WO2023186159A1 - Chip device and instrument for rapid nucleic acid detection, and application thereof - Google Patents

Chip device and instrument for rapid nucleic acid detection, and application thereof Download PDF

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Publication number
WO2023186159A1
WO2023186159A1 PCT/CN2023/085818 CN2023085818W WO2023186159A1 WO 2023186159 A1 WO2023186159 A1 WO 2023186159A1 CN 2023085818 W CN2023085818 W CN 2023085818W WO 2023186159 A1 WO2023186159 A1 WO 2023186159A1
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Prior art keywords
sample
chamber
nucleic acid
amplification reaction
sample chamber
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PCT/CN2023/085818
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French (fr)
Chinese (zh)
Inventor
赵海峰
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恒泰医疗有限公司
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Publication of WO2023186159A1 publication Critical patent/WO2023186159A1/en

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    • 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
    • C12M1/34—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
    • 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
    • C12M1/38—Temperature-responsive control
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • 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.
  • nucleic acids 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.
  • 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.
  • infectious diseases such as viruses, sexually transmitted diseases, and influenza.
  • the role of genetic testing in cancer treatment is becoming clear.
  • automation of applications employing PCR and RT-PCR, especially small-scale automation, is not easy.
  • 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).
  • the invention provides a chip device for nucleic acid detection in a sample, which has a substrate, a sample chamber, and an amplification reaction chamber disposed in the substrate, wherein the sample chamber It is connected to the amplification reaction chamber through a liquid flow channel provided in the substrate,
  • the sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample
  • the sample chamber has a pressure mechanism for increasing the gas pressure in the sample chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
  • the amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
  • the pressure mechanism of the sample chamber is a mechanical mechanism, such as a piston mechanism.
  • the movement of the piston mechanism can be realized by push rods, spiral structures or springs.
  • a chip device for nucleic acid detection in a sample which has a substrate, a sample chamber and a cap of the sample chamber, and an amplification reaction chamber disposed in the substrate, wherein , the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the substrate,
  • the sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample
  • the cap is used to increase the gas pressure in the sample chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
  • the amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
  • the amplification reaction chamber of the chip device includes a plurality of nucleic acid amplification units, for example, 2-24 units.
  • the cap and the sample chamber are separate components.
  • the cap is a piston-type cap, which has a piston fixedly connected to the top of the cap through a connecting rod.
  • the piston When the piston is inserted into the sample chamber, it forms a piston mechanism with the tube wall of the sample chamber. .
  • the cap has internal threads that can cooperate with external threads on the outside of the upper part of the sample chamber.
  • a reagent solution for preserving and extracting nucleic acids in the sample can be added to or preset in the sample chamber.
  • the reagent solution is a reagent for nucleic acid extraction from samples using a direct extraction method.
  • a filter cavity may be provided in the liquid flow channel.
  • materials for nucleic acid amplification reaction can be preset in the amplification reaction chamber.
  • 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.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • CRCA cascade Rolling circle amplification
  • LAMP circle-mediated DNA isothermal amplification
  • ICAN isothermal chimeric primer-initiated nucleic acid amplification
  • HDA target-based helicase-dependent amplification
  • TMA Transcription-mediated amplification
  • the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
  • LAMP loop-mediated DNA isothermal amplification
  • 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.
  • 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.
  • the present invention also provides an instrument for nucleic acid detection in a sample, such as a POCT instrument, which includes the aforementioned chip device of the present invention.
  • the chip device has a substrate, a sample chamber and a cap of the sample chamber, and is provided with an amplification reaction chamber in the substrate, wherein the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the substrate,
  • the sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample
  • the pressure mechanism of the sample chamber is used to increase the gas pressure in the sample chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
  • the amplification reaction chamber is used for amplification reaction of nucleic acid in the solution.
  • the pressure mechanism of the sample chamber is a piston-type cap, which has a piston fixedly connected to the top of the cap through a connecting rod.
  • the piston When the piston is inserted into the sample chamber, it is connected with the tube of the sample chamber.
