CN115704049A - Nucleic acid detection method and detection reactor - Google Patents

Abstract

The invention provides a nucleic acid detection method and a detection reactor, wherein the method comprises the following steps: s1, preparing a reaction system and a sample preservation solution and adding the reaction system and the sample preservation solution into a reactor; s2, adding a sample to be detected into the sample preservation solution to obtain a sample solution; s3, pressing the sample liquid into a reaction system through an external force; and S4, carrying out amplification reaction on the reaction system to read a reaction result, and judging the detection result through colorimetry or fluorescence. The reactor comprises a sample adding part, a sample part and a reaction part, wherein the sample adding part and the sample part and the reaction part are movably connected to realize a sealing state; the sample adding part moves towards the reaction part through external force to press sample liquid into the reaction part through the micropores in a sealed state. The reaction process of the invention does not need to open the cover and add liquid for many times, and the whole nucleic acid detection can be realized without a PCR laboratory and aerosol pollution.

Description

Nucleic acid detection method and detection reactor

Technical Field

The invention relates to the technical field of nucleic acid detection (DNA or RNA), in particular to a nucleic acid detection method and a detection reactor.

Background

Nucleic acid detection, as a method having high sensitivity and specificity, has been widely used in many fields such as disease diagnosis, food safety, infectious disease control, and the like. Detection of specific nucleic acid sequences in a simple manner can confer greater value in point-of-care (point-of-care) diagnostics and in point-of-care pathogen detection.

PCR (polymerase chain reaction) is a molecular biological technique for amplifying and amplifying a specific DNA fragment, and can be regarded as special DNA replication in vitro. However, PCR, a classical nucleic acid detection method, has inherent denaturation-renaturation-extension cycles, which require that a thermal cycler apparatus be used as a support, and a professional laboratory is one of the necessary conditions because of aerosol contamination. Currently, the PCR extension technology platform, particularly the quantitative PCR (qPCR) method, is the most widely used pathogen detection method and is considered a new gold standard test. qPCR provides a much shorter sample-to-result time (3 to 5 hours). However, although qPCR is widely accepted, it is limited by relying on standard reference substances (standard curves) for quantification. Unreliable and inconsistent commercial standard reference materials may also affect the accuracy of qPCR quantification. In addition, qPCR is susceptible to inhibition by naturally occurring substances in environmental samples (e.g., heavy metals and organic matter), leading to inaccurate or false negative results in target quantification. Therefore, the application of PCR in the fields of point-of-care rapid diagnosis (POCT), on-site rapid detection and the like is greatly limited. Compared to qPCR, recent digital PCR techniques have proven to be more robust solutions for the detection of microbial pathogens in environmental samples. Digital PCR is based on partitioning (partioning) and poisson statistics, so there is no need to compare external quantification standards to quantify samples of unknown concentration. However, implementing digital PCR methods in point-of-use applications can be challenging. This is because digital PCR requires expensive instrumentation (i.e., bio-rad droplet digital PCR), a fully equipped laboratory environment, and trained technicians to perform the assays. These factors severely limit the accessibility and applications of digital PCR in resource-limited contexts.

To overcome these disadvantages, a large class of new methods for isothermal nucleic acid amplification has emerged, with LAMP being the most interesting and promising method.

Loop-mediated isothermal amplification (LAMP) is an alternative PCR nucleic acid amplification method developed by Nippon Rongyan chemical company, 2000. It is characterized by that 4 specific primers are designed according to 6 regions of target gene, under the action of strand displacement DNA polymerase (Bst DNA polymerase), the amplification is implemented at constant temp. of 60-65 deg.C for about 15-60 min ⁹ ～10 ¹⁰ The nucleic acid amplification is simple to operate, strong in specificity, easy to detect products and the like. LAMP, as a molecular biology detection technology, has the characteristics of high specificity, high sensitivity, simplicity, convenience and low cost, and is widely used for diagnosis of clinical diseases, qualitative and quantitative detection of epidemic bacteria or viruses, sex identification of animal embryos and gene chips.

Thus, LAMP is expected to be a rapid, simplified, low cost assay for detecting microorganisms to provide molecular assays outside of a centralized laboratory, for example, where on-site point-of-use testing of environmental water in resource-limited locations is required.

LAMP detection is performed under isothermal conditions, which may be maintained in different instruments, such as a thermal cycler and a water bath, or LAMP detection device as used herein. The apparatus enables amplification of DNA/cDNA from a sample by heating a detection chamber inside the device to detect pathogens.

In the assays developed on the devices mentioned in the present application, in general, an aliquot of the biological sample under analysis is added to the reagent and the set (set) is heated in the LAMP detection chamber. These samples are then monitored during the test to identify a possible color change (e.g., from purple to sky blue when using hydroxynaphthol blue reagent) that indicates a positive reaction of the sample with respect to the reagent used. Various configurations of LAMP detection chambers are known from the prior art and will be described in the following paragraphs.

The article "A novel CMOS image sensor System for quantitative loop-mediated isothermal amplification assay to detect food-borne pathogens" published by Wang, TT on "Journal of microbiological methods" proposes a low cost CMOS image sensor system for LAM detection to detect food-borne pathogens. The described system monitors in real time the photon changes caused by color changes during the amplification process. The article finally indicates that a simple, compact, low cost and low power design represents a significant advance in the development of portable, sensitive, easy to use, real-time quantitative analysis tools for field diagnostics.

Ahmed, ME in "BMC on the vehicle Research" Development and evaluation of real-time loop-mediated isothermal amplification assay for Rapid detection of cystic echinococcosis "describes the Development and evaluation of real-time LMAP detection for the rapid detection of cystic echinococcosis. The detection was performed at constant temperature (63 ℃) with real-time monitoring using amplification and detection instruments and fluorescent dyes. After amplification cycles in a water bath, the LAMP product was visually observed to detect a color change and visualized under a UV light source.

