with the amplification reaction chamber,
其中，in,
所述样品腔用于容纳待检测样品以及将样品中的核酸分离，The sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample,
所述增压腔用于使得腔体内气体压力增大，从而将样品腔中的溶液通过液体流道挤压到扩增反应腔中，The pressurized chamber is used to increase the gas pressure in the chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
所述扩增反应腔用于溶液中的核酸进行扩增反应。The amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
在本发明的其中一个方面，所述所述芯片装置的扩增反应腔包括多个核酸扩增单元，例如2-24个。In one aspect of the present invention, the amplification reaction chamber of the chip device includes a plurality of nucleic acid amplification units, for example, 2-24 units.
在本发明的其中一个方面，所述样品腔的顶部具有可打开或封闭的盖。在本发明的其中又一个方面，所述盖为螺帽，其具有内螺纹，与样品腔外部的外螺纹形成配合。In one aspect of the invention, the top of the sample chamber has an openable or closable cover. In yet another aspect of the present invention, the cover is a nut having internal threads that cooperate with external threads outside the sample chamber.
在本发明的其中一个方面，所述样品腔中可加入或预置用于保存和提取样品中的核酸的试剂溶液。In one aspect of the present invention, a reagent solution for preserving and extracting nucleic acids in the sample can be added to or preset in the sample chamber.
在本发明的其中一个方面，所述试剂溶液为采用直提法对样品进行核酸提取的试剂。In one aspect of the present invention, the reagent solution is a reagent for nucleic acid extraction from samples using a direct extraction method.
在本发明的其中一个方面，所述增压腔通过化学方法在其腔体内产生气体。在本发明的其中一个方面，所述化学方法为产生气体的化学反应，例如酸碱反应等。在本发明的其中一个方面，所述气体可以为二氧化碳、氢气、氧气等，优选的，所述气体为在核酸检测反应中没有活性(反应惰性)的气体，例如二氧化碳等。在本发明的一种实施方式中，可用于产生二氧化碳的化学反应包括采用碳酸盐或碳酸氢盐等盐类与酸的反应。In one aspect of the invention, the pressurized chamber generates gas in its chamber through chemical methods. In one aspect of the present invention, the chemical method is a chemical reaction that generates gas, such as an acid-base reaction. In one aspect of the present invention, the gas may be carbon dioxide, hydrogen, oxygen, etc. Preferably, the gas is a gas that is inactive (reactive inert) in the nucleic acid detection reaction, such as carbon dioxide, etc. In one embodiment of the present invention, chemical reactions that can be used to produce carbon dioxide include the reaction of salts such as carbonates or bicarbonates with acids.
在本发明的其中又一个方面，在增压腔产生的气体的量为样品腔和增压腔中原有的气体的量的约1-50倍，例如为约5-20倍。在本发明的其中又一个方面，所述增压腔内设置隔档层。In yet another aspect of the present invention, the amount of gas generated in the pressurized chamber is about 1-50 times, for example, about 5-20 times the amount of original gas in the sample chamber and the pressurized chamber. In yet another aspect of the present invention, a barrier layer is provided in the pressurizing chamber.
在本发明的其中一个方面，所述增压腔通过热膨胀的方式增加其腔体内的气压。在本发明中，热膨胀一般是指压力保持不变时，由于温度的改变，造成固体或液体材料发生长度或体积变化的现象。在本发明的其中一个方面，热膨胀可为物理热膨胀增压或化学热膨胀增压的方式。In one aspect of the present invention, the pressurized chamber increases the air pressure in the chamber through thermal expansion. In the present invention, thermal expansion generally refers to the phenomenon that the length or volume of a solid or liquid material changes due to a change in temperature when the pressure remains constant. In one aspect of the invention, the thermal expansion may be in the form of physical thermal expansion pressurization or chemical thermal expansion pressurization.
在本发明的其中一个方面，所述空气流道中或其与样品腔或增压腔的
开口处设置过滤件。例如为设置在空气流道与增压腔的开口处的滤片膜，或为设置在空气流道内的滤柱。In one aspect of the present invention, in the air flow channel or between it and the sample chamber or the pressurized chamber A filter is installed at the opening. For example, it is a filter membrane disposed at the opening of the air flow channel and the pressurization chamber, or a filter column disposed in the air flow channel.
在本发明的其中一个方面，所述液体流道中可设置过滤腔。In one aspect of the present invention, a filter cavity may be provided in the liquid flow channel.
在本发明的其中一个方面，扩增反应腔内可预置核酸扩增反应的材料。In one aspect of the present invention, materials for nucleic acid amplification reaction can be preset in the amplification reaction chamber.
在本发明中，所述核酸扩增反应为变温的或等温的扩增方法，如聚合酶链反应(PCR)、链置换扩增(SDA)、基于核酸序列的扩增(NASBA)、级联滚环扩增(CRCA)、环介导的DNA等温扩增(LAMP)、等温和嵌合引物-起始的核酸扩增(ICAN)、基于靶的解旋酶依赖性扩增(HDA)、转录介导的扩增(TMA)等。In the present invention, the nucleic acid amplification reaction is a variable temperature or isothermal amplification method, such as polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), cascade Rolling circle amplification (CRCA), circle-mediated DNA isothermal amplification (LAMP), isothermal chimeric primer-initiated nucleic acid amplification (ICAN), target-based helicase-dependent amplification (HDA), Transcription-mediated amplification (TMA), etc.
在本发明的其中一个方面，所述核酸扩增反应为等温的扩增方法，例如为环介导的DNA等温扩增(LAMP)。In one aspect of the invention, the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
在本发明的其中一个方面，所述扩增反应腔的下游还具有分压腔，所述分压腔设置在芯片的基板内，通过分压气体通道与扩增反应腔连通。在本发明的其中又一个方面，所述分压气体通道与在扩增反应腔和分压腔连接的开口设置在扩增反应腔和分压腔的顶部位置。In one aspect of the present invention, there is a pressure dividing chamber downstream of the amplification reaction chamber. The pressure dividing chamber is arranged in the substrate of the chip and communicates with the amplification reaction chamber through a pressure dividing gas channel. In yet another aspect of the present invention, the opening connecting the partial pressure gas channel to the amplification reaction chamber and the pressure partial pressure chamber is disposed at the top of the amplification reaction chamber and the pressure partial pressure chamber.