  • the walls form the piston mechanism.
  • the cap and the sample chamber are separate components.
  • the instrument has a chip device receiving system.
  • the instrument has a signal detection module device for detecting nucleic acid amplification products, such as a fluorescence detection device.
  • 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.
  • 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 is disposed in the substrate an amplification reaction chamber, wherein the sample chamber is connected to the amplification reaction chamber through a liquid flow channel provided in the substrate,
  • the sample chamber is used to accommodate the sample to be detected and separate the nucleic acid in the sample
  • the pressure mechanism of the sample chamber is used to increase the gas pressure in the sample chamber, thereby squeezing the solution in the sample chamber into the amplification reaction chamber through the liquid flow channel,
  • the amplification reaction chamber is used for amplification reaction of nucleic acids in the solution
  • the method includes the following steps:
  • the pressure mechanism of the sample chamber in the device is a piston-type cap, which has a piston fixedly connected to the top of the cap through a connecting rod, and has internal threads that can be connected with the upper part of the sample chamber.
  • the external external threads form a fit, the cap is capped and rotated to cause the cap and piston to move downward in the sample chamber.
  • 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
  • identifiable labels carried by the amplified nucleic acids are detected, including but not limited to fluorescence or other forms (such as chemiluminescence, bioluminescence, radioluminescence, electroluminescence, electroluminescence, Chemiluminescence, mechanoluminescence, crystallographic luminescence, thermoluminescence, sonoluminescence, phosphorescence and photoluminescence, etc.) luminescence, enzymatic reaction, radioactivity, etc.
  • fluorescence or other forms such as chemiluminescence, bioluminescence, radioluminescence, electroluminescence, electroluminescence, Chemiluminescence, mechanoluminescence, crystallographic luminescence, thermoluminescence, sonoluminescence, phosphorescence and photoluminescence, etc.
  • 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 structural diagram of the cap of the sample chamber of the exemplary chip device used for nucleic acid detection in samples provided by the present invention.
  • Figure 2A is a three-dimensional perspective view of the cover.
  • Figure 2B is a schematic working diagram of a cross-sectional view of the cover.
  • Figure 3 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.
  • Figure 3A shows a schematic diagram of the chip device provided by the present invention for nucleic acid detection in samples after configuring reagents and adding samples.
  • FIG. 3B is a schematic diagram of the exemplary chip device after the cover is screwed on.
  • the present invention provides a chip device for quickly, simply, easily and effectively detecting target nucleic acids from samples.
  • the chip device is usually used to detect the presence and amount of nucleic acid in a sample after extracting and amplifying it.
  • Nucleic acid samples used in the methods described herein can be from any source.
  • a sample may be biological material isolated from its natural environment and containing polynucleotides.
  • the sample may consist of purified or isolated polynucleotides, or may comprise a biological sample such as a tissue sample, biological fluid sample, or cell sample that contains the 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.
  • Figure 1 is a three-dimensional perspective view of an exemplary chip device for nucleic acid detection in samples provided by the present invention, which includes a substrate 1, a sample chamber 2 vertically arranged on the substrate and a cover 3 of the sample chamber, and An amplification reaction chamber 4 is provided in the chip device substrate.
  • Figure 2 is a structural diagram of the cap 3 of the sample chamber of the exemplary chip device used for nucleic acid detection in samples provided by the present invention.
  • Figure 2A is a three-dimensional perspective view of the cap.
  • FIG. 2B is a schematic working diagram of a cross-sectional view of the cap.
  • Figure 3 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.
  • Figure 3A shows a schematic diagram of configuring reagents and adding samples to the chip device for nucleic acid detection in samples provided by the present invention.
  • FIG. 3B is a schematic diagram of the exemplary chip device after the cap is
  • the chip device provided by the present invention for detecting nucleic acids in samples includes a substrate 1, a sample chamber 2 vertically arranged on the substrate, a cover 3 of the sample chamber, and a cap 3 arranged in the substrate of the chip device.