The article "A microfluidic lab-on-a-disc integrated loop-mediated isothermal amplification for food pathogen detection" published by Sayad, AA on "Sensors And Actuators B-Chemical" reports a LAMP amplification centrifugal microfluidic device for detection of food pathogens in which a forced convection LAMP heating source is used to drive a wax valve And temperature heating for amplification.

Document WO2013043203 relates to a container for LAMP assay of isothermal and non-isothermal nucleic acids comprising a body having an inner/outer surface and an open end in addition to a plug-in lid (plug-in lid) having a body open end and a flexible material extending through a channel. According to this document, the developed container guarantees greater flexibility compared to the known microfluidic chips, due to its inherent flexibility of handling small and large volumes of fluid. Another significant advantage is that the disclosed container also allows for sample extraction and fluid transfer functionality.

The document US20160231324 describes a high performance multiplex system for detecting targets comprising encapsulating a biological sample with a detection system or sensor comprising, for example, a dnase capable of producing a detectable signal and detecting the signal, wherein the detection system or sensor comprises LAMP-based detection. According to this document, the sample to be analyzed can be heated, wherein the system comprises light emitting diodes and detectors in addition to data analysis software, visual displays for transmitting information, electronics connections, and other features.

Document WO2011150115 relates to a LAMP method and device for in situ detection of nucleic acids in a sample. Methods used are described including introducing nucleic acid amplification reagents and heating the nucleic acid amplification reagents. According to this document, the heating step is performed using a disposable heater, and the detecting step includes detecting a color change of a colorimetric dye in fluid communication with the nucleic acid amplification reagent. Little detail is provided about the construction of the device.

Document US20140356874 discloses methods and devices for portable nucleic acid amplification and detection, wherein the instrument preferably uses isothermal nucleic acid amplification techniques, such as LAMP. Detection of target amplification may be achieved, for example, by detecting a shift in color or fluorescence of a dye added to the amplification reaction. According to this document, the device disclosed comprises a heating chamber for heating the sample and a sensor for detecting a color change of the sample, wherein the elements can be controlled by a central control system.

Document US9476836 discloses a method for Detecting Nucleic Acids (DNA) in a sample (e.g. blood) by the LAMP method, which comprises contacting the nucleic acids with a detection reagent within a closed system and observing the color change of the nucleic acids and/or the detection reagent. It is further described that the observation of the color change of visible light can also be performed by measuring the absorbance of the sample solution in the visible light range. However, little information is provided about the overall construction details of the device, such as the arrangement of sensors and light sources, display devices, etc.

Document US20130331298 discloses a test cartridge for detecting a variety of analytes, including pathogens, comprising an injection port, a central channel, a processing chamber, a reagent container and a waste chamber. It is described that the detection may be of the LAMP type. The device also includes a series of elements such as heaters, heater controllers, optical sensors for measuring flow in the cartridge. The use of sensors to identify color changes in the sample under analysis is not described.

The document CN 109355429A discloses a loop-mediated isothermal amplification (LAMP) -based micro-fluidic chip based circulating nucleic acid detection kit and an application method thereof, and the invention develops a new high-sensitivity circulating nucleic acid analysis technology which takes functionalized microspheres under microscopic conditions as a sensing element by taking circulating nucleic acid as an analysis object, taking a micro-fluidic dynamic micro-array technology as a support, taking a loop-mediated isothermal amplification technology, a rolling loop amplification technology, a material transfer enhancement effect based on micro-fluidic and high fluorescence quantum yield characteristics of graphene quantum dots as a signal amplification means. The kit can better solve the problem that the characteristics of high flux and high sensitivity are difficult to coexist under the condition of trace samples in the technical field of trace medical analysis or the fields of minimally invasive and noninvasive diagnosis. The technology only needs 2 mu L of virus DNA samples, can realize the detection of 10amol/L of EB virus DNA, namely 10 EB virus DNA can be detected in a single experiment. However, in the detection of EB virus DNA outside a chip, the kit still needs to pretreat a sample, needs to be manually added into a sample inlet hole, is not a closed reaction environment, still has the possibility of aerosol pollution, and always needs to be operated in a professional laboratory.

The document CN 111902212A discloses a LAMP detection apparatus including a heating chamber adapted to accommodate a support rail of at least one sample, wherein the support rail is inserted into the heating chamber through a sample insertion opening, and further, the heating chamber includes: at least one internal heating element; a light emitting element circuit on the front wall or the rear wall; a photosensor circuit located on a wall opposite the light emitting element circuit. The device achieves major cost reductions and differences by temperature control using thermal inertia alone, as opposed to commercial systems that use removal and/or cooling techniques to control temperature, thereby increasing equipment costs and energy consumption. However, the device does not realize the traditional operation of sample-adding-free in the experimental process, and still adopts the mode of eight connecting pipes for operation.

Because of the limitations of nucleic acid detection reagents and detection equipment based on amplification, the problem of extraction of nucleic acid or other samples to be detected cannot be solved by amplification operation in the existing detection, multiple uncovering is also needed in the amplification process, especially when an eight-connected tube is used as a reactor, and operation in a professional PCR laboratory is also needed to avoid pollution, so that the nucleic acid detection in the prior art cannot realize field sampling and field detection, especially no reactor capable of directly completing reaction at one time after directly adding a sample is available, the traditional eight-connected tube or EP tube (centrifugal tube) is still adopted, which is an important toggle for the nucleic acid detection not to be well applied to POCT and the development and application of pathogenic microorganisms.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the nucleic acid detection method is more convenient, low in cost and accurate, and the nucleic acid detection method and the detection reactor can realize direct POCT or on-site quick detection without depending on various limitations such as professional PCR laboratories, laboratory personnel operation and the like.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a method for detecting nucleic acids, the method comprising the steps of:

s1, preparing a detection reagent: selecting an amplification method according to an object to be detected and adaptively designing a detection reagent, in other words, an isothermal amplification reaction system is adopted for isothermal amplification, and a variable temperature amplification reaction system (namely PCR) is adopted for variable temperature amplification; preparing a detection reagent (not containing a sample to be detected) and then adding the detection reagent into the reactor in advance; the detection reagent comprises a reaction system and a sample preservation solution, and the reaction system and the sample preservation solution are respectively and independently arranged in the same reactor;

s2, taking a sample to be detected and adding the sample to be detected into a sample preservation solution to obtain a sample solution, wherein the sample is a sample source from which nucleic acid can be obtained;

s3, adding a sample liquid, pressing the sample liquid into a reaction system through an external force, wherein the addition amount of the sample liquid is less than 100 microliters;

s4, carrying out amplification reaction on the reaction system, reading a reaction result to complete detection, and judging the detection result through colorimetry or fluorescence;

wherein:

the reaction system is 10-200 microliter, and at least comprises: reaction enzyme, primer, dNTP, buffer and dye, wherein the reaction system of S1 is in an unreacted state;

adaptively designing a specific primer sequence according to the nucleic acid to be detected,

the sample is selected from: nasopharyngeal secretions, intestinal secretions, respiratory secretions, genital secretions, or blood samples.

In a preferred embodiment, when the nucleic acid to be detected is RNA, the reactive enzyme comprises RNA-reverse transcriptase; and when the isothermal amplification reaction system is adopted, the reaction enzyme also comprises BST polymerase; or when the temperature-variable amplification reaction system is adopted, the reaction enzyme also comprises Taq polymerase.

In a preferred embodiment, when the nucleic acid to be detected is DNA, the reaction enzyme of the isothermal amplification reaction system includes BST polymerase, and the reaction enzyme of the temperature-variable amplification reaction system includes Taq polymerase.

As a preferred embodiment, the dye is a chromogenic dye or a fluorescent dye, wherein the chromogenic dye is preferably: HNB, neutral red dye, calcein, acid Chrome Blue K (ACBK), ammonium molybdate or visible light dyes; the fluorescent dye is preferably: SYBR Green, SYBR Gold, SYBR-safe, evaGreen, RTGreen, SYTO-9, gel Green, gel Red, EB dyes or Gold View I dyes.

As a preferred embodiment, the sampling means is preferably swab sampling; after sampling, putting the swab with the nucleic acid into a reaction tube added with a sample preservation solution to obtain a sample solution.

As a preferred embodiment, the transportation and storage requirements of the reaction system in the detection method are reduced, normal-temperature transportation can be realized, and the reaction system in S1 is in a freeze-dried state; in order to further reduce the environmental sensitivity of the reaction system, at least one component of the reaction enzyme, the primer and the dNTP in the reactor in the S1 and the other two components are independently and respectively added into the same cavity of the reactor through a preheating meltable sealing layer, in other words, after the S4 is heated, the reaction system is in a reaction liquid state after the sealing layer is melted, and the amplification reaction is carried out.

In a preferred embodiment, the sealing layer has a melting point of 50 to 70 ℃ and a freezing point of 0 to 40 ℃, and is preferably paraffin, silicone oil, octadecane or the like, and preferably has a density less than that of water.

As a preferred embodiment, the reaction system further comprises a lyoprotectant, preferably, a mixture of one or more of the following: trehalose, sucrose, polysucrose, dextran, sorbitol, polyvinylpyrrolidone, polyethylene glycol, mannitol, dextran, glutamic acid, glycine, histidine, glycerol or human serum albumin.

The invention also provides a reactor for realizing the nucleic acid detection method, which comprises a sample adding part, a sample part and a reaction part which are connected in sequence, wherein the sample adding part, the sample part and the reaction part are movably connected to realize a sealing state, the sample adding part is of a piston structure, a sample preserving fluid is pre-filled in the sample part, and a reaction system is filled in the reaction part; micropores are arranged at the joint of the reaction part and the sample part, and the aperture of the micropores is not more than the capillary length of the sample liquid or the sample preservation liquid (at this time, the sample liquid or the sample preservation liquid of the sample part cannot flow into the reaction part under the natural state, namely under the action of gravity or without the action of external force); in other words, the surface tension of the liquid at the micropores is greater than its gravity, and the pore diameter of the micropores is 0.3 to 0.6mm; under the sealed state of the reactor, the sample adding part moves towards the reaction part by external force to realize that the sample liquid passes through the micropores and is pressed into the reaction part under the sealed state.

As a preferred embodiment, a plurality of reaction chambers are arranged in the reaction portion, an independent reaction system is preset in each reaction chamber, so that simultaneous detection of a plurality of detection items for the same sample can be realized, the micropores are arranged at the openings of the reaction chambers, and each reaction chamber is provided with one micropore, so that liquid cannot flow into the reaction chambers from the sample portion without external force. The reaction part can be provided with a reaction cavity and a plurality of reaction cavities, each reaction cavity corresponds to a micropore, and the micropores are matched with the reaction cavities.

As a preferred embodiment, the reaction part is provided with a shunt plug, the shunt plug is made of a flexible material, the shunt plug is located at the joint of the reaction part and the sample part, the reaction chamber is independently sealed by the shunt plug, and the micro-hole is formed in the shunt plug.

As a preferred embodiment, one surface of the shunting plug, which faces the reaction cavity, protrudes to close the cavity opening, the micro-hole penetrates through the protrusion, and one surface of the shunting plug, which faces the sample part, is a smooth surface.

In a preferred embodiment, the reaction part is provided with an inner reaction tube and an outer sleeve which are sleeved, the inner reaction tube is clamped in the outer sleeve, and the outer sleeve is provided with a thread which is in coordination threaded connection with the sample part.

In a preferred embodiment, the inner reaction tube is provided with a plurality of reaction cavities, and the outer sleeve is provided with threads.