本发明还提供了一种用于样品中核酸检测的仪器，例如为POCT仪器)，其包括前述本发明的芯片装置，其中所述芯片装置具有基板，样品腔和增压腔，以及设置在所述基板内的扩增反应腔，其中，所述样品腔和增压腔之间通过空气流道连通，以及样品腔通过设置在基板内的液体流道与所述扩增反应腔连通，The present invention also provides an instrument for nucleic acid detection in samples, such as a POCT instrument, which includes the aforementioned chip device of the present invention, wherein the chip device has a substrate, a sample chamber and a pressurized chamber, and is disposed on the The amplification reaction chamber in the substrate, wherein the sample chamber and the pressurizing chamber are connected through an air flow channel, and the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the base plate,
其中，in,
所述样品腔用于容纳待检测样品以及将样品中的核酸分离，The sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample,
所述增压腔用于使得腔体内气体压力增大，从而将样品腔中的溶液通过液体流道挤压到扩增反应腔中，The pressurized chamber is used to increase the gas pressure in the chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
所述扩增反应腔用于溶液中的核酸进行扩增反应。The amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
在本发明的其中又一个方面，所述仪器具有芯片装置接收系统。In yet another aspect of the invention, the instrument has a chip device receiving system.
在本发明的其中又一个方面，所述仪器具有检测核酸扩增产物的信号检测模块装置，例如为荧光检测装置。
In yet another aspect of the present invention, the instrument has a signal detection module device for detecting nucleic acid amplification products, such as a fluorescence detection device.
在本发明的其中又一个方面，所述仪器具有对所述芯片的核酸扩增区域进行温控的系统。In yet another aspect of the present invention, the instrument has a system for temperature controlling the nucleic acid amplification area of the chip.
在本发明的其中又一个方面，所述仪器具有核酸扩增结果分析和/或输出系统。In yet another aspect of the present invention, the instrument has a nucleic acid amplification result analysis and/or output system.
本发明还提供了一种用于样品中核酸检测的方法，其包括采用前述本发明的芯片装置或前述本发明的仪器，其中所述芯片装置具有基板，样品腔和增压腔，以及设置在所述基板内的扩增反应腔，其中，所述样品腔和增压腔之间通过空气流道连通，以及样品腔通过设置在基板内的液体流道与所述扩增反应腔连通，The present invention also provides a method for nucleic acid detection in a sample, which includes using the aforementioned chip device of the present invention or the aforementioned instrument of the present invention, wherein the chip device has a substrate, a sample chamber and a pressurized chamber, and is disposed in The amplification reaction chamber in the substrate, wherein the sample chamber and the pressurizing chamber are connected through an air flow channel, and the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the base plate,
其中，in,
所述样品腔用于容纳待检测样品以及将样品中的核酸分离，The sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample,
所述增压腔用于使得腔体内气体压力增大，从而将样品腔中的溶液通过液体流道挤压到扩增反应腔中，The pressurized chamber is used to increase the gas pressure in the chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
所述扩增反应腔用于溶液中的核酸进行扩增反应。The amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
在本发明的其中又一个方面，所述方法包括以下步骤：In yet another aspect of the invention, the method includes the following steps:
(1).将样品加入到所述芯片装置的样品腔中，使样品中的核酸分离和进入到溶液中；(1). Add the sample into the sample chamber of the chip device to separate the nucleic acids in the sample and enter the solution;
(2).使得所述增压腔的腔体内气体压力增大，将样品腔中的溶液通过液体流道挤压到扩增反应腔中；以及(2). Increase the gas pressure in the pressurized chamber and squeeze the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel; and
(3).在扩增反应腔对溶液中的核酸进行扩增反应。(3). Perform amplification reaction on the nucleic acid in the solution in the amplification reaction chamber.
在本发明的其中一个方面，所述方法中在所述增压腔的腔体内通过化学方法产生气体。优选的，在增压腔产生的气体的量为样品腔和增压腔中原有的气体的量的约1-50倍，例如为约5-20倍。In one aspect of the present invention, the method includes chemically generating gas within the cavity of the pressurized chamber. Preferably, the amount of gas generated in the pressurized chamber is about 1-50 times the amount of original gas in the sample chamber and the pressurized chamber, for example, about 5-20 times.
在本发明的其中一个方面，所述方法中在所述增压腔的腔体内通过热膨胀的方式增加气压，例如为物理热膨胀增压或化学热膨胀增压的方式。In one aspect of the present invention, in the method, the air pressure is increased in the cavity of the pressurizing chamber through thermal expansion, such as physical thermal expansion pressurization or chemical thermal expansion pressurization.
在本发明的其中一个方面，所述方法的核酸扩增反应为等温的扩增方法，例如为环介导的DNA等温扩增(LAMP)。In one aspect of the present invention, the nucleic acid amplification reaction of the method is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
在本发明的其中一个方面，所述方法中对扩增的核酸携带的可识别的标记进行检测，包括但不限于荧光或其它形式的(例如化学发光，生物发
光，辐射发光，电发光，电化学发光，机械发光，结晶发光，热致发光，声致发光，磷光和光致发光等)发光，酶促反应，放射性等。In one aspect of the invention, in the method, identifiable labels carried by the amplified nucleic acids are detected, including but not limited to fluorescence or other forms (such as chemiluminescence, bioluminescence). Light, radioluminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallographic luminescence, thermoluminescence, sonoluminescence, phosphorescence and photoluminescence, etc.) luminescence, enzymatic reactions, radioactivity, etc.
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.
图1为一种示例性的本发明提供的用于样品中核酸检测的芯片装置的立体透视图。Figure 1 is a three-dimensional perspective view of an exemplary chip device for detecting nucleic acids in samples provided by the present invention.
图2为示例性的本发明提供的用于样品中核酸检测的芯片装置配置试剂和加入样品后工作的示意图。其中图2A为示例性的本发明提供的用于样品中核酸检测的芯片装置配置试剂后的剖面示意图。图2B为所述示例性的芯片装置加入样品后的工作示意图。Figure 2 is a schematic diagram of an exemplary chip device for detecting nucleic acids in samples provided by the present invention, configuring reagents and working after adding samples. FIG. 2A is a schematic cross-sectional view of an exemplary chip device for nucleic acid detection in samples provided by the present invention after reagents are configured therein. Figure 2B is a schematic diagram of the operation of the exemplary chip device after adding a sample.
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的区间。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.
实施例1Example 1
本发明提供了一种用于样品中核酸检测的芯片装置。所述芯片装置通常用于对样品中的核酸进行提取和扩增后检测其是否存在和存在的量。The invention provides a chip device for detecting nucleic acids in samples. The chip device is usually used to detect the presence and amount of nucleic acid in a sample after extracting and amplifying it.
