CN116891800A

CN116891800A - Chip device and instrument for detecting nucleic acid and application thereof

Info

Publication number: CN116891800A
Application number: CN202310330468.6A
Authority: CN
Inventors: 赵海峰
Original assignee: Hengtai Medical Co ltd
Current assignee: Hengtai Medical Co ltd
Priority date: 2022-03-31
Filing date: 2023-03-30
Publication date: 2023-10-17
Also published as: CN220703691U; WO2023186084A1

Abstract

The application provides a chip device for detecting nucleic acid in a sample, which is provided with a substrate, a sample cavity, a pressurizing cavity and an amplification reaction cavity arranged in the substrate. The application also provides an instrument for detecting nucleic acid in a sample, in particular a POCT instrument, which adopts the chip device. The application also provides a method for detecting nucleic acid in a sample by using the chip device or the instrument.

Description

Chip device and instrument for detecting nucleic acid and application thereof

The present application claims priority from the following chinese patent applications: the application number 202210329941.4, the application entitled "chip set and apparatus for detecting nucleic acids," and uses thereof, filed on 3/31/2022, are incorporated herein by reference in their entirety.

Technical Field

The application relates to the application fields of biotechnology and equipment, in particular to a chip device and an instrument for detecting nucleic acid in a sample and application of the chip device and the instrument in biological sample detection.

Background

Detection of nucleic acids is central to many fields such as clinical trials, identification of pathogenic microorganism types, and the like. By nucleic acid extraction, amplification and detection analysis, various diseases such as cancer, microbial infection and gene labeling can be detected.

The methods such as PCR and real-time PCR are effective methods for exponentially amplifying and detecting genes. The gene detection market using PCR/real-time PCR devices is rapidly expanding in gene detection of infectious diseases such as viral, sexually transmitted diseases, and influenza. The role of gene detection in cancer therapy has become apparent. However, automation, particularly small automation, of applications employing PCR and RT-PCR is not easy.

Thus, there is also a need in the art for nucleic acid amplification and detection devices and apparatus that are fast, real-time, small and easy to operate, particularly chip devices and apparatus suitable for point-of-care testing (POCT).

Disclosure of Invention

The application provides a chip device for detecting nucleic acid in a sample, which is provided with a substrate, a sample cavity, a pressurizing cavity and an amplification reaction cavity arranged in the substrate, wherein the sample cavity and the pressurizing cavity are communicated through an air flow channel, the sample cavity is communicated with the amplification reaction cavity through a liquid flow channel arranged in the substrate,

wherein, the liquid crystal display device comprises a liquid crystal display device,

the sample cavity is used for containing a sample to be detected and separating nucleic acid in the sample,

the pressurizing cavity is used for increasing the pressure of gas in the cavity so as to squeeze the solution in the sample cavity into the amplification reaction cavity through the liquid flow channel,

the amplification reaction cavity is used for carrying out amplification reaction on nucleic acid in the solution.

In one aspect of the application, the amplification reaction chamber of the chip device comprises a plurality of nucleic acid amplification units, e.g., 2-24.

In one aspect of the application, the top of the sample chamber has a lid that can be opened or closed. In yet another aspect of the present application, the cap is a screw cap having internal threads that mate with external threads on the exterior of the sample chamber.

In one aspect of the application, a reagent solution for preserving and extracting nucleic acids in a sample may be added or preset to the sample chamber.

In one aspect of the application, the reagent solution is a reagent for extracting nucleic acid from a sample by direct extraction.

In one aspect of the application, the plenum chamber chemically generates a gas within its chamber. In one aspect of the application, the chemical process is a chemical reaction that employs a gas-generating reaction, such as an acid-base reaction, or the like. In one aspect of the present application, the gas may be carbon dioxide, hydrogen, oxygen, or the like, and preferably, the gas is a gas that is inactive (reaction-inert) in the nucleic acid detection reaction, such as carbon dioxide or the like. In one embodiment of the application, the chemical reaction that may be used to produce carbon dioxide includes a reaction with an acid using salts such as carbonates or bicarbonates.

In yet another aspect of the application, the amount of gas generated in the plenum is about 1-50 times, such as about 5-20 times, the amount of gas present in the sample and plenum. In yet another aspect of the present application, a barrier layer is disposed within the plenum.