As a better implementation mode, the sample adding part comprises a sleeved concave plug and a sample adding cap, a hard convex part inserted into the concave plug and sleeved is arranged in the sample adding cap, the sample adding cap is also provided with a threaded connection section in coordination and screw connection with the sample part, so that the sealed connection between the sample adding part and the sample part can be realized, and the sample liquid can enter the reaction part by pressurizing the sample part through unidirectional rotation.

As a further preferred mode, the sample adding part is further provided with a limiting part, and the limiting part is used for limiting the rotation of the sample adding part; in other words, the sample addition part can be rotated after the stopper is removed before the sample addition part is pressurized. Preferably, the locating part is a tear ring, the tear ring is connected with application of sample cap and sample end port respectively, and it can realize spacing between application of sample portion and the sample portion to tear the ring this moment.

The port of the sample part near the reaction part is closed, and is in an independent sealing state when no sample is added, and the port is closed through a sealing plug or a sealing film.

In order to further ensure the sealing effect between the sample part and the sample adding part and reduce the production cost, the sample part is a coaxial sleeve, wherein the inner tube is a hollow tube, the inner diameter of the inner tube is in sealing fit connection with the concave plug, and one end of the outer tube is provided with a thread which is in coordination threaded connection with the sample adding part; when the reactor is sealed, one section of the inner tube is positioned in the gap between the concave plug and the sample adding part; the sample preservation solution is pre-arranged in the cavity of the inner tube before reaction.

In addition, in order to further guarantee the sealed effect and reduce manufacturing cost simultaneously between sample portion and reaction portion, the one end that the outer tube of sample portion is close to the reaction portion is equipped with the screw thread with reaction portion coordination spiro union, reaction portion and outer tube do not communicate, and the outer tube and the seamless laminating of inner tube of screw thread department.

And in order to avoid misassembly or misoperation in the production process and the detection process, a limiting ring extends outwards from one end port of the inner tube of the sample part, which is close to the reaction part.

As a preferred embodiment, in the method for detecting nucleic acid using the above-mentioned reaction vessel, S3 further comprises the steps of:

s3-1, adding the sampled swab into the sample part, sealing the reaction part and the sample part, and applying external force to uniformly mix the sample liquid; the specific operation is as follows: unscrewing a sample part and a reaction part of the reactor, adding the sampled swab into the sample part, screwing the reaction part and the sample part, and shaking and uniformly mixing the reactor;

s3-2, pressurizing and sampling the sample part through the sampling cap under the condition of not damaging the sealing of the reactor; the specific operation is as follows: tearing the tearing ring (easy to tear), and rotating the sample adding cap to the sample part to add sample under pressure;

s3-3, applying a force to the reactor, wherein the force is in the same direction as the force of the reactor, so that the sample liquid hung on the top of the reaction part enters the reaction cavity, and the specific operation is as follows: the reactor was then shaken (flicked) in a downward direction so that the sample solution hanging from the top of the reaction part entered the bottom of the reaction chamber.

As another preferred embodiment, S4 further includes the following steps:

s4-1, putting the reactor into a quick detection device or a heating device, heating, then sealing, melting a reaction system by a barrier layer, mixing the reaction system with a sample solution to obtain a reaction solution,

s4-2, maintaining the temperature in the reactor to the amplification temperature, and oscillating or rotating the reactor to fully and uniformly mix the reaction liquid for amplification reaction; to ensure the reaction is complete, the reactor may be oscillated or rotated repeatedly.

Compared with the prior art, the nucleic acid detection method and the reactor provided by the invention have the following advantages:

(1) The nucleic acid detection method and the reactor can solve the problems of pollution, complex operation and the like in the amplification method in the prior art, and the method can realize the pre-addition of the reaction system in the amplification operation, and the pre-added reaction system not only avoids the limitation of the field configuration of the reaction system on the environment, simplifies the system configuration steps before detection, but also can ensure the rapid detection and the simple and convenient use; the subsequent amplification only needs to add a sample to be detected, the reaction can be directly carried out after the amplification reaction conditions such as temperature are reached, liquid adding again is not needed, so that the reactor only needs to be opened once to add the sample to be detected in the detection process, the sample is not in contact with other components in the reaction system, the reaction can be directly carried out through full contact in the reaction process, the cover opening is not needed again, the whole nucleic acid detection process can be realized, the detection conditions are basically unlimited, no aerosol pollution exists, and the result can be obtained by processing the reactor after the detection reaction is finished.

(2) The nucleic acid detection method can design specific primers aiming at the nucleic acid to be detected, and can simultaneously detect a plurality of nucleic acids to be detected in one sample to be detected by pre-burying different reaction systems when one tube of reaction chambers are multiple, so that the detection efficiency is improved, the detection results of the related nucleic acids can be jointly considered, and a more accurate gene level suggestion is provided for clinical judgment.

(3) According to the nucleic acid detection method and the reactor, the special design of the sample adding part can control the adding amount of the sample preserving fluid, so that mutual pollution can not be caused while the reaction in the reaction cavity is fully carried out.

(4) The pore diameter of the reactor micropore is set, so that the sample liquid or the sample preservation liquid does not enter the reaction cavity in the processes of storage, transportation, adding of a sample to be detected and the like, and the sample liquid can smoothly enter the reaction cavity for full reaction under the condition of applying external force.

(5) The nucleic acid detection method and the reactor have the advantages of simple overall structure, low production cost, environment-friendly material and strong adaptability of the cylindrical pipe body, can be used for various types of heating equipment, and are convenient for applying external force to ensure full reaction.

(6) According to the nucleic acid detection method and the reactor, the melting temperature of the material of the sealing layer is matched with the heating reaction temperature, the amplification reaction system is not influenced, the sealing layer is melted after heating, the reaction system is fully mixed under the condition of not opening the cover, the melted material of the sealing layer floats on the reaction system due to the small density, the material of the sealing layer further forms the sealing layer on the reaction system, and double guarantee is provided for fully carrying out the reaction.