本文中所描述的芯片装置应用于分析任何用途所用的任何含有核酸的样本，包括但不限于用于人类基因的遗传测试和各种传染性疾病的临床测试。应用于本文所述方法的核酸样品可来自于任何来源。通常，样品可以是与其天然环境所相分离并包含多核苷酸的生物材料。样品可由纯化的
或分离的多核苷酸组成，或可包含生物样品例如包含多核苷酸的组织样品、生物流体样品或细胞样品。生物流体包括作为非限制性例子的血液、血浆、痰液、尿、脑脊液、灌洗液样品。核酸样品可来自于植物、动物、细菌或病毒来源。样品可获得自不同来源，包括但不限于来自不同个体、相同或不同个体的不同发育阶段、不同患病个体、正常个体、相同或不同个体的不同疾病阶段、进行了不同疾病处理的个体、处于不同环境因素的个体、或具有易患病体质的个体、或暴露于传染性疾病介质的个体的样品。The chip device described herein should be used to analyze any nucleic acid-containing sample for any purpose, including but not limited to genetic testing of human genes and clinical testing of various infectious diseases. Nucleic acid samples used in the methods described herein can be from any source. Typically, a sample may be biological material isolated from its natural environment and containing polynucleotides. Samples can be purified from or consisting of an isolated polynucleotide, or may comprise a biological sample such as a tissue sample, biological fluid sample or cell sample containing a polynucleotide. Biological fluids include, as non-limiting examples, blood, plasma, sputum, urine, cerebrospinal fluid, and lavage fluid samples. Nucleic acid samples can be from plant, animal, bacterial or viral sources. Samples may be obtained from different sources, including but not limited to, from different individuals, different developmental stages of the same or different individuals, different diseased individuals, normal individuals, different disease stages of the same or different individuals, individuals treated for different diseases, individuals under Samples from individuals with different environmental factors, individuals with predisposition to disease, or individuals exposed to infectious disease agents.
图1为一种示例性的本发明提供的用于样品中核酸检测的芯片装置的立体透视图。图2为示例性的本发明提供的用于样品中核酸检测的芯片装置配置试剂和加入样品后工作的示意图。其中图2A为示例性的本发明提供的用于样品中核酸检测的芯片装置配置试剂后的剖面示意图。图2B为所述示例性的芯片装置加入样品后的工作示意图。Figure 1 is a three-dimensional perspective view of an exemplary chip device for detecting nucleic acids in samples provided by the present invention. Figure 2 is a schematic diagram of an exemplary chip device for detecting nucleic acids in samples provided by the present invention, configuring reagents and working after adding samples. FIG. 2A is a schematic cross-sectional view of an exemplary chip device for nucleic acid detection in samples provided by the present invention after reagents are configured therein. Figure 2B is a schematic diagram of the operation of the exemplary chip device after adding a sample.
如图1和图2所示，本发明提供的用于样品中核酸检测的芯片装置包括具有基板1，垂直设置在基板上的样品腔2和与样品腔2平行设置的增压腔3，以及设置在芯片装置基板内的扩增反应腔4。样品腔2和增压腔3为圆柱状的腔体，由与所述底板垂直的壁围成。样品腔2和增压腔3之间通过空气流道5连通。样品腔2通过设置在其下方的基板内的液体流道6与所述扩增反应腔4连通。样品腔2底部具有与设置在所述底板中的液体流道6相通的开孔。As shown in Figures 1 and 2, the chip device for nucleic acid detection in samples provided by the present invention includes a substrate 1, a sample chamber 2 arranged vertically on the substrate, and a pressurized chamber 3 arranged parallel to the sample chamber 2, and An amplification reaction chamber 4 is provided in the chip device substrate. The sample chamber 2 and the pressurizing chamber 3 are cylindrical chambers surrounded by walls perpendicular to the bottom plate. The sample chamber 2 and the pressurizing chamber 3 are connected through an air flow channel 5 . The sample chamber 2 is connected to the amplification reaction chamber 4 through the liquid flow channel 6 provided in the substrate below it. The bottom of the sample chamber 2 has an opening communicating with the liquid flow channel 6 provided in the bottom plate.
所述扩增反应腔4设置在芯片的基板内，其可容纳用于核酸扩增反应的反应物或反应体系，以及来自样品腔的包含已分离的核酸溶液，并且可以在合适的条件下进行扩增反应。在本发明的其它方面，所述芯片装置的扩增反应腔包括多个核酸扩增单元，例如2-24个。The amplification reaction chamber 4 is arranged in the substrate of the chip, which can accommodate reactants or reaction systems for nucleic acid amplification reactions, as well as solutions containing separated nucleic acids from the sample chamber, and can be performed under appropriate conditions. amplification reaction. In other aspects of the invention, the amplification reaction chamber of the chip device includes a plurality of nucleic acid amplification units, for example, 2-24 units.
样品腔2用于容纳待检测样品以及将样品中的核酸分离。所述样品腔的顶部具有可打开或封闭的盖，例如为如图1所示的螺帽21，其具有内螺纹，与样品腔外部的外螺纹形成配合。在另一种实施方式中，所述样品腔的盖为压盖，其具有与样品腔固定连接的柄。The sample chamber 2 is used to accommodate the sample to be detected and to separate nucleic acids in the sample. The top of the sample chamber has an openable or closable cover, such as a nut 21 as shown in FIG. 1 , which has internal threads and is matched with external threads outside the sample chamber. In another embodiment, the cover of the sample chamber is a gland, which has a handle fixedly connected to the sample chamber.
在需要加入样品时，可将所述样品腔的盖打开，将样品加入到样品腔后用盖封闭，避免生物样本的泄漏和由此造成的污染。
When it is necessary to add a sample, the cover of the sample chamber can be opened, and the sample is added to the sample chamber and closed with the cover to avoid leakage of biological samples and resulting contamination.
样品腔中可加入或预置用于保存(避免待测核酸发生不希望的分解)和提取样品中的核酸的试剂溶液。A reagent solution for preserving (to avoid unwanted decomposition of the nucleic acid to be tested) and extracting the nucleic acid in the sample can be added or preset in the sample chamber.