  • the sample chamber 2 is a cylindrical chamber surrounded by walls perpendicular to the bottom plate.
  • 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 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.
  • the amplification reaction chamber of the chip device includes a plurality of nucleic acid amplification units, for example, 2-24 units.
  • the sample chamber 2 is used to accommodate the sample to be detected and to separate nucleic acids in the sample.
  • the sample chamber The top of the sample chamber has a detachable cap 3 so that the sample chamber can be opened or closed.
  • the cap 3 has an internal thread 31 that can cooperate with the external thread on the upper side of the sample chamber 2 .
  • the cap can provide a tight seal to the sample chamber to prevent gas or liquid leakage.
  • the cap of the sample chamber can be opened, and the sample is added to the sample chamber and sealed with the cap to avoid leakage of biological samples and resulting contamination.
  • 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.
  • 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.
  • 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.
  • the nucleic acid detection chip device when there is a solution in the sample chamber, air pressure is applied above the solution, so that the liquid in the sample chamber is squeezed into the amplification reaction chamber through the liquid flow channel 6 4 in.
  • the purpose of increasing the gas pressure is achieved by forming a closed space above the solution and applying pressure to the gas in the space.
  • a piston mechanism is used to form a closed space above the solution and apply pressure to the gas in the space.
  • the cap 3 of the sample chamber is a piston cap.
  • the cap 3 of the sample chamber has a piston 32 inside, which is fixedly connected to the top of the cap through a connecting rod.
  • the outer periphery of the piston is made of elastic material.
  • the piston When inserted into the sample chamber, the piston forms a close fit with the inner wall of the sample chamber, that is, it forms a piston mechanism with the tube wall of the sample chamber.
  • the bottom of the piston is substantially flush with the bottom of the cap.
  • the piston 32 moves toward the bottom in the sample chamber, forming air pressure in the sample chamber, thereby squeezing the solution in the sample chamber. , so that it is transferred to the amplification reaction chamber 4 through the liquid flow channel 6 .
  • the distance that the piston moves in the amplification reaction chamber so that the target volume of solution is transferred from the sample chamber to the amplification reaction chamber, that is, the position at which the piston stops in the amplification reaction chamber, can be determined based on calculations or experiments.
  • 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 .
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • CRCA cascade rolling circle amplification
  • LAMP loop-mediated DNA isothermal amplification
  • ICAN isother
  • 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.
  • 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 passes through the pressure dividing gas Lane 42 is connected with the amplification reaction chamber 4 .
  • 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.
  • 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.
  • the workflow of the chip device for nucleic acid detection in samples is: open the cap of the sample chamber, and put the sample to be tested (such as a throat swab) into , and a nucleic acid separation reaction is performed in the sample preservation solution, and the nucleic acid is released into the solution; the cap is capped and moved downwards to the sample chamber by rotation.
  • the sample to be tested such as a throat swab
  • the sample preservation solution containing nucleic acid moves through the liquid channel 6 to In the amplification reaction chamber 4, the amplification raw materials pre-disposed in the amplification reaction chamber are reconstituted, for example, freeze-dried particles containing one or more of nucleic acid amplification enzymes, substrates and buffers; originally present in The air in the flow channel and reaction chamber will be compressed; then a nucleic acid amplification reaction is carried out in the amplification reaction chamber, the characteristic nucleic acid of the detected pathogen is copied (amplified), and a corresponding optical signal is generated; finally, it is observed with the naked eye Or it can be judged by optical device detection and other methods.
  • the amplification raw materials pre-disposed in the amplification reaction chamber are reconstituted, for example, freeze-dried particles containing one or more of nucleic acid amplification enzymes, substrates and buffers; originally present in The air in the flow channel and reaction chamber will be compressed; then a nucleic acid amplification reaction is carried out in the amplification reaction
  • the substrate and sample chamber are made of rigid materials.
  • 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.
  • 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.
  • 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 Light, etc.) luminescence, enzymatic reactions, radioactivity, etc.
  • fluorescence or other forms such as chemiluminescence
  • 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.