In one aspect of the application, the plenum increases the air pressure within its cavity by way of thermal expansion. In the present application, thermal expansion generally refers to a phenomenon in which a solid or liquid material changes in length or volume due to a change in temperature while pressure is maintained. In one aspect of the application, the thermal expansion may be by way of physical thermal expansion pressurization or chemical thermal expansion pressurization.

In one aspect of the application, a filter is disposed in the air flow path or at an opening thereof to the sample or plenum chamber. For example, a filter membrane arranged at the openings of the air flow passage and the pressurizing cavity, or a filter column arranged in the air flow passage.

In one aspect of the application, a filter chamber may be disposed in the liquid flow path.

In one aspect of the present application, the amplification reaction chamber may be provided with nucleic acid amplification reaction materials.

In the present application, the nucleic acid amplification reaction is a temperature-changing or isothermal amplification method, such as Polymerase Chain Reaction (PCR), strand Displacement Amplification (SDA), nucleic acid sequence-based amplification (NASBA), cascading Rolling Circle Amplification (CRCA), loop-mediated isothermal amplification (LAMP) of DNA, isothermal and chimeric primer-initiated nucleic acid amplification (ICAN), target-based helicase-dependent amplification (HDA), transcription-mediated amplification (TMA), and the like.

In one aspect of the application, the nucleic acid amplification reaction is an isothermal amplification method, such as loop-mediated isothermal amplification of DNA (LAMP).

In one aspect of the application, a partial pressure chamber is further provided at the downstream of the amplification reaction chamber, and the partial pressure chamber is disposed in the substrate of the chip and is in communication with the amplification reaction chamber through a partial pressure gas channel. In still another aspect of the present application, the partial pressure gas channel and the opening connecting the amplification reaction chamber and the partial pressure chamber are provided at top positions of the amplification reaction chamber and the partial pressure chamber.

The application also provides an instrument for detecting nucleic acid in a sample, such as POCT instrument, comprising the chip device of the application, wherein the chip device is provided with a substrate, a sample cavity and a pressurizing cavity, and an amplification reaction cavity arranged in the substrate, wherein the sample cavity and the pressurizing cavity are communicated through an air flow channel, and the sample cavity is communicated with the amplification reaction cavity through a liquid flow channel arranged in the substrate,

In yet another aspect of the present application, the instrument has a chip device receiving system.

In yet another aspect of the application, the instrument has a signal detection module device, such as a fluorescence detection device, for detecting nucleic acid amplification products.

In yet another aspect of the application, the instrument has a system for temperature control of the nucleic acid amplification region of the chip.

In yet another aspect of the application, the apparatus has a nucleic acid amplification result analysis and/or output system.

The application also provides a method for detecting nucleic acid in a sample, comprising employing the chip device of the application or the instrument of the application, wherein the chip device is provided with a substrate, a sample cavity and a pressurizing cavity, and an amplification reaction cavity arranged in the substrate, wherein the sample cavity and the pressurizing cavity are communicated through an air flow channel, and the sample cavity is communicated with the amplification reaction cavity through a liquid flow channel arranged in the substrate,

In yet another aspect of the present application, the method includes the steps of:

(1) Adding a sample into a sample chamber of the chip device, separating nucleic acids in the sample and entering into a solution;

(2) The gas pressure in the cavity of the pressurizing cavity is increased, and the solution in the sample cavity is extruded into the amplification reaction cavity through the liquid flow channel; and

(3) And (3) performing an amplification reaction on the nucleic acid in the solution in the amplification reaction chamber.

In one aspect of the application, the method chemically generates a gas within the cavity of the plenum. Preferably, the amount of gas generated in the plenum is about 1-50 times, for example about 5-20 times, the amount of gas present in the sample and plenum.

In one aspect of the application, the method increases the air pressure within the cavity of the plenum by thermal expansion, such as physical thermal expansion pressurization or chemical thermal expansion pressurization.

In one aspect of the application, the nucleic acid amplification reaction of the method is an isothermal amplification method, such as loop-mediated isothermal amplification of DNA (LAMP).

In one aspect of the application, the detection of the identifiable label carried by the amplified nucleic acid in the method includes, but is not limited to, fluorescence or other forms of luminescence (e.g., chemiluminescence, bioluminescence, radiant luminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallization luminescence, thermoluminescence, sonoluminescence, phosphorescence, photoluminescence, etc.), enzymatic reactions, radioactivity, and the like.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions of the prior art, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it will be obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort to a person skilled in the art.