本实施例中的核酸检测的芯片装置特别适于采用直提法对样品进行核酸提取，即样本与核酸提取试剂接触和反应后获得的溶液中含有可用于后续扩增反应的核酸。适合采用直提法进行核酸提取和扩增的样品包括人工合成的克隆菌液、体外转录RNA、质粒，血清、血浆、尿液、棉拭子洗脱物、痰液、肺泡灌洗液等样本。用于提取上述样本的核酸提取试剂通常含有裂解剂，包括各种表面活性剂如SDS、Triton、NP-40等，以及其它化学试剂如缓冲剂、蛋白酶抑制剂、还原剂等，以及各种裂解细胞壁或细胞膜中成分的酶，例如Labiase裂解酶、溶葡球菌酶、鸡蛋蛋白来源溶菌酶、人源溶菌酶、消色肽酶、球孢链霉菌源变溶菌素、几丁质酶、立枯丝核菌源裂解酶、藤黄节杆菌源裂解酶、哈茨木霉源裂解酶、酿脓链球菌源链球菌溶血素O、破伤风杆菌源破伤风菌溶血素等。所述核酸提取试剂的主要要功能为：(1)利用去污剂破坏脂质双分子层，破裂细胞；(2)溶解蛋白；(3)促进蛋白变性；(4)抑制蛋白酶和核酸酶的活性。可商购的直提式核酸提取试剂例如中国圣湘生物科技股份有限公司提供的样本释放剂(型号S1014)。The chip device for nucleic acid detection in this embodiment is particularly suitable for nucleic acid extraction from samples using the direct extraction method, that is, the solution obtained after the sample contacts and reacts with the nucleic acid extraction reagent contains nucleic acids that can be used for subsequent amplification reactions. Samples suitable for nucleic acid extraction and amplification using the direct extraction method include synthetic clones, in vitro transcribed RNA, plasmids, serum, plasma, urine, cotton swab eluates, sputum, alveolar lavage fluid and other samples . Nucleic acid extraction reagents used to extract the above samples usually contain lysis reagents, including various surfactants such as SDS, Triton, NP-40, etc., as well as other chemical reagents such as buffers, protease inhibitors, reducing agents, etc., as well as various lysis agents. Enzymes that are components of the cell wall or cell membrane, such as Labiase lyase, lysostaphin, egg protein-derived lysozyme, human lysozyme, achromopeptidase, Streptomyces sporozoites-derived mutolysin, chitinase, and Rhizoctonia lytic enzyme, Arthrobacter luteus-derived lytic enzyme, Trichoderma harzianum-derived lytic enzyme, Streptococcus pyogenes-derived streptococcal hemolysin O, Tetanus tetanus-derived tetanus hemolysin, etc. The main functions of the nucleic acid extraction reagent are: (1) using detergents to destroy lipid bilayers and rupture cells; (2) dissolve proteins; (3) promote protein denaturation; (4) inhibit proteases and nucleases. active. Commercially available direct-extraction nucleic acid extraction reagents include sample release reagent (model S1014) provided by China Shengxiang Biotechnology Co., Ltd.
增压腔3用于产生气体并由此使得样品腔2和增压腔3形成的体系中的气体压力增大，从而将样品腔中的液体被通过液体流道6挤压到扩增反应腔4中。The pressurizing chamber 3 is used to generate gas and thereby increase the gas pressure in the system formed by the sample chamber 2 and the pressurizing chamber 3, so that the liquid in the sample chamber is squeezed into the amplification reaction chamber through the liquid flow channel 6 4 in.
样品腔2和增压腔3之间通过空气流道5连通。所述空气流道5与样品腔2和增压腔3相连的开口位于所述样品腔2和增压腔3的上部。空气流道的开口位置需高于加入/预置在样品腔2的核酸提取溶液的液面的上方，由此增压腔3提供的增大的气体压力可将样品腔中的液体挤压，并通过液体流道6转移至扩增反应腔4中。The sample chamber 2 and the pressurizing chamber 3 are connected through an air flow channel 5 . The opening connecting the air flow channel 5 to the sample chamber 2 and the pressurizing chamber 3 is located at the upper part of the sample chamber 2 and the pressurizing chamber 3 . The opening position of the air flow channel needs to be higher than the liquid level of the nucleic acid extraction solution added/preset in the sample chamber 2. The increased gas pressure provided by the booster chamber 3 can squeeze the liquid in the sample chamber. And transferred to the amplification reaction chamber 4 through the liquid flow channel 6 .
在本发明的其中一个方面，可通过化学方法在增压腔3产生气体。例如通过化学反应生成二氧化碳、氧气等气体，优选为对核酸提取反应和核酸分子的生物活性没有影响的惰性气体。例如为通过盐酸或硫酸等强酸与具有碳酸根或碳酸氢根的固体化合物反应产生二氧化碳。在本发明的其中
一个方面，在增压腔3内还设置隔档层。所述隔档层可供气体通过而对液体起部分阻挡作用。如果生成气体的化学反应过于剧烈，会导致液体被顶起，隔档层可用于限制液体升起过高。In one aspect of the invention, gas can be generated in the pressurized chamber 3 by chemical methods. For example, gases such as carbon dioxide and oxygen are generated through chemical reactions, and are preferably inert gases that have no effect on the nucleic acid extraction reaction and the biological activity of the nucleic acid molecules. For example, carbon dioxide is generated by reacting a strong acid such as hydrochloric acid or sulfuric acid with a solid compound having carbonate or bicarbonate radicals. among the present invention On the one hand, a barrier layer is also provided in the pressurizing chamber 3 . The barrier layer allows gas to pass through and acts as a partial barrier to liquid. Barriers can be used to limit the liquid from rising too high if the chemical reaction that produces the gas is too violent, causing the liquid to be lifted up.
在本发明的其中又一个方面，在增压腔3产生的气体的量为样品腔2和增压腔3原有的气体的量(即体积，通常不包括加入样品腔的核酸处理溶液的体积)的约1-50倍，优选为约5-20倍。In another aspect of the present invention, the amount of gas generated in the pressurized chamber 3 is the amount of original gas (i.e., volume) in the sample chamber 2 and the pressurized chamber 3, usually excluding the volume of the nucleic acid processing solution added to the sample chamber. ) about 1-50 times, preferably about 5-20 times.
在本发明的其中一个方面，可通过热膨胀的方式增加增压腔3内的气压，包括物理热膨胀增压或化学热膨胀增压的方式。In one aspect of the present invention, the air pressure in the pressurizing chamber 3 can be increased through thermal expansion, including physical thermal expansion pressurization or chemical thermal expansion pressurization.
在本发明的其中一个方面，在空气流道5中或其与样品腔2或增压腔3的开口处设置用于过滤气体的不期望的物质(例如酸性气体或液体)的过滤件。在一种实施方式中，所述过滤件为设置在空气流道与增压腔3的开口处的滤片膜51。在另一种实施方式中，所述过滤件为设置在空气流道内的滤柱，更优选的，所述空气流道和滤柱的形状设置为从增压腔一端向样品腔一端逐步缩小，由此可避免因气体压力导致滤柱在空气流道中的移动。在本发明中，所述过滤件采用可吸收酸性和/或碱性物质的过滤材料制备而得。In one aspect of the present invention, a filter element for filtering undesired substances (such as acidic gas or liquid) of the gas is provided in the air flow channel 5 or at its opening with the sample chamber 2 or the pressurized chamber 3 . In one embodiment, the filter element is a filter membrane 51 disposed at the opening of the air flow channel and the pressurizing chamber 3 . In another embodiment, the filter element is a filter column disposed in the air flow channel. More preferably, the shapes of the air flow channel and the filter column are set to gradually shrink from one end of the pressurizing chamber to one end of the sample chamber. This prevents the filter column from moving in the air flow path due to gas pressure. In the present invention, the filter element is made of filter material that can absorb acidic and/or alkaline substances.