  • the top or bottom of the amplification reaction chamber can be made of materials that do not absorb the fluorescent signal generated by the amplification reaction.
  • 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.
  • 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 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) are 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 sample preservation solution is squeezed into the amplification reaction chamber through the flow channel, and the amplification raw materials pre-disposed in the amplification reaction chamber are reconstituted.
  • 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 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.
  • 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 rapid nucleic acid detection of a sample, said device being provided with a substrate, a sample chamber, and 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

快速检测核酸的芯片装置和仪器以及其应用Chip devices and instruments for rapid detection of nucleic acids and their applications
本申请要求以下中国专利申请的优先权：2022年4月2日提交的、申请号为202210340886.9、发明名称为“快速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 April 2, 2022, with application number 202210340886.9, and the invention title is "Chip device and instrument for rapid detection of nucleic acids and applications thereof", the entire contents of which are incorporated herein by reference. Applying.
技术领域Technical field
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.
背景技术Background technique
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
在本发明的其中一个方面，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，以及设置在所述基板内的扩增反应腔，其中，所述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，In one aspect of the invention, the invention provides a chip device for nucleic acid detection in a sample, which has a substrate, a sample chamber, and an amplification reaction chamber disposed in the substrate, wherein the sample chamber It 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,
所述样品腔具有压力机构，用于使得样品腔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 sample chamber has a pressure mechanism for increasing the gas pressure in the sample 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，所述样品腔的压力机构为机械机构，例如为活塞机构。所述活塞机构的运动可通过推杆、螺旋结构或弹簧等方式实现。In one aspect of the invention, the pressure mechanism of the sample chamber is a mechanical mechanism, such as a piston mechanism. The movement of the piston mechanism can be realized by push rods, spiral structures or springs.
在本发明的其中一个方面，提供了一种用于样品中核酸检测的芯片装置，其具有基板，样品腔和所述样品腔的盖帽，以及设置在所述基板内的扩增反应腔，其中，所述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，In one aspect of the present invention, a chip device for nucleic acid detection in a sample is provided, which has a substrate, a sample chamber and a cap of the sample chamber, and an amplification reaction chamber disposed in the substrate, wherein , 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,
所述盖帽用于使得样品腔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 cap is used to increase the gas pressure in the sample 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，所述盖帽与所述样品腔为分离的部件。In one aspect of the invention, the cap and the sample chamber are separate components.
在本发明的其中一个方面，所述盖帽为活塞式盖帽，其具有通过连接杆与所述盖帽的顶部固定连接的活塞，所述活塞在***样品腔时，与样品腔的管壁形成活塞机构。In one aspect of the present invention, the cap is a piston-type cap, which has a piston fixedly connected to the top of the cap through a connecting rod. When the piston is inserted into the sample chamber, it forms a piston mechanism with the tube wall of the sample chamber. .
在本发明的其中一个方面，所述盖帽具有内螺纹，可以与样品腔上部的外侧的外螺纹形成配合。In one aspect of the present invention, the cap has internal threads that can cooperate with external threads on the outside of the upper part of 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，所述芯片装置具有基板，样品腔和所述样品腔的盖帽，以及设置在所述基板内的扩增反应腔，其中，所述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 a sample, such as a POCT instrument, which includes the aforementioned chip device of the present invention. The chip device has a substrate, a sample chamber and a cap of the sample chamber, and is provided with an amplification reaction chamber in the substrate, wherein 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,
所述样品腔的压力机构用于使得样品腔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 pressure mechanism of the sample chamber is used to increase the gas pressure in the sample 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，所述样品腔的压力机构为活塞式盖帽，其具有通过连接杆与所述盖帽的顶部固定连接的活塞，所述活塞在***样品腔时，与样品腔的管壁形成活塞机构。In one aspect of the invention, the pressure mechanism of the sample chamber is a piston-type cap, which has a piston fixedly connected to the top of the cap through a connecting rod. When the piston is inserted into the sample chamber, it is connected with the tube of the sample chamber. The walls form the piston mechanism.