Fig. 1 is a perspective view of an exemplary chip device for detecting nucleic acids in a sample according to the present application.

FIG. 2 is a schematic diagram of an exemplary chip set-up for nucleic acid detection in a sample according to the present application configured with reagents and working after sample addition. FIG. 2A is a schematic cross-sectional view of an exemplary chip device for detecting nucleic acids in a sample according to the present application after the chip device is configured with reagents. Fig. 2B is a schematic diagram of the operation of the exemplary chip device after sample addition.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present application, are intended to be within the scope of the present application.

Example 1

The application provides a chip device for detecting nucleic acid in a sample. The chip device is typically used to detect the presence and amount of nucleic acids in a sample after extraction and amplification.

The chip devices described herein are applied to analyze any nucleic acid-containing sample used for any purpose, including, but not limited to, genetic testing for human genes and clinical testing for various infectious diseases. The nucleic acid sample for use in the methods described herein may be from any source. In general, a sample may be a biological material that is separated from its natural environment and that comprises 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 comprising polynucleotides. Biological fluids include, as non-limiting examples, blood, plasma, sputum, urine, cerebrospinal fluid, lavage fluid samples. The nucleic acid sample may be from a plant, animal, bacterial or viral source. The samples may be obtained from different sources, including but not limited to samples from different individuals, different stages of development of the same or different individuals, different diseased individuals, normal individuals, different stages of disease of the same or different individuals, individuals undergoing different disease treatments, individuals under different environmental factors, or individuals with predisposition to disease, or individuals exposed to infectious disease agents.

Fig. 1 is a perspective view of an exemplary chip device for detecting nucleic acids in a sample according to the present application. FIG. 2 is a schematic diagram of an exemplary chip set-up for nucleic acid detection in a sample according to the present application configured with reagents and working after sample addition. FIG. 2A is a schematic cross-sectional view of an exemplary chip device for detecting nucleic acids in a sample according to the present application after the chip device is configured with reagents. Fig. 2B is a schematic diagram of the operation of the exemplary chip device after sample addition.

As shown in fig. 1 and 2, the chip device for detecting nucleic acid in a sample provided by the present application comprises a substrate 1, a sample chamber 2 vertically arranged on the substrate, a pressurizing chamber 3 arranged in parallel with the sample chamber 2, and an amplification reaction chamber 4 arranged in the substrate of the chip device. The sample cavity 2 and the pressurizing cavity 3 are cylindrical cavities and are surrounded by walls perpendicular to the bottom plate. The sample cavity 2 is communicated with the pressurizing cavity 3 through an air flow channel 5. The sample chamber 2 communicates with the amplification reaction chamber 4 through a liquid flow path 6 provided in the substrate therebelow. The bottom of the sample chamber 2 has an opening communicating with a liquid flow channel 6 provided in the bottom plate.

The amplification reaction chamber 4 is provided in a substrate of the chip, and can accommodate a reactant or a reaction system for a nucleic acid amplification reaction, and a solution containing the separated nucleic acid from the sample chamber, and can perform the amplification reaction under appropriate conditions. In other aspects of the application, the amplification reaction chamber of the chip device comprises a plurality of nucleic acid amplification units, e.g., 2-24.

The sample chamber 2 is used for accommodating a sample to be detected and separating nucleic acids in the sample. The top of the sample chamber has an openable or closable lid, such as a screw cap 21 as shown in fig. 1, which has an internal thread that mates with an external thread on the outside of the sample chamber. In another embodiment, the cover of the sample chamber is a gland having a stem fixedly connected to the sample chamber.

When a sample needs to be added, the cover of the sample cavity can be opened, and the sample is closed by the cover after being added into the sample cavity, so that the leakage of the biological sample and the pollution caused by the leakage are avoided.

Reagent solutions for preserving (avoiding unwanted decomposition of the nucleic acids to be tested) and extracting the nucleic acids from the sample may be added or preset in the sample chamber.