(7) The nucleic acid detection method and the nucleic acid detection reactor can be realized by matching simple heating equipment (even a vacuum cup) with the nucleic acid detection method and the nucleic acid detection reactor aiming at public health events, do not need to be operated by professionals, have clear and easily-judged results, are suitable for various medical detection scene requirements at home and abroad at present, and can greatly improve the molecular diagnosis capability of the nucleic acid detection method and the nucleic acid detection reactor especially in relatively laggard areas.

Drawings

FIG. 1 is a schematic diagram of a reactor configuration according to one embodiment of the present invention.

Fig. 2 is a schematic diagram of an exploded structure of a reactor according to an embodiment of the present invention.

Fig. 3 is a schematic view of a diverter plug according to an embodiment of the present invention.

Fig. 4 is a schematic structural diagram of a fast inspection apparatus according to an embodiment of the present invention.

Fig. 5 is an exploded view of a rapid inspection apparatus according to an embodiment of the present invention.

FIG. 6 is a flow chart of a detection method according to an embodiment of the invention.

FIG. 7 is a graph showing the results of the detection method according to one embodiment of the present invention.

The reference numbers in the figures illustrate:

1. a sample-bearing portion; 11. a load bearing cavity; 2. a heating section; 21. heating plates; 22. a ceramic heating plate; 3. a vibrating section; 31. a vibration deformation plate; 4. a drive section; 41. a vibration motor; 42. an in-out motor; 5. a photographing part; 51. a camera; 52. a photographing box; 53. a slider; 54. a slide rail; 55. a connecting plate; 6. a housing; 7. installing a frame body; 71. an upper mounting seat; 72. a support; 73. a base; 74. a connecting seat. 8. A reactor; 81. a sample section; 811. a sample section outer tube; 812. a sample section inner tube; 813. a limiting ring; 814. a seal member; 82. a sample addition part; 821. a concave plug; 822. a sample adding cap; 823. a limiting member; 83. a reaction section; 831. a reaction section outer tube; 832. a reaction chamber; 833. a reaction chamber; 834. a shunt plug; 8341. a shunt cap; 8342. a connecting projection; 8343. and (4) micro-pores.

Detailed Description

The method, the reactor and the rapid detection device for detecting nucleic acid by isothermal amplification and color development according to the present invention will be described in detail and in full with reference to the following examples. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

A detection reactor comprises an adding part 82, a sample part 81 and a reaction part 83 which are connected in sequence, wherein the adding part 82 and the sample part 81 are movably connected, and the sample part 81 and the reaction part 83 are movably connected to realize a sealing state.

The sample portion 81 is a coaxial sleeve, the outer tube and the inner tube are hollow tubes, respectively, a sample portion outer tube 811 and a sample portion inner tube 812, and preferably, the sample portion outer tube 811 and the sample portion inner tube 812 are cylinders. The outer diameter of the sample portion inner tube 812 is smaller than the inner diameter of the sample portion outer tube 811. The sample portion outer tube 811 has an external thread at one end and an internal thread at the other end. The sample portion inner tube 812 is a smooth cylinder, and a stop ring 813 extends outwards from a port at one end of the sample portion inner tube 812. In other words, the retainer 813 is annular, and the inner ring of the retainer 813 is connected to the outer surface of the end port of the sample part inner tube 812, forming a structure protruding out of the outer surface of the sample part inner tube 812. Preferably, the stop collar 813 is perpendicular to the sample section inner tube 812. The outer diameter of the stopper ring 813 is larger than the inner diameter of the sample portion outer tube 811 and not larger than the outer diameter of the sample portion outer tube 811 at which the external thread end port is provided, so that the stopper between the sample portion inner tube 812 and the sample portion outer tube 811 is realized without affecting the connection of the external thread and other components. Specifically, the sample portion inner tube 812 is located the sample portion outer tube 811, and the end face of the one end of the retainer ring 813 close to the sample portion inner tube 812 contacts the end face of the sample portion outer tube 811 where the external thread is arranged, so that the seamless fit between the one end of the sample portion inner tube 812 and the one end of the sample portion outer tube 811 is realized.

The sample adding part 82 is a piston structure, and the sample adding part 82 includes a stopper 823, a sleeved concave plug 821, and a sample adding cap 822. The concave plug 821 is made of elastic rubber. The concave plug 821 is located in the sample part inner tube 812, the concave plug 821 is a cylinder, and the outer diameter of the concave plug 821 of the cylinder is not smaller than the inner diameter of the sample part inner tube 812, so that when the concave plug 821 is located in the sample part inner tube 812, the outer surface of the concave plug 821 is attached to the inner surface of the sample part inner tube 812, that is, the sample part inner tube 812 and the concave plug 821 are in sealing and attaching connection, sealing of one end of the sample part inner tube 812 is achieved, spaces on two sides of the concave plug 821 in the sample part inner tube 812 cannot be communicated, and meanwhile, the concave plug 821 can move along the axial direction of the sample part inner tube 812, so that the concave plug 821 has a piston function. One end of the concave plug 821 is provided with a blind hole, the opening of the blind hole is arranged on one end surface of the concave plug 821, and the length direction of the blind hole is parallel to or coincided with the central line of the concave plug 821.