在本发明的其中一个方面，所述盖帽与所述样品腔为分离的部件。In one aspect of the invention, the cap and the sample chamber are separate components.
在本发明的其中又一个方面，所述仪器具有芯片装置接收***。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***。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***。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，以及设置在所述基板内的扩增反应腔，其中，所述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 is disposed in the substrate an amplification reaction chamber, wherein 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,
所述样品腔的压力机构用于使得样品腔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 pressure mechanism of the sample chamber is used to increase the gas pressure in the sample 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,
所述方法包括以下步骤：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)用所述样品腔的压力机构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) Use the pressure mechanism of the sample chamber to 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) Amplify the nucleic acid in the solution in the amplification reaction chamber.
在本发明的其中一个方面，所述装置中的样品腔的压力机构为活塞式盖帽，其具有通过连接杆与所述盖帽的顶部固定连接的活塞，以及具有内螺纹，可以与样品腔上部的外侧的外螺纹形成配合，盖上盖帽并通过旋转使得盖帽以及活塞在样品腔腔体内向下移动。In one aspect of the present invention, the pressure mechanism of the sample chamber in the device is a piston-type cap, which has a piston fixedly connected to the top of the cap through a connecting rod, and has internal threads that can be connected with the upper part of the sample chamber. The external external threads form a fit, the cap is capped and rotated to cause the cap and piston to move downward in the sample chamber.
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, radioluminescence, electroluminescence, electroluminescence, Chemiluminescence, mechanoluminescence, crystallographic luminescence, thermoluminescence, sonoluminescence, phosphorescence and photoluminescence, etc.) luminescence, enzymatic reaction, radioactivity, etc.
附图说明Description of drawings
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为所述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样品腔的盖帽的结构图。其中图2A为所述盖子的立体透视图。图2B为所述盖子的剖面图的工作示意图。Figure 2 is a structural diagram of the cap of the sample chamber of the exemplary chip device used for nucleic acid detection in samples provided by the present invention. Figure 2A is a three-dimensional perspective view of the cover. Figure 2B is a schematic working diagram of a cross-sectional view of the cover.
图3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3A显示本发明提供的用于样品中核酸检测的芯片装置配置试剂，加入样品后的示意图。图3B为所述示例性的芯片装置拧上盖子后的示意图。Figure 3 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. Figure 3A shows a schematic diagram of the chip device provided by the present invention for nucleic acid detection in samples after configuring reagents and adding samples. FIG. 3B is a schematic diagram of the exemplary chip device after the cover is screwed on.
具体实施方式Detailed ways
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快速、简单、易行和有效地从样品中检测目标核酸的芯片装置。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 present invention provides a chip device for quickly, simply, easily and effectively detecting target nucleic acids from 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 use of various infectious diseases. test. 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. The sample may consist of purified or isolated polynucleotides, or may comprise a biological sample such as a tissue sample, biological fluid sample, or cell sample that contains the 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，其包括基板1，垂直设置在基板上的样品腔2和所述样品腔的盖帽3，以及设置在芯片装置基板内的扩增反应腔4。图2为所述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样品腔的盖帽3的结构图。其中图2A为所述盖帽的立体透视图。图2B为所述盖帽的剖面图的工作示意图。图3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3A显示本发明提供的用于样品中核酸检测的芯片装置配置试剂，加入样品的示意图。图3B为所述示例性的芯片装置拧上盖帽后的示意图。Figure 1 is a three-dimensional perspective view of an exemplary chip device for nucleic acid detection in samples provided by the present invention, which includes a substrate 1, a sample chamber 2 vertically arranged on the substrate and a cover 3 of the sample chamber, and An amplification reaction chamber 4 is provided in the chip device substrate. Figure 2 is a structural diagram of the cap 3 of the sample chamber of the exemplary chip device used for nucleic acid detection in samples provided by the present invention. Figure 2A is a three-dimensional perspective view of the cap. FIG. 2B is a schematic working diagram of a cross-sectional view of the cap. Figure 3 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. Figure 3A shows a schematic diagram of configuring reagents and adding samples to the chip device for nucleic acid detection in samples provided by the present invention. FIG. 3B is a schematic diagram of the exemplary chip device after the cap is screwed on.