The chip device for nucleic acid detection in this embodiment is particularly suitable for extracting nucleic acid from a sample by a direct extraction method, i.e., a solution obtained after the sample is contacted and reacted with a nucleic acid extraction reagent contains nucleic acid that can be used in a subsequent amplification reaction. Samples suitable for nucleic acid extraction and amplification by direct extraction include artificially synthesized clone bacterial solution, in vitro transcribed RNA, plasmid, serum, plasma, urine, cotton swab eluate, sputum, alveolar lavage solution and the like. Nucleic acid extraction reagents for extracting such samples generally contain a lysing agent including various surfactants such as SDS, triton, NP-40 and the like, and other chemical reagents such as buffers, protease inhibitors, reducing agents and the like, and various enzymes that lyse components in cell walls or cell membranes, such as Labiase lyase, lysostaphin, egg-derived lysozyme, human-derived lysozyme, achromatopeptidase, streptomyces globosus-derived mutanolysin, chitinase, rhizoctonia solani-derived lyase, arthrobacter gambogi-derived lyase, trichoderma harzianum-derived lyase, streptococcus pyogenes-derived streptolysin O, tetanus-derived tetanus-hemolysin and the like. The main functions of the nucleic acid extraction reagent are as follows: (1) Disrupting the lipid bilayer with a detergent, disrupting the cells; (2) solubilising the protein; (3) promote protein denaturation; (4) inhibiting the activity of proteases and nucleases. Commercially available direct-extraction nucleic acid extraction reagents such as sample releasing agent (model S1014) available from Santa Clara Biotechnology Co., ltd.

The pressurizing chamber 3 serves to generate gas and thereby increase the pressure of the gas in the system formed by the sample chamber 2 and the pressurizing chamber 3, thereby squeezing the liquid in the sample chamber into the amplification reaction chamber 4 through the liquid flow path 6.

The sample cavity 2 is communicated with the pressurizing cavity 3 through an air flow channel 5. The openings of the air flow path 5 connecting with the sample chamber 2 and the pressurizing chamber 3 are located at the upper portions of the sample chamber 2 and the pressurizing chamber 3. The opening position of the air flow channel is required to be higher than the liquid level of the nucleic acid extracting solution added/preset in the sample chamber 2, whereby the increased gas pressure provided by the pressurizing chamber 3 can squeeze the liquid in the sample chamber and transfer it to the amplification reaction chamber 4 through the liquid flow channel 6.

In one aspect of the application, the gas may be chemically generated in the plenum 3. For example, a gas such as carbon dioxide or oxygen is generated by a chemical reaction, and an inert gas which does not affect the nucleic acid extraction reaction and the biological activity of the nucleic acid molecule is preferable. For example, by reacting a strong acid such as hydrochloric acid or sulfuric acid with a solid compound having carbonate or bicarbonate to produce carbon dioxide. In one aspect of the application, a barrier layer is also provided within the plenum 3. The barrier layer may provide a partial barrier to the passage of gas therethrough. If the chemical reaction that generates the gas is too severe, which can cause the liquid to be lifted, the barrier layer can be used to limit the liquid from lifting too high.

In yet another aspect of the present application, the amount of gas generated in the pressurizing chamber 3 is about 1 to 50 times, preferably about 5 to 20 times, the amount of gas (i.e., volume, typically excluding the volume of the nucleic acid processing solution added to the sample chamber) that is present in the sample chamber 2 and pressurizing chamber 3.

In one aspect of the present application, the air pressure within the plenum 3 may be increased by thermal expansion, including physical thermal expansion pressurization or chemical thermal expansion pressurization.

In one aspect of the application, a filter for filtering undesirable substances of the gas (e.g. acid gases or liquids) is provided in the air flow channel 5 or at its opening to the sample chamber 2 or the pressurizing chamber 3. In one embodiment, the filter element is a filter membrane 51 disposed at the opening of the air flow channel and plenum 3. In another embodiment, the filter element is a filter column arranged in an air flow channel, more preferably the air flow channel and the filter column are shaped to taper from one end of the plenum chamber to one end of the sample chamber, whereby movement of the filter column in the air flow channel due to gas pressure is avoided. In the present application, the filter is made of a filter material that absorbs acidic and/or basic substances.

In one aspect of the present application, a filter chamber 61 for filtering unwanted substances (e.g., cells, cell debris, or large protein molecules, etc.) in the solution containing the sample nucleic acid may be provided in the liquid flow channel 6. In the present application, the filter chamber contains a filter material having a pore size that allows nucleic acids (including genomic nucleic acids or fragments thereof, etc.) in solution to pass freely therethrough, trapping tissue fragments, cells and cell fragments or large protein molecules, etc. In the present application, the filter material employed in the filter chamber does not physically adsorb or substantially physically adsorb nucleic acids in solution, and does not react or inhibit nucleic acids.