The sample-adding cap 822 comprises an insert rod and a cap head, wherein the insert rod is rod-shaped, one end of the insert rod is connected with the middle part of one end of the cap head, the other end of the insert rod stretches, and the insert rod forms a hard protruding part on the cap head. The rod can be inserted into the blind hole of the concave plug 821, and preferably, the outer diameter of the rod is not smaller than the aperture of the blind hole of the concave plug 821, so as to realize the stable connection between the rod and the blind hole. The cap head is provided with an external thread, and the external thread on the cap head and the internal thread at one end of the sample part outer tube 811 can be in coordination screw connection. After assembly, the concave plug 821 is located in the sample part inner tube 812, and one end provided with the blind hole is farther from the stopper ring 813 of the sample part inner tube 812 than the other end. The insert rod of the sample application cap 822 is inserted into the blind hole of the concave plug 821, and the cap head is screwed with the internal thread at one end of the sample part outer tube 811. When the cap head is rotated in one direction, the concave plug 821 is pushed by the sample application cap 822 to move in the sample part inner tube 812. In this way, one end of the sample portion inner tube 812 is connected to the sample portion outer tube 811 via the cap head, and the other end is restricted by the stopper ring 813 and the sample portion outer tube 811, thereby achieving stable connection between the sample portion inner tube 812 and the sample portion outer tube 811.

The stopper 823 is for restricting the rotation of the cap head of the sample application cap 822. The locating part 823 is a tearing ring which can be torn, the two ends of the tearing ring are respectively connected with the end port, provided with the internal thread, of the cap head and the sample part outer tube 811, the tearing ring is used for limiting between the sample adding part 82 and the sample part 81 at the moment, the cap head cannot continue to rotate, and after the tearing ring is torn down, the limiting effect is relieved, and the cap head can continue to rotate.

The sample portion inner tube 812 is used for containing a sample preservation solution and receiving an added sample, and the sample solution is formed after the added sample is received. When no sample is added, the sample section inner tube 812 is in an independent sealed state, one end is sealed by the concave plug 821, the port of the other end is sealed by the sealing member 814, and the sealing member 814 is a sealing plug or a sealing film. Obviously, the end surface of the other end of the sample part inner tube 812 is the end surface of the limiting ring 813, the end surface of one end of the limiting ring 813 contacts the end surface of one end of the sample part outer tube 811, and the end surface of the other end of the limiting ring 813 contacts the sealing member 814 to achieve sealing. Thus, the sample-holding liquid can be effectively and completely held in the sample portion inner tube 812. When the sample is required to be added, the sealing plug is taken down or the sealing film is torn.

The reaction part 83 is a sleeve, which comprises an inner reaction tube and an outer sleeve 831, wherein the inner reaction tube is detachably arranged in the outer sleeve and is in interference fit with the outer sleeve 831, so that the inner reaction tube is clamped in the outer sleeve 831. The outer sleeve 831 is a hollow tube, preferably a cylinder, and one end of the outer sleeve 831 is provided with an internal thread which is in coordination screw connection with an external thread at one end of the sample portion outer tube 811.

The inner reaction tube includes a reaction chamber 832 and a shunt plug 834. The reaction cavity 832 comprises a plurality of reaction cavities 833, the plurality of reaction cavities 833 are formed by arranging a plurality of parallel and spaced blind holes on the reaction cavity 832, an opening of each reaction cavity 833 is arranged on one end face of the reaction cavity 832, and the other end face of the reaction cavity 832 is closed. The reaction chambers 833 are used for holding reaction systems, and a plurality of reaction chambers 833 can hold the same or different reaction systems. When the inner reaction tube is arranged in the outer sleeve 831, the central line of each reaction cavity is parallel to the central line of the outer sleeve 831, and the closed end face of the reaction cavity 832 is flush with the end face of the outer sleeve 831, which is not provided with the internal thread, so that the end face of one end of the outer sleeve 831 is sealed. Preferably, the outer surface of the reaction cavity 832 is provided with a positioning protrusion, the inner surface of the outer sleeve 831 is provided with a positioning groove, the positioning protrusion and the positioning groove are both parallel to the axis of the outer sleeve 831, and the positioning protrusion and the positioning groove are connected in a coordination manner. During installation, the positioning protrusion of the reaction cavity 832 is aligned with and inserted into the positioning groove of the outer sleeve 831, so that the reaction cavity 832 can be accurately inserted into the outer sleeve 831.

A shunt plug 834 is used to close off reaction chamber 833 and shunt fluid. The shunt plug 834 is made of a flexible material, preferably, the shunt plug 834 is made of rubber, and the outer diameter of the shunt plug 834 is not larger than the outer sleeve 831 so as to be capable of being sleeved in the outer sleeve 831. Diverter plug 834 includes a diverter cap 8341 and a plurality of attachment tabs 8342. The connecting protrusions 8342 are connected to the diversion caps 8341, the outer diameter of the connecting protrusions 8342 is not smaller than the aperture of the reaction cavities 833, the number of the connecting protrusions 8342 is the same as the number of the reaction cavities 833, and the connecting protrusions 8342 can be inserted into the reaction cavities 833 respectively and keep not falling off to close the cavity openings of the reaction cavities 833. Meanwhile, the shunt plug 834 is provided with a plurality of micropores 8343, and the plurality of micropores 8343 are respectively communicated with the plurality of reaction cavities 833, so that the reaction cavities 833 can be communicated with the outside. Obviously, the micro holes 8343 pass through the connection protrusions 8342 and the diversion caps 8341. The pore size of the micropores 8343 is not larger than the capillary length of the sample liquid or the sample preservation liquid, in other words, the surface tension of the liquid at the micropores is larger than the gravity thereof. The aperture of the micropores 8343 is 0.3-0.6 mm. As such, when the sample fluid or the sample preservation fluid is not subjected to a force other than gravity, the sample fluid or the sample preservation fluid cannot pass through the micropores 8343; when the sample liquid or the sample preservation liquid is subjected to acting force exceeding a certain magnitude except gravity, the sample liquid or the sample preservation liquid can pass through the micropores 8343, and the shunt plug 834 can shunt the sample liquid or the sample preservation liquid.