如图1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和所述样品腔的盖帽3，以及设置在芯片装置基板内的扩增反应腔4。样品腔2为圆柱状的腔体，由与所述底板垂直的壁围成。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 Figure 1, the chip device provided by the present invention for detecting nucleic acids in samples includes a substrate 1, a sample chamber 2 vertically arranged on the substrate, a cover 3 of the sample chamber, and a cap 3 arranged in the substrate of the chip device. Amplification reaction chamber 4. The sample chamber 2 is a cylindrical chamber surrounded by walls perpendicular to the bottom plate. 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 的顶部具有分离式的盖帽3，由此样品腔可打开或封闭。如图2所示，盖帽3具有内螺纹31，可以与样品腔2上部的外侧的外螺纹形成配合。盖帽可对所述样品腔提供严密的封闭性，避免发生气体性或液体性泄漏。The sample chamber 2 is used to accommodate the sample to be detected and to separate nucleic acids in the sample. The sample chamber The top of the sample chamber has a detachable cap 3 so that the sample chamber can be opened or closed. As shown in FIG. 2 , the cap 3 has an internal thread 31 that can cooperate with the external thread on the upper side of the sample chamber 2 . The cap can provide a tight seal to the sample chamber to prevent gas or liquid leakage.
在需要加入样品时，可将所述样品腔的盖帽打开，将样品加入到样品腔后用盖帽封闭，避免生物样本的泄漏和由此造成的污染。When a sample needs to be added, the cap of the sample chamber can be opened, and the sample is added to the sample chamber and sealed with the cap 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.
在本发明提供的核酸检测的芯片装置中，在样品腔中存在溶液时，通过在所述溶液的上方施加气压，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。In the nucleic acid detection chip device provided by the present invention, when there is a solution in the sample chamber, air pressure is applied above the solution, so that the liquid in the sample chamber is squeezed into the amplification reaction chamber through the liquid flow channel 6 4 in.
在本发明的其中一个方面，通过在所述溶液上方形成封闭空间后给予空间内的气体施加压力，由此达到增加气压的目的。In one aspect of the present invention, the purpose of increasing the gas pressure is achieved by forming a closed space above the solution and applying pressure to the gas in the space.
在本发明的其中一个方面，通过活塞机构在所述溶液上方形成封闭空间后给予空间内的气体施加压力。 In one aspect of the invention, a piston mechanism is used to form a closed space above the solution and apply pressure to the gas in the space.
在本发明的其中一个方面，所述样品腔的盖帽3为活塞式盖帽。在本发明的其中一种实施方式中，如图2A和2B所示，所述样品腔的盖帽3内具有活塞32，其通过连接杆与所述盖帽的顶部固定连接。所述活塞的外周由弹性材料制成，在***样品腔时，所述活塞与样品腔内壁形成紧密贴合，即与样品腔的管壁形成活塞机构。在本发明的其中又一种实施方式中，所述活塞的底部基本与所述管帽的底部平齐。In one aspect of the present invention, the cap 3 of the sample chamber is a piston cap. In one embodiment of the present invention, as shown in Figures 2A and 2B, the cap 3 of the sample chamber has a piston 32 inside, which is fixedly connected to the top of the cap through a connecting rod. The outer periphery of the piston is made of elastic material. When inserted into the sample chamber, the piston forms a close fit with the inner wall of the sample chamber, that is, it forms a piston mechanism with the tube wall of the sample chamber. In yet another embodiment of the present invention, the bottom of the piston is substantially flush with the bottom of the cap.