The nucleic acid amplification reaction can be performed in the amplification reaction chamber 4 of the chip device for detecting nucleic acid in a sample provided by the application. Various nucleic acid amplification methods using primers known in the art can be used in the present application, including variable temperature or isothermal amplification methods such as Polymerase Chain Reaction (PCR), strand Displacement Amplification (SDA), nucleic Acid Sequence Based Amplification (NASBA), cascading Rolling Circle Amplification (CRCA), loop-mediated isothermal amplification of DNA (LAMP), isothermal and chimeric primer-initiated nucleic acid amplification (ICAN), target-based Helicase Dependent Amplification (HDA), transcription Mediated Amplification (TMA), and the like.

In one embodiment of the application, isothermal amplification methods such as LAMP are used. The chip device further comprises a temperature control unit, for example with a temperature regulator, for the amplification reaction chamber 4, so that the amplification reaction chamber is kept at a constant temperature.

In one aspect of the present application, a partial pressure chamber 41 is further provided downstream of the amplification reaction chamber 4 (downstream in the gas flow direction). The partial pressure cavity is arranged in the substrate of the chip and is communicated with the amplification reaction cavity 4 through a partial pressure gas channel 42. The partial pressure chamber shares the pressure when the air in the amplification chamber is compressed on the one hand, avoids the adverse effect on the chip caused by the overhigh pressure, and on the other hand can avoid the overlarge area of the amplification reaction chamber to influence the nucleic acid amplification efficiency and signal observation. The partial pressure gas channel 42 and the opening connecting the amplification reaction chamber and the partial pressure chamber are provided at the top positions of the amplification reaction chamber and the partial pressure chamber.

As shown in fig. 2A, in an exemplary working scenario, the initial state of the chip device for nucleic acid detection in a sample is: a sample preservation solution (namely nucleic acid extracting solution) 71 is preset in the sample cavity 2, and the liquid level of the sample preservation solution 71 is lower than the air flow passage 5 between the sample cavity 2 and the pressurizing cavity 3; a first reactant 83 (e.g., sulfuric acid) and a second reactant 84 (e.g., sodium bicarbonate) that generate a chemical reaction-generating gas upon contact are preset in the pressurizing chamber 3, with a separation medium 82 (e.g., paraffin that melts at a higher temperature) between the first reactant 83 and the second reactant 84 that is meltable or decomposable under appropriate conditions. The first reactant and/or the second reactant disposed within the plenum 3 may also have a separation medium 81 disposed above it that may melt or decompose under suitable conditions. In addition, a reactant or reaction system 95 for the nucleic acid amplification reaction, such as lyophilized particles comprising one or more of a nucleic acid amplification enzyme, a substrate, and a buffer, is preset in the amplification reaction chamber 4.

As shown in fig. 2B, in an exemplary working scenario, the workflow of the chip device for nucleic acid detection in a sample is: opening a cover of the sample cavity, putting a sample to be detected (such as a throat swab) into the sample cavity, and performing nucleic acid separation reaction on a sample preservation solution to release nucleic acid into the solution; heating the pressurizing chamber 3, which causes the separation medium 81/83 to melt, the first reactant 83 (e.g., sulfuric acid) and the second reactant 84 (e.g., sodium bicarbonate) to contact and react, and generate a gas (e.g., carbon dioxide), the increased gas pressure acting through the air flow path onto the sample-holding liquid of the sample chamber; along with the increase of the air pressure, the sample preservation solution containing nucleic acid moves into the amplification reaction cavity 4 through the liquid flow channel 4, and the amplification raw materials preset in the amplification reaction cavity are redissolved; air originally existing in the flow channels and the reaction cavity is compressed; then, performing nucleic acid amplification reaction in an amplification reaction chamber, copying (amplifying) the characteristic nucleic acid of the detected pathogen, and generating a corresponding optical signal; finally, discrimination is carried out by means of visual inspection or optical device detection.

In this embodiment, the substrate, sample chamber and plenum chamber are made of rigid materials. Including but not limited to silica, silicon, quartz, glass, or polymeric materials (e.g., PDMS, plastic, etc.). The liquid flow channels provided in the substrate are typically microfluidic channels having dimensions on the order of millimeters, for example having a cross-section of the flow channels of about 0.1-5mm.