Based on above-mentioned structure, when sample portion outer tube 811 and outer tube 831 coordination spiro union, sample portion 81 and application of sample portion 82 are spacing through tearing the pull ring, just reaction chamber 833 is built-in to have the reaction system, and when sample portion inner tube 812 is equipped with sample preservation liquid in, sample preservation liquid one end is sealed through concave stopper 821, and the other end seals through sealing member 814, and the reaction system separates through reposition of redundant personnel stopper 834 and the external world. When the device is used, the sample portion outer tube 811 and the outer sleeve 831 are unscrewed, the sealing member 814 is removed, a sample to be measured is placed in a sample preservation solution to form a sample solution, the sample portion outer tube 811 and the outer sleeve 831 are screwed to form a closed shell by the sample portion outer tube 811 and the outer sleeve 831, and at this time, the shunt plug 834 realizes the function of separating the reaction cavity 833 from the sample solution of the sample portion 81. Then the tearing ring is torn off, the cap head of the sample adding cap 822 is rotated in one direction, so that the concave plug 821 is pushed to move towards the direction close to the reaction part 83, the sample liquid is pressurized, and the sample liquid can enter the reaction cavity 833 through the micro-pores 8343.

In summary, the sample addition part 82 can realize the sealing connection between one end of the sample part 81 and the outside, and can also realize the addition of the sample liquid into the reaction part 83 by pressurizing the sample part 81 through the unidirectional rotation of the sample addition part 82. Meanwhile, an independent reaction system is arranged in the reaction cavity 833, so that simultaneous detection of a plurality of detection items of the same sample can be realized.

The embodiment also provides a multi-person nucleic acid rapid detection device matched with the reactor. The nucleic acid rapid detection equipment comprises a reactor 8 and a reaction instrument, wherein the reactor is provided with a plurality of bearing cavity positions 11, a sample to be detected is placed in the reactor 8, the sealing of the reactor 8 is not damaged in the detection process of the reactor, and the reaction instrument can simultaneously carry out mixed detection on different samples.

In this embodiment, the device includes a bearing part 1, a heating part 2, a vibrating part 3, a driving part 4, a photographing part 5, a mounting frame body 7 and a housing 6, and the bearing part 1, the heating part 2, the vibrating part 3, the driving part 4 and the photographing part 5 are arranged in the housing 6 through the mounting frame body 7. The mounting frame body 7 is of a special-shaped plate structure, the bearing part 1, the heating part 2 and the vibrating part 3 are mounted at the upper end of the special-shaped plate, and the driving part 4 is mounted in a cavity of the special-shaped plate and is positioned below the vibrating part 3; the photographing part 5 is installed at one side of the special-shaped plate.

In this embodiment, the mounting frame body 7 includes an upper mounting seat 71, a base 73 and two supports 72, four corner ends of the upper mounting seat 71 are respectively provided with a protruding mounting block 75, and the upper mounting seat 71 is provided with a mounting hole. Two supports 72 are respectively arranged at two sides of the upper mounting seat 71, and the bottom of the support 72 is fixed on the base 73.

In this embodiment, the bearing portion 1 is located to bearing chamber position 11, and bearing portion 1 is equipped with a loading board and a plurality of location section of thick bamboo, and a location section of thick bamboo is fixed in on the loading board, and loading board and a location section of thick bamboo adopt heat conduction and non-flexible material preparation, and bearing chamber position 11 is enclosed by a loading board and a location section of thick bamboo and establishes and form, and bearing chamber position 11 arranges according to the order array, and a reactor 8 can be placed to every bearing chamber position 11. The housing 6 is provided with a cover 61, the cover 61 being located above the carrier 1, the cover 61 being openable for insertion or removal of the reactor 8.

In this embodiment, heating portion 2 includes heating plate 21 and a plurality of heating plate, and the heating plate adopts ceramic heating plate in this embodiment, and ceramic heating plate is located heating plate 21 below, and heating plate 21 is fixed in the bottom of loading board, and ceramic heating plate passes through loading board and a location section of thick bamboo to 8 bulk heating of reactor, can the even heating, control reaction temperature that simultaneously can be more accurate.

In this embodiment, the vibrating portion 3 is provided with the vibration deformation plate 31, the ceramic heating sheet is mounted on the vibration deformation plate 31, the vibration deformation plate 31 is mounted on the upper mounting seat 71, the vibration deformation plate 31 is provided with four mounting plates, and each mounting plate is fixedly connected with one mounting block 75. A gap is provided between the side surface of the vibration deformation plate 31 and the mounting plate, so that the impact of the vibration deformation plate 31 on the upper mounting seat 71 can be reduced.

In this embodiment, the driving portion 4 includes a vibration motor 41 and an in-out motor 42, an eccentric vibrator is disposed on a driving shaft of the vibration motor 41, the vibration motor 41 is mounted below the upper mounting seat 71 and fixed on the base 73, the driving shaft of the vibration motor 41 passes through the mounting hole of the upper mounting seat 71, and the eccentric vibrator drives the vibration deformation plate 31 to drive the bearing portion 1 to vibrate. The in-out motor 42 is fixed to the base 73 and drives the photographing section 5.

In this embodiment, the photographing part 5 includes a camera 51, a photographing box 52, a sliding block 53, a sliding rail 54 and a connecting plate 55, the upper mounting plate is provided with a connecting seat 74, the sliding block 53 is fixedly connected with the connecting seat 74, and the sliding block 53 is matched with the sliding rail 54 and can slide relatively. The connecting plate 55 is fixed to the bottom end of the sliding rail 54, the connecting plate 55 is provided with a sliding slot, and the driving shaft of the in-out motor 42 is fixedly connected with an eccentric connecting rod which can slide in the sliding slot of the connecting plate 55. The camera 51 is fixed on the base 73 through a camera mount, and the photographing box 52 is fixedly mounted on one end of the slide rail 54. The in-and-out motor 42 moves the slide rail 54 relative to the slide block 53 through the eccentric link to drive the photographing box 52 to extend out of the housing 6. The housing 6 is provided with a corresponding openable and closable opening. The photographing box 52 is provided with a slot for placing a reaction tube. The photographing box 52 is moved out of the housing 6 by a distance that exposes the housing 6 in the slot position, so that the reaction tube can be placed.