在本发明的其中一个方面，在液体流道6中可设置用于过滤含有样品核酸的溶液中不需要的物质(例如细胞、细胞碎片或大蛋白质分子等)的过滤腔61。在本发明中，所述过滤腔内包含过滤材料，所述过滤材料的孔径可使得溶液中的核酸(包括基因组核酸或其片段等)自由通过，截留组织碎片、细胞和细胞碎片或大蛋白质分子等。在本发明中，过滤腔中采用的过滤材料对溶液中的核酸不产生物理吸附或基本上不产生物理吸附，且不与核酸发生反应或产生抑制。In one aspect of the present invention, a filter chamber 61 for filtering unwanted substances (such as cells, cell debris or large protein molecules, etc.) in a solution containing sample nucleic acid may be provided in the liquid flow channel 6 . In the present invention, the filter cavity contains filter material, and the pore size of the filter material can allow nucleic acids (including genomic nucleic acids or fragments thereof, etc.) in the solution to pass freely and intercept tissue fragments, cells and cell fragments or large protein molecules. wait. In the present invention, the filter material used in the filter chamber does not physically adsorb or substantially does not physically adsorb the nucleic acid in the solution, and does not react with or inhibit the nucleic acid.
在本发明提供的用于样品中核酸检测的芯片装置的扩增反应腔4内可进行核酸扩增反应。本领域已知的各种使用引物的核酸扩增方法均可用于本发明，包括变温的或等温的扩增方法，如聚合酶链反应(PCR)、链置换扩增(SDA)、基于核酸序列的扩增(NASBA)、级联滚环扩增(CRCA)、环介导的DNA等温扩增(LAMP)、等温和嵌合引物-起始的核酸扩增(ICAN)、基于靶的解旋酶依赖性扩增(HDA)、转录介导的扩增(TMA)等。
Nucleic acid amplification reactions can be performed in the amplification reaction chamber 4 of the chip device for nucleic acid detection in samples provided by the present invention. Various nucleic acid amplification methods using primers known in the art can be used in the present invention, including variable temperature or isothermal amplification methods, such as polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), cascade rolling circle amplification (CRCA), loop-mediated DNA isothermal amplification (LAMP), isothermal and chimeric primer-initiated nucleic acid amplification (ICAN), target-based unwinding Enzyme-dependent amplification (HDA), transcription-mediated amplification (TMA), etc.
在本发明的其中一种实施方式中，采用等温的扩增方法如LAMP。所述芯片装置还包括对所述扩增反应腔4的控温单元，例如具有温度调节器，使得扩增反应腔保持恒温。In one embodiment of the invention, an isothermal amplification method such as LAMP is used. The chip device also includes a temperature control unit for the amplification reaction chamber 4, such as a temperature regulator, so that the amplification reaction chamber maintains a constant temperature.
在本发明的其中一个方面，扩增反应腔4的下游(气体流动方向的下游)还具有分压腔41。所述分压腔设置在芯片的基板内，通过分压气体通道42与扩增反应腔4连通。分压腔一方面分担扩增腔中的空气被压缩时的压力，避免因为压力过高对芯片造成的不利影响，另一方面可避免扩增反应腔的面积过大，影响核酸扩增效率和信号观察。分压气体通道42与在扩增反应腔和分压腔连接的开口设置在扩增反应腔和分压腔的顶部位置。In one aspect of the present invention, there is also a partial pressure chamber 41 downstream of the amplification reaction chamber 4 (downstream in the gas flow direction). The pressure dividing chamber is arranged in the substrate of the chip and communicates with the amplification reaction chamber 4 through the pressure dividing gas channel 42 . On the one hand, the pressure dividing chamber shares the pressure when the air in the amplification chamber is compressed to avoid adverse effects on the chip due to excessive pressure. On the other hand, it prevents the area of the amplification reaction chamber from being too large, which affects the nucleic acid amplification efficiency and Signal observation. The opening connecting the partial pressure gas channel 42 to the amplification reaction chamber and the pressure partial pressure chamber is disposed at the top of the amplification reaction chamber and the pressure partial pressure chamber.
如图2A所示，在示例性的工作情形中，所述用于样品中核酸检测的芯片装置的初始状态为：所述样品腔2中预置样品保存液(也即为核酸提取液)71，样品保存液71的液面低于样品腔2和增压腔3之间的空气流道5；在所述增压腔3中预置在接触后产生化学反应生成气体的第一反应物83(例如为硫酸)和第二反应物84(例如为碳酸氢钠)，第一反应物83和第二反应物84之间具有在合适条件下可融化或分解的分隔介质82(如在较高温度下可融化的石蜡)。设置在增压腔3内的第一反应物和/或第二反应物的上方也可具有在合适条件下可融化或分解的分隔介质81。另外，在扩增反应腔4内预置用于核酸扩增反应的反应物或反应体系95，如为包含核酸扩增酶、底物和缓冲液中的一项或多项的冻干颗粒。As shown in Figure 2A, in an exemplary working situation, the initial state of the chip device for nucleic acid detection in samples is: a sample preservation solution (that is, a nucleic acid extraction solution) 71 is preset in the sample chamber 2 , the liquid level of the sample preservation liquid 71 is lower than the air flow channel 5 between the sample chamber 2 and the pressurizing chamber 3; the first reactant 83 that generates a chemical reaction to generate gas after contact is preset in the pressurizing chamber 3 (for example, sulfuric acid) and the second reactant 84 (for example, sodium bicarbonate). There is a separation medium 82 between the first reactant 83 and the second reactant 84 that can melt or decompose under appropriate conditions (such as at a higher temperature). paraffin that melts at temperatures). There may also be a separation medium 81 that can melt or decompose under appropriate conditions above the first reactant and/or the second reactant disposed in the pressurized chamber 3 . In addition, a reactant or reaction system 95 for a nucleic acid amplification reaction is preset in the amplification reaction chamber 4, such as a freeze-dried particle containing one or more of a nucleic acid amplification enzyme, a substrate, and a buffer.