In one aspect of the application, since the nucleic acid is detected by detecting visible or fluorescent signals carried by the amplified nucleic acid, the walls of the cylindrical cavity and the floor may be fabricated from materials that fully or substantially absorb the target signal (e.g., fluorescence) in order to avoid or minimize interference with signals from adjacent nucleic acid amplification regions. While the top/bottom of the amplification reaction chamber is made of or closed with a material that does not absorb at all or substantially absorb the signal to be detected. Thus, the signal detection module provided in the chip or an external detection system can detect the fluorescent 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 nucleic acid detection in a sample provided by the present application may employ any means for detecting identifiable labels carried by nucleic acids, including but not limited to fluorescent or other forms of luminescence (e.g., chemiluminescence, bioluminescence, radiant luminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallization luminescence, thermoluminescence, sonoluminescence, phosphorescence, photoluminescence, etc.), luminescence, enzymatic reactions, radioactivity, and the like.

In one aspect of the application, detection of amplified nucleic acids is by detection of fluorescent signals carried by the nucleic acids. The primers and oligonucleotides contained in the reaction system of the amplification reaction chamber may be detectably labeled, either radioactive, fluorescent or non-radioactive, by methods well known to those skilled in the art. The commonly used fluorescent dyes and their signal-related wavelengths are shown in table 1 below.

TABLE 1 fluorescent dyes

Dye name	Excitation wavelength	Emission wavelength
			FAM/SYBGREEN	492nm	516nm
HEX/JOE/VIC	535nm	555nm
			ROX/TEXRED	585nm	610nm
CY5	635nm	665nm

In one aspect of the application, the top or bottom of the amplification reaction chamber may be of a material that does not absorb fluorescent signals generated by the amplification reaction. In addition, the detection result can be obtained by observing or collecting the optical signals generated by each amplification reaction unit through human eyes or various known optical systems (including optical filters, cameras and the like).

Example 2 test

The nucleic acid detection chip shown in FIG. 1 is prepared by taking PDMS as a material of a substrate, a sample cavity and a pressurizing cavity. Wherein the thickness of the substrate 1 is about 2.0mm; the cavity volume of the sample cavity 2 is about 1.6ml, and the inner diameter is about 7.0mm; the volume of the pressurizing 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 about 25.0ul and each with a volume of about 6.0ul. The cross-section of the liquid flow channel has a size of about 0.4mm x0.5mm.

About 500ul of sample releasing agent (Changzhou Jin Maige Biotechnology Co., ltd., su Changxie/20200123) was added to the sample chamber. According to the instructions of the reagent provider, the test principle of the sample releasing agent is that a protein denaturing agent and a biochemical reagent are utilized to quickly destroy the viral capsid structure in a throat swab sample, so that viruses are cracked and nucleic acid is released; meanwhile, the nucleic acid stabilizer can effectively prevent the degradation of nucleic acid, thereby achieving the effects of extracting and preserving nucleic acid. The method is applicable to the types of specimens: a pharyngeal swab sample or a body fluid sample.

The bottom of the pressurizing chamber 3 is provided with about 126mg NaHCO ₃ Paraffin is added for sealing and wrapping, then about 65ul of 10N HCl is added, and paraffin is added on the HCl to form a separation layer.

The reagent provided by SARS-CoV-2 novel coronavirus RT-LAMP kit (N gene) of Beijing Bai Oy Lai Bo technology Co., ltd.) is used as test reagent, which comprises SARS-CoV-2N gene RT-LAMP positive control.

The SARS-CoV-2N gene RT-LAMP primer mixture and 2 xLAMP MagicMix in the kit are processed to form freeze-dried powder, and then added into an amplification reaction cavity.

Opening the nut above the sample cavity, and stretching the nasopharyngeal swab adsorbed with the SARS-CoV-2N gene RT-LAMP positive control into the sample cavity to break, so that the swab head falls into the sample preservation solution. After the screw cap is covered, the liquid in the sample cavity is subjected to ultrasonic treatment. Viral nucleic acids are shed from the swab head and into solution.

The pressurizing chamber is heated to about 70 ℃, paraffin is melted, HCl and NaHCO are melted ₃ React and produce CO ₂ And (3) gas. Along with the increase of air pressure, the sample preservation solution is extruded into the amplification reaction cavity through the flow channel, and the amplification raw materials preset in the amplification reaction cavity are reconstituted.