在本发明的其中一个方面，当所述管帽通过螺纹向样品腔底部运动时，所述活塞32在样品腔内向其底部运动，在样品腔内形成气压，从而将样品腔中的溶液挤压，使其通过液体流道6转移到扩增反应腔4中。可根据计算或实验确定使得目标体积的溶液从样品腔转移到扩增反应腔的活塞在扩增反应腔内移动的距离，也即活塞在扩增反应腔内停止的位置。In one aspect of the present invention, when the tube cap moves toward the bottom of the sample chamber through the thread, the piston 32 moves toward the bottom in the sample chamber, forming air pressure in the sample chamber, thereby squeezing the solution in the sample chamber. , so that it is transferred to the amplification reaction chamber 4 through the liquid flow channel 6 . The distance that the piston moves in the amplification reaction chamber so that the target volume of solution is transferred from the sample chamber to the amplification reaction chamber, that is, the position at which the piston stops in the amplification reaction chamber, can be determined based on calculations or experiments.
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 passes through the pressure dividing gas Lane 42 is connected with the amplification reaction chamber 4 . 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3A和3B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：打开样品腔的盖帽，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，核酸释放到溶液中；盖上盖帽并通过旋转向样品腔下移动，随着气压的增大，含有核酸的样本保存液通过液体流道6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，例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；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 Figures 3A and 3B, in an exemplary working situation, the workflow of the chip device for nucleic acid detection in samples is: open the cap of the sample chamber, and put the sample to be tested (such as a throat swab) into , and a nucleic acid separation reaction is performed in the sample preservation solution, and the nucleic acid is released into the solution; the cap is capped and moved downwards to the sample chamber by rotation. As the air pressure increases, the sample preservation solution containing nucleic acid moves through the liquid channel 6 to In the amplification reaction chamber 4, the amplification raw materials pre-disposed in the amplification reaction chamber are reconstituted, for example, freeze-dried particles containing one or more of nucleic acid amplification enzymes, substrates and buffers; originally present in The air in the flow channel and reaction chamber will be compressed; then a nucleic acid amplification reaction is carried out in the amplification reaction chamber, the characteristic nucleic acid of the detected pathogen is copied (amplified), and a corresponding optical signal is generated; finally, it is observed with the naked eye Or it can be judged by optical device detection and other methods.
在本实施例中，所述基板和样品腔由刚性材料制备。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 and sample 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***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 Light, etc.) luminescence, enzymatic reactions, 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
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***(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 materials that do not absorb the fluorescent signal generated by the amplification reaction. 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作为基板和样品腔的材料，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。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 and sample cavity, 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. 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.
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) are 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.
拧开样本腔上方的盖帽，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中搅动，病毒核酸从拭子头上脱落并进入到溶液中。Unscrew the cap above the sample chamber, insert the nasopharyngeal swab adsorbed with the SARS-CoV-2N gene RT-LAMP positive control into the sample chamber and stir, the viral nucleic acid will fall off the swab head and enter the solution.
取走鼻咽拭子，将盖帽拧上至底部到达预设位置(盖帽无法再拧动)。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。Remove the nasopharyngeal swab and twist the cap to the bottom to reach the preset position (the cap cannot be twisted any further). As the air pressure increases, the sample preservation solution is squeezed into the amplification reaction chamber through the flow channel, and the amplification raw materials pre-disposed in the amplification reaction chamber are reconstituted.
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***，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***。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***。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***。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 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 (15)

  1. 一种用于样品中核酸检测的芯片装置，其具有基板，样品腔和所述样品腔的活塞式盖帽，以及设置在所述基板内的扩增反应腔，其中，所述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 base plate, a sample chamber and a piston cap of the sample chamber, and an amplification reaction chamber arranged in the base plate, wherein the sample chamber is configured by The liquid flow channel in the substrate 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,
    所述活塞式盖帽用于使得样品腔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 piston cap is used to increase the gas pressure in the sample 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.
  2. 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.
  3. 根据权利要求1所述的芯片装置，其特征在于，所述活塞式盖帽具有活塞，其通过连接杆与所述盖帽的顶部固定连接，所述活塞在***样品腔时，与样品腔的管壁形成活塞机构。The chip device according to claim 1, wherein the piston-type cap has a piston, which is fixedly connected to the top of the cap through a connecting rod. When the piston is inserted into the sample chamber, it is in contact with the wall of the sample chamber. Form a piston mechanism.