如图2B所示，在示例性的工作情形中，所述用于样品中核酸检测的芯片装置的工作流程为：打开样品腔的盖子，将待测样品(如咽拭子)放入，并在样品保存液进行核酸分离的反应，核酸释放到液体中；对增压腔3进行加热，加热导致分隔介质81/83融化，第一反应物83(例如为硫酸)和第二反应物84(例如为碳酸氢钠)接触和反应，并生成气体(例如二氧化碳)，增加的气体压力通过空气流道作用到样品腔的样本保存液上；随着气压的增大，含有核酸的样本保存液通过液体流道4运动至扩增反应腔4中，复溶预置于扩增反应腔中的扩增原料；原本存在于流道和反应腔中的空气会被压缩；然后在扩增反应腔中进行核酸扩增反应，对被检测病原
体的特征核酸进行复制(扩增)，并产生相应的光学信号；最后通过肉眼观察或者光学装置探测等方式进行判别。As shown in Figure 2B, in an exemplary working situation, the workflow of the chip device for nucleic acid detection in samples is: open the cover of the sample chamber, put the sample to be tested (such as a throat swab), and A nucleic acid separation reaction is performed in the sample preservation solution, and the nucleic acid is released into the liquid; the pressurized chamber 3 is heated, and the heating causes the separation medium 81/83 to melt, and the first reactant 83 (for example, sulfuric acid) and the second reactant 84 ( For example, sodium bicarbonate) contacts and reacts, and generates gas (such as carbon dioxide). The increased gas pressure acts on the sample preservation solution in the sample chamber through the air flow channel; as the air pressure increases, the sample preservation solution containing nucleic acid passes through The liquid flow channel 4 moves into the amplification reaction chamber 4 to redissolve the amplification raw materials pre-placed in the amplification reaction chamber; the air originally existing in the flow channel and reaction chamber will be compressed; and then in the amplification reaction chamber Carry out nucleic acid amplification reaction to detect the pathogen The characteristic nucleic acid of the body is replicated (amplified) and a corresponding optical signal is generated; finally, the nucleic acid is identified through naked eye observation or optical device detection.
在本实施例中，所述基板、样品腔和增压腔由刚性材料制备。所述材料包括但不限于硅石、硅、石英、玻璃或聚合材料(例如PDMS、塑料等)。设置在所述基板内的液体流道通常为微流体通道，其尺寸在毫米级别，例如其流道的横截面的宽度为约0.1-5mm。In this embodiment, the substrate, sample chamber and pressurization chamber are made of rigid materials. Such materials include, but are not limited to, silica, silicon, quartz, glass or polymeric materials (eg PDMS, plastic, etc.). The liquid flow channel provided in the substrate is usually a microfluidic channel, the size of which is on the millimeter level, for example, the width of the cross-section of the flow channel is about 0.1-5 mm.
在本发明的其中一个方面，由于对核酸的检测是通过对扩增的核酸携带的可见光或荧光信号进行检测，为了避免或尽量消除相邻核酸扩增区的信号产生干扰，可采用完全吸收或基本吸收目标信号(例如荧光)的材料制备所述圆柱状腔体的壁和所述底板。而扩增反应腔的顶部/底部以完全不吸收或基本不吸收待检测信号的材料制备或封闭。由此，设置在芯片中的信号检测模块或是外部的检测系统可通过扩增反应腔的顶部/底部检测各个扩增反应单元内产生的荧光信号。In one aspect of the present invention, since the detection of nucleic acids is based on the detection of visible light or fluorescence signals carried by amplified nucleic acids, in order to avoid or try to eliminate interference from signals in adjacent nucleic acid amplification regions, complete absorption or The walls of the cylindrical cavity and the floor are made of a material that substantially absorbs the target signal (eg, fluorescence). The top/bottom of the amplification reaction chamber is prepared or sealed with a material that does not absorb at all or basically does not absorb the signal to be detected. Therefore, the signal detection module provided in the chip or the external detection system can detect the fluorescence signal generated in each amplification reaction unit through the top/bottom of the amplification reaction chamber.
本发明提供的用于样品中核酸检测的芯片装置的核酸扩增和信号检测可采用任何方式对核酸携带的可识别的标记进行检测的方法，包括但不限于荧光或其它形式的(例如化学发光，生物发光，辐射发光，电发光，电化学发光，机械发光，结晶发光，热致发光，声致发光，磷光和光致发光等)发光，酶促反应，放射性等。The nucleic acid amplification and signal detection of the chip device for detecting nucleic acids in samples provided by the present invention can adopt any method to detect identifiable labels carried by nucleic acids, including but not limited to fluorescence or other forms (such as chemiluminescence). , bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallographic luminescence, thermoluminescence, sonoluminescence, phosphorescence and photoluminescence, etc.) luminescence, enzymatic reaction, radioactivity, etc.
在本发明的其中一个方面，对扩增的核酸的检测是通过对核酸携带的荧光信号进行检测。在扩增反应腔的反应体系中包含的引物和寡核苷酸可通过本领域技术人员所熟知的方法进行放射性、荧光或非放射性的可检测标记。常用的荧光染料和其信号相关波长如下表1所示。In one aspect of the invention, the amplified nucleic acid is detected by detecting a fluorescent signal carried by the nucleic acid. The primers and oligonucleotides contained in the reaction system of the amplification reaction chamber can be radioactive, fluorescent or non-radioactive detectably labeled by methods well known to those skilled in the art. Commonly used fluorescent dyes and their signal-related wavelengths are shown in Table 1 below.
表1荧光染料
Table 1 Fluorescent dyes
Table 1 Fluorescent dyes
在本发明的其中一个方面，所述扩增反应腔的顶部或底部可采用不吸
收扩增反应产生的荧光信号的材料。另外，可通过人眼或已知的各种光学系统(包括滤光片、相机等)对各个扩增反应单元产生的光信号进行观察或采集，进而得到检测结果。In one aspect of the present invention, the top or bottom of the amplification reaction chamber can be made of non-aspirated Materials that collect fluorescent signals generated by amplification reactions. In addition, the light signals generated by each amplification reaction unit can be observed or collected through the human eye or various known optical systems (including filters, cameras, etc.) to obtain detection results.
实施例2测试Example 2 Test
以PDMS作为基板、样品腔和增压腔的材料，制备如图1所示的核酸检测芯片。其中基板1的厚度为约2.0mm；样品腔2的腔体体积约为1.6ml，内径为约7.0mm；增压腔3的腔体体积约为1.2ml，内径为约7.0mm。芯片上具有4个扩增腔单元，每个体积为约25.0ul，每个分压腔的体积约为6.0ul。所述液体流道的横截面的尺寸为约0.4mm x 0.5mm。Using PDMS as the material of the substrate, sample chamber and pressurization chamber, a nucleic acid detection chip as shown in Figure 1 was prepared. The thickness of substrate 1 is about 2.0mm; the cavity volume of sample chamber 2 is about 1.6ml, and the inner diameter is about 7.0mm; the cavity volume of booster chamber 3 is about 1.2ml, and the inner diameter is about 7.0mm. There are 4 amplification chamber units on the chip, each with a volume of approximately 25.0ul, and each partial pressure chamber with a volume of approximately 6.0ul. The size of the cross-section of the liquid flow channel is approximately 0.4mm x 0.5mm.