Nasopharyngeal swabs, to which SARS nucleic acid was not adsorbed, were added to the other chip as controls.

The amplification reaction chamber was heated to 61℃and observed after 1 hour of reaction.

Nasopharyngeal swabs, which did not adsorb SARS nucleic acid, were added to the other chip as controls, and the rest of the procedure was the same as that of the experimental group.

The result shows that the amplification reaction chamber of the positive detection chip is blue. The amplification reaction chamber of the negative control exhibited a very light blue color.

Example 3 apparatus for detection of nucleic acids in samples

In one embodiment, the application provides an apparatus for detecting nucleic acids in a sample, which is a POCT apparatus, comprising the chip device defined and described in example 1.

The apparatus has a chip device receiving and motion control system for receiving the chip device and performing various processes on the chip, including heat treatment.

The instrument may also have a signal detection module, such as a fluorescent detection system, for detecting nucleic acid amplification products.

The instrument has a system for temperature control of the nucleic acid amplification region of the chip.

The apparatus has a nucleic acid amplification result analysis and/or output system.

The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the application.

Claims

1. A chip device for detecting nucleic acid in a sample is provided with a substrate, a sample cavity, a pressurizing cavity and an amplification reaction cavity arranged in the substrate, wherein the sample cavity and the pressurizing cavity are communicated through an air flow channel, the sample cavity is communicated with the amplification reaction cavity through a liquid flow channel arranged in the substrate,

the sample chamber is used for containing a sample to be detected and separating nucleic acid in the sample, optionally, a reagent solution for preserving and extracting the nucleic acid in the sample can be added or preset in the sample chamber,

the amplification reaction chamber is used for performing an amplification reaction of nucleic acids in solution, and optionally comprises a plurality of nucleic acid amplification units, e.g. 2-24.

2. The chip device of claim 1, wherein the top of the sample chamber has a lid that can be opened or closed.

3. Chip device according to claim 1, wherein the pressurizing chamber generates gas in its chamber by chemical means, preferably in an amount of about 1-50 times, such as about 5-20 times, the amount of gas present in the sample chamber and pressurizing chamber.

4. Chip device according to claim 1, wherein the pressurizing cavity increases the air pressure in its cavity by means of thermal expansion, such as by means of physical thermal expansion pressurizing or chemical thermal expansion pressurizing.

5. Chip device according to claim 1, wherein a filter element is arranged in the air flow channel or at the opening of the air flow channel and the sample chamber or the pressurizing chamber, for example a filter membrane arranged at the opening of the air flow channel and the pressurizing chamber or a filter column arranged in the air flow channel.

6. The chip device according to claim 1, wherein a material for nucleic acid amplification reaction, preferably isothermal amplification method such as loop-mediated isothermal amplification of DNA (LAMP), can be preset in the amplification reaction chamber.

7. The chip device according to claim 1, wherein a partial pressure chamber is further provided downstream of the amplification reaction chamber, the partial pressure chamber being provided in the substrate of the chip and communicating with the amplification reaction chamber through a partial pressure gas passage.

8. An instrument for the detection of nucleic acids in a sample, preferably a POCT instrument, comprising a chip device according to any of claims 1-7, wherein the chip device 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 in communication via an air flow channel, and the sample chamber is in communication with the amplification reaction chamber via a liquid flow channel arranged in the substrate,

the amplification reaction cavity is used for carrying out amplification reaction on nucleic acid in the solution,

optionally, it further comprises one of:

a chip device receiving system;

signal detection module means for detecting nucleic acid amplification products, such as fluorescent detection means;

a system for temperature controlling the nucleic acid amplification region of the chip;

and (3) analyzing and/or outputting a nucleic acid amplification result.

9. A method for detecting nucleic acid in a sample, comprising employing the chip device of any one of claims 1 to 7 or the instrument of claim 8, wherein the chip device has a substrate, a sample chamber and a pressurizing chamber, and an amplification reaction chamber provided in the substrate, wherein the sample chamber and the pressurizing chamber are communicated with each other through an air flow path, and the sample chamber is communicated with the amplification reaction chamber through a liquid flow path provided in the substrate,

the method comprises the following steps:

10. A method according to claim 9, wherein the gas is generated chemically in the cavity of the pressurizing cavity, preferably in an amount of about 1-50 times, such as about 5-20 times, the amount of gas present in the sample cavity and pressurizing cavity.