In this embodiment, the photographing box 52 extends out of the housing 6 during photographing, the reaction tube after reaction is placed in the photographing box 52, the photographing box 52 is moved into the housing 6, the camera 51 photographs, the photographing box 52 extends out of the housing 6 after photographing, the reaction tube after photographing is taken out, and the reaction tube after photographing is placed in the next reaction tube for continuing photographing. In fact, the photographing and the reaction can be performed simultaneously, that is, after the reaction is performed for the first time, the second batch of reaction tubes can be placed in the bearing part 1, so that the detection speed is increased.

The quick detection device of the embodiment is also provided with a cooling unit for cooling the motor and a host machine for controlling the operation of each part. The cooling unit is provided with a fan for cooling the motor. The quick detection equipment is provided with a control system, and the control system comprises a detection module, a conversion module, a processing module and a display module. The detection module is provided with a temperature sensor and is used for detecting the temperature of the bearing part 1 and transmitting detection data to the processing module; the conversion module comprises a DC-DC converter; the processing module is used for displaying the detected temperature data on the display module. The camera 51 is electrically connected to the processing module and the picture is displayed on the display module. The cooling unit and the control system are arranged according to the mechanical structure part of the equipment, and conventional components capable of achieving corresponding control effects are adopted.

In order to verify the detection method provided by the present invention, in this embodiment, a reaction system is designed according to the disclosure of CN112442555A, and the specific preparation process of the reagent refers to the prior art, which is not described herein again.

The detection method for detecting nucleic acid based on the detection reactor and the multiple-person nucleic acid rapid detection equipment comprises the following steps:

s1, preparing a detection reagent: obtaining a reaction system corresponding to the new coronavirus according to the literature, wherein the reaction system comprises a primer, reverse transcriptase, bst DNA polymerase, dNTP, buffer and neutral red, and the reaction system is prepared into a freeze-dried powder preparation which is added into a reactor 8 in advance; the sample-storing solution is commercially available (manufactured by da gene), the reaction system and the sample-storing solution are separately contained in the same reactor 8, the sample-storing solution is contained in the sample portion inner tube 812 of the reactor 8, one end of the sample portion inner tube 812 is sealed by the concave plug 821, and the other end is sealed by the sealing member 814, and the sample-storing solution is stored in the sample portion inner tube 812 in a sealed manner. The reaction system is loaded in reaction chamber 833 of reactor 8, and then a shunt plug 834 is inserted. After the sample portion outer tube 811 and the outer sleeve 831 are screwed, the shunt plug 834 separates the reaction system from the outside.

In order to ensure the stability of the reaction system, a layer of paraffin wax is sealed on the freeze-dried reagent in the reaction cavity, and in addition, the reaction system can be divided into a plurality of layers by the paraffin wax, such as a layer of dye and dNTP, a layer of reverse transcriptase, bst DNA polymerase and primer, and a layer of Buffer.

S2, taking a sample to be detected and adding the sample to be detected into a sample preservation solution to obtain a sample solution, wherein the sample is a nasal cavity swab for sampling, and the swab after sampling is placed into a reaction tube added with the sample preservation solution to obtain the sample solution.

And S3, adding a sample liquid, pressing the sample liquid into the reaction system through an external force, wherein the addition amount of the sample liquid is less than 100 microliters.

Specifically, step S3 includes the following three steps:

s3-1, adding the sampled swab into the sample part 81, sealing the reaction part 83 and the sample part 81, and applying external force to uniformly mix the sample liquid.

The specific operation is as follows: unscrew the sample portion outer tube 811 and the outer tube 831 of reactor 8, take off the sealing plug of sample portion 81 or tear the sealing membrane down, add the swab after the sample in sample portion inner tube 812, again with outer tube 831 and sample portion outer tube 811 spiro union screw up, during the spiro union, with the open end of sample portion inner tube 812 up in order to prevent that the sample liquid from falling out. Finally the reactor 8 is shaken and mixed.

S3-2, the sample portion 81 is pressurized and applied with the application of a sample by the application cap 822 without breaking the seal of the reaction vessel 8.

The specific operation is as follows: the tearing ring (easy-to-tear) is torn off, and the sample cap 822 is rotated toward the sample part 81 to apply pressure to the sample. Specifically, the cap head of the sample addition cap 822 is rotated to push the concave plug 821, the concave plug 821 moves in the sample part inner tube 812 in a direction close to the reaction part 83, and the sample liquid in the sample part inner tube 812 is pressurized and can enter the reaction cavity 833 of the reaction part 83 through the micropores 8343.

S3-3, applying a force to the reactor 8 in the same direction as the gravity of the reactor 8, so that the sample solution hanging on the top of the reaction portion 83 enters the reaction chamber 833, specifically: the reactor 8 is shaken (flicked) in a downward direction so that the sample solution hanging from the top of the reaction section 83 enters the bottom of the reaction chamber 833.

And S4, carrying out amplification reaction on the reaction system, reading the reaction result to finish detection, and judging the detection result through colorimetry or fluorescence.

Specifically, step S4 includes the following three steps:

s4-1, putting the reactor 8 into the quick detection equipment or heating equipment (a water bath kettle, a metal bath or a common PCR instrument), heating, sealing, separating, melting a reaction system, mixing the reaction system with a sample solution to obtain a reaction solution,

s4-2, maintaining the temperature in the reactor 8 to the amplification temperature, melting paraffin at the moment, adding the sample liquid into the reaction system, floating the paraffin to the reaction system containing the sample liquid, forming a sealing layer effect, further sealing to ensure a reaction result, and oscillating the reactor 8 to fully mix the reaction liquid to carry out amplification reaction; to ensure a sufficient reaction, the reactor 8 may be oscillated or rotated repeatedly.

S4-3, photographing the reactor 8 after color development, wherein the