在样品腔内加入约500ul样品释放剂(常州金麦格生物技术有限公司，苏常械备20200123号)。根据试剂提供商的说明书，所述样品释放剂的检验原理为利用蛋白变性剂和生化试剂快速破坏咽拭子样本中的病毒衣壳结构，从而使病毒裂解并释放出核酸；同时包含的核酸稳定剂可以有效防止核酸降解，从而达到提取和保存核酸的作用。适用标本类型：咽拭子样本或体液样本。Add about 500ul of sample release agent (Changzhou Jinmag Biotechnology Co., Ltd., Su Chang Mechanical Preparation No. 20200123) into the sample chamber. According to the instructions of the reagent provider, the testing principle of the sample release agent is to use protein denaturants and biochemical reagents to quickly destroy the virus capsid structure in the throat swab sample, thereby causing the virus to cleave and release the nucleic acid; at the same time, the nucleic acid contained is stable The agent can effectively prevent the degradation of nucleic acids, thereby achieving the function of extracting and preserving nucleic acids. Applicable specimen types: throat swab samples or body fluid samples.
增压腔3的底部设置约126mg NaHCO3,加入石蜡进行封闭包裹，然后加入约65ul 10N HCl，在HCl上加石蜡形成隔离层。Set approximately 126 mg NaHCO 3 at the bottom of the pressurized chamber 3, add paraffin wax for sealing and wrapping, then add approximately 65 ul 10N HCl, and add paraffin wax on the HCl to form an isolation layer.
采用北京百奥莱博科技有限公司的SARS-CoV-2新型冠状病毒RT-LAMP试剂盒(N基因)提供的试剂作为测试用试剂，其中包括SARS-CoV-2N基因RT-LAMP阳性对照。The reagents provided by Beijing Biolab Technology Co., Ltd.'s SARS-CoV-2 novel coronavirus RT-LAMP kit (N gene) were used as test reagents, including SARS-CoV-2N gene RT-LAMP positive control.
将上述试剂盒中的SARS-CoV-2N基因RT-LAMP引物混合液以及2×LAMP MagicMix经处理形成冻干粉末，加入到扩增反应腔体内。The SARS-CoV-2N gene RT-LAMP primer mixture and 2×LAMP MagicMix in the above kit are processed to form a freeze-dried powder and added to the amplification reaction chamber.
打开样本腔上方的螺帽，将吸附了SARS-CoV-2N基因RT-LAMP阳性对照的鼻咽拭子伸入到样本腔折断，让拭子头落入到样本保存液中。盖上螺盖后，对样本腔内的液体进行超声。病毒核酸从拭子头上脱落并进入到溶液中。Open the screw cap above the sample chamber, insert the nasopharyngeal swab adsorbed with the SARS-CoV-2N gene RT-LAMP positive control into the sample chamber, break it, and let the swab head fall into the sample preservation solution. After closing the screw cap, ultrasonicate the liquid in the sample chamber. Viral nucleic acid breaks off the swab head and enters the solution.
对增压腔加热至约70℃，石蜡融化，HCl和NaHCO3发生反应并生成CO2气体。随着气压的增大，样本保存液通过流道被挤压至扩增反应腔中，
复溶预置于扩增反应腔中的扩增原料。The pressurized chamber is heated to about 70°C, the paraffin melts, HCl and NaHCO react to generate CO gas . As the air pressure increases, the sample preservation solution is squeezed into the amplification reaction chamber through the flow channel. Reconstitute the amplification raw materials pre-placed in the amplification reaction chamber.
在另一芯片中加入未吸附SARS核酸的鼻咽拭子作为对照。A nasopharyngeal swab without SARS nucleic acid adsorption was added to another chip as a control.
对扩增反应腔进行加温至61℃，反应1小时后观察。Warm the amplification reaction chamber to 61°C and observe after 1 hour of reaction.
在另一芯片中加入未吸附SARS核酸的鼻咽拭子作为对照，其余步骤同实验组。A nasopharyngeal swab without SARS nucleic acid adsorption was added to another chip as a control, and the remaining steps were the same as in the experimental group.
结果显示，阳性检测芯片的扩增反应腔呈现蓝色。阴性对照的扩增反应腔呈现非常浅的蓝色。The results showed that the amplification reaction chamber of the positive detection chip turned blue. The amplification reaction chamber of the negative control appears very light blue.
实施例3用于样品中核酸检测的仪器Example 3 Instrument for nucleic acid detection in samples
在其中一种实施方式中，本发明提供了一种用于样品中核酸检测的仪器，其为POCT仪器，其中包括实施例1中定义和描述的芯片装置。In one embodiment, the present invention provides an instrument for detecting nucleic acids in samples, which is a POCT instrument, including the chip device defined and described in Embodiment 1.
所述仪器具有芯片装置接收和运动控制系统，用于接纳上述芯片装置和对所述芯片进行各种处理，包括加热处理。The instrument has a chip device receiving and motion control system for receiving the chip device and performing various treatments on the chip, including heating treatment.
所述仪器还可具有检测核酸扩增产物的信号检测模块，例如为荧光检测系统。The instrument may also have a signal detection module for detecting nucleic acid amplification products, such as a fluorescence detection system.
所述仪器具有对所述芯片的核酸扩增区域进行温控的系统。The instrument has a system for temperature controlling the nucleic acid amplification area of the chip.
所述仪器具有核酸扩增结果分析和/或输出系统。The instrument has a nucleic acid amplification result analysis and/or output system.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection.
Claims (14)
- 一种用于样品中核酸检测的芯片装置，其具有基板，样品腔和增压腔，以及设置在所述基板内的扩增反应腔，其中，所述样品腔和增压腔之间通过空气流道连通，以及样品腔通过设置在基板内的液体流道与所述扩增反应腔连通，A chip device for nucleic acid detection in a sample, which has a substrate, a sample chamber and a pressurized chamber, and an amplification reaction chamber arranged in the substrate, wherein air is passed between the sample chamber and the pressurized chamber. The flow channels are connected, and the sample chamber is connected with the amplification reaction chamber through a liquid flow channel provided in the substrate,其中，in,所述样品腔用于容纳待检测样品以及将样品中的核酸分离，The sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample,所述增压腔用于使得腔体内气体压力增大，从而将样品腔中的溶液通过液体流道挤压到扩增反应腔中，所述增压腔通过化学反应方法在其腔体内产生气体，The pressurized chamber is used to increase the gas pressure in the chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel. The pressurized chamber generates gas in its cavity through a chemical reaction method. ,所述扩增反应腔用于溶液中的核酸进行扩增反应。The amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
- 根据权利要求1所述的芯片装置，其特征在于，所述芯片装置的扩增反应腔包括多个核酸扩增单元，例如2-24个。The chip device according to claim 1, wherein the amplification reaction chamber of the chip device includes a plurality of nucleic acid amplification units, such as 2-24.
- 根据权利要求1所述的芯片装置，其特征在于，所述样品腔的顶部具有可打开或封闭的盖。The chip device according to claim 1, wherein the top of the sample chamber has an openable or closable cover.
- 根据权利要求1所述的芯片装置，其特征在于，所述样品腔中可加入或预置用于保存和提取样品中的核酸的试剂溶液。The chip device according to claim 1, wherein a reagent solution for preserving and extracting nucleic acids in the sample can be added to or preset in the sample chamber.