  4. 根据权利要求1-3中任一项所述的芯片装置，其特征在于，所述盖帽具有内螺纹，可以与样品腔上部的外侧的外螺纹形成配合。The chip device according to any one of claims 1 to 3, characterized in that the cap has an internal thread that can cooperate with the external thread of the upper part of the sample chamber.
  5. 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.
  6. 根据权利要求1所述的芯片装置，其特征在于，所述液体流道中可设置过滤腔。The chip device according to claim 1, characterized in that a filter cavity can be provided in the liquid flow channel.
  7. 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，优选的，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 1, wherein materials for nucleic acid amplification reaction can be preset in the amplification reaction chamber. Preferably, the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated amplification. DNA isothermal amplification (LAMP).
  8. 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, characterized in that there is also a pressure dividing chamber downstream of the amplification reaction chamber, and 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. Connected.
  9. 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1-8中任一项所述的芯片装置，其中所述芯片装置具有基板，样品腔和所述样品腔的活塞式盖帽，以及设置在所述基板内的扩增反应腔，其中，所述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 for nucleic acid detection in samples (preferably a POCT instrument), which includes The chip device according to any one of claims 1 to 8, wherein the chip device has a base plate, a sample chamber and a piston cover of the sample chamber, and an amplification reaction chamber disposed in the base plate, wherein , 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,
    所述活塞式盖帽用于使得样品腔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 piston cap is used to increase the gas pressure in the sample 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.
  10. 根据权利要求9所述的仪器，其特征在于，其具有芯片装置接收***。The instrument according to claim 9, characterized in that it has a chip device receiving system.
  11. 根据权利要求9所述的仪器，其特征在于，其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。The instrument according to claim 9, characterized in that it has a signal detection module device for detecting nucleic acid amplification products, such as a fluorescence detection device.
  12. 根据权利要求9所述的仪器，其特征在于，其具有对所述芯片的核酸扩增区域进行温控的***。The instrument according to claim 9, characterized in that it has a system for temperature controlling the nucleic acid amplification area of the chip.
  13. 一种用于样品中核酸检测的方法，其包括采用权利要求1-8中任一项所述的芯片装置或权利要求9-12中任一项所述的仪器，其中所述芯片装置具有基板，样品腔和所述样品腔的活塞式盖帽，以及设置在所述基板内的扩增反应腔，其中，所述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 1-8 or the instrument according to any one of claims 9-12, wherein the chip device has a substrate , a sample chamber and a piston cap of the sample chamber, and an amplification reaction chamber provided in the substrate, wherein 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,
    所述活塞式盖帽用于使得样品腔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 piston cap is used to increase the gas pressure in the sample 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，优选的，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 amplification reaction chamber is used for amplification reaction of nucleic acid in solution. Preferably, the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated DNA isothermal amplification (LAMP).
    所述方法包括以下步骤：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)用所述活塞式盖帽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) Use the piston cap to 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) Amplify the nucleic acid in the solution in the amplification reaction chamber.
  14. 根据权利要求13所述的方法，其特征在于，所述盖帽具有内螺纹，可以与样品腔上部的外侧的外螺纹形成配合，盖上盖帽并通过旋转使得盖帽以及活塞在样品腔腔体内向下移动。The method according to claim 13, characterized in that the cap has internal threads that can be matched with external threads on the outside of the upper part of the sample chamber. The cap is capped and rotated so that the cap and the piston move downward in the sample chamber. move.
  15. 根据权利要求13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 13, 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.
PCT/CN2023/085818 2022-04-02 2023-04-02 Chip device and instrument for rapid nucleic acid detection, and application thereof WO2023186159A1 (en)

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CN111647498A (en) * 2020-05-25 2020-09-11 清华大学 Integrated self-service nucleic acid detection device and use method thereof
CN113388513A (en) * 2021-06-28 2021-09-14 清华大学深圳国际研究生院 Micro-fluidic chip for nucleic acid extraction, amplification and detection
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