- 根据权利要求1所述的芯片装置，其特征在于，在增压腔产生的气体的量为样品腔和增压腔中原有的气体的量的约1-50倍，例如为约5-20倍。The chip device according to claim 1, wherein the amount of gas generated in the pressurized chamber is about 1-50 times, for example, about 5-20 times the amount of original gas in the sample chamber and the pressurized chamber. .
- 根据权利要求1所述的芯片装置，其特征在于，所述空气流道中或其与样品腔或增压腔的开口处设置过滤件，例如为设置在空气流道与增压腔的开口处的滤片膜，或为设置在空气流道内的滤柱。The chip device according to claim 1, characterized in that a filter element is provided in the air flow channel or at an opening between the air flow channel and the sample chamber or the pressurization chamber, for example, a filter element is provided at the opening of the air flow channel and the pressurization chamber. Filter membrane, or filter column installed in the air flow channel.
- 根据权利要求1所述的芯片装置，其特征在于，扩增反应腔内可预置核酸扩增反应的材料。The chip device according to claim 1, wherein materials for nucleic acid amplification reaction can be preset in the amplification reaction chamber.
- 根据权利要求7所述的芯片装置，其特征在于，所述核酸扩增反应为等温的扩增方法，例如为环介导的DNA等温扩增(LAMP)。The chip device according to claim 7, wherein the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
- 根据权利要求1所述的芯片装置，其特征在于，所述扩增反应腔 的下游还具有分压腔，所述分压腔设置在芯片的基板内，通过分压气体通道与扩增反应腔连通。The chip device according to claim 1, wherein the amplification reaction chamber There is also a pressure dividing chamber downstream of the chip. The pressure dividing chamber is arranged in the substrate of the chip and is connected to the amplification reaction chamber through a pressure dividing gas channel.
- 一种用于样品中核酸检测的仪器(优选为POCT仪器)，其中包括权利要求1-9中任一项所述的芯片装置，其中所述芯片装置具有基板，样品腔和增压腔，以及设置在所述基板内的扩增反应腔，其中，所述样品腔和增压腔之间通过空气流道连通，以及样品腔通过设置在基板内的液体流道与所述扩增反应腔连通，An instrument (preferably a POCT instrument) for nucleic acid detection in samples, which includes the chip device according to any one of claims 1 to 9, wherein the chip device has a substrate, a sample chamber and a pressurized chamber, and An amplification reaction chamber provided in the substrate, wherein the sample chamber and the pressurizing chamber are connected through an air flow channel, and the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the substrate ,其中，in,所述样品腔用于容纳待检测样品以及将样品中的核酸分离，The sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample,所述增压腔用于使得腔体内气体压力增大，从而将样品腔中的溶液通过液体流道挤压到扩增反应腔中，The pressurized chamber is used to increase the gas pressure in the chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,所述扩增反应腔用于溶液中的核酸进行扩增反应，The amplification reaction chamber is used for amplification reaction of nucleic acids in the solution,任选的，其还包括以下之一：Optionally, it also includes one of the following:芯片装置接收系统；chip device receiving system;检测核酸扩增产物的信号检测模块装置，例如为荧光检测装置；A signal detection module device for detecting nucleic acid amplification products, such as a fluorescence detection device;对所述芯片的核酸扩增区域进行温控的系统；A system for temperature controlling the nucleic acid amplification area of the chip;核酸扩增结果分析和/或输出系统。Nucleic acid amplification result analysis and/or output system.
- 一种用于样品中核酸检测的方法，其包括采用权利要求1-9中任一项所述的芯片装置或权利要求10所述的仪器，其中所述芯片装置具有基板，样品腔和增压腔，以及设置在所述基板内的扩增反应腔，其中，所述样品腔和增压腔之间通过空气流道连通，以及样品腔通过设置在基板内的液体流道与所述扩增反应腔连通，A method for nucleic acid detection in a sample, which includes using the chip device according to any one of claims 1-9 or the instrument according to claim 10, wherein the chip device has a substrate, a sample chamber and a pressurization chamber, and an amplification reaction chamber provided in the substrate, wherein the sample chamber and the pressurizing chamber are connected through an air flow channel, and the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the substrate. The reaction chamber is connected,其中，in,所述样品腔用于容纳待检测样品以及将样品中的核酸分离，The sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample,所述增压腔用于使得腔体内气体压力增大，从而将样品腔中的溶液通过液体流道挤压到扩增反应腔中，The pressurized chamber is used to increase the gas pressure in the chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,所述扩增反应腔用于溶液中的核酸进行扩增反应，The amplification reaction chamber is used for amplification reaction of nucleic acids in the solution,所述方法包括：The methods include:(1).将样品加入到所述芯片装置的样品腔中，使样品中的核酸分离 和进入到溶液中；(1). Add the sample into the sample chamber of the chip device to separate the nucleic acids in the sample. and enter the solution;(2).通过化学反应方法在其腔体内产生气体，使得所述增压腔的腔体内气体压力增大，将样品腔中的溶液通过液体流道挤压到扩增反应腔中；以及(2). Generate gas in its cavity through a chemical reaction method, so that the gas pressure in the cavity of the pressurized cavity increases, and the solution in the sample cavity is squeezed into the amplification reaction cavity through the liquid flow channel; and(3).在扩增反应腔对溶液中的核酸进行扩增反应。(3). Perform amplification reaction on the nucleic acid in the solution in the amplification reaction chamber.
- 根据权利要求11所述的方法，其特征在于，在增压腔产生的气体的量为样品腔和增压腔中原有的气体的量的约1-50倍，例如为约5-20倍。The method according to claim 11, wherein the amount of gas generated in the pressurized chamber is about 1-50 times, for example, about 5-20 times the amount of original gas in the sample chamber and the pressurized chamber.
- 根据权利要求11所述的方法，其特征在于，所述核酸扩增反应为等温的扩增方法，例如为环介导的DNA等温扩增(LAMP)。The method according to claim 11, wherein the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
- 根据权利要求11所述的方法，其特征在于，其中对扩增的核酸携带的可识别的标记进行检测，包括但不限于荧光或其它形式的(例如化学发光，生物发光，辐射发光，电发光，电化学发光，机械发光，结晶发光，热致发光，声致发光，磷光和光致发光等)发光，酶促反应，放射性等。 The method according to claim 11, wherein the identifiable label carried by the amplified nucleic acid is detected, including but not limited to fluorescence or other forms (such as chemiluminescence, bioluminescence, radioluminescence, electroluminescence , electrochemiluminescence, mechanoluminescence, crystal luminescence, thermoluminescence, sonoluminescence, phosphorescence and photoluminescence, etc.) luminescence, enzymatic reaction, radioactivity, etc.
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| CN220703691U (en) | 2024-04-02 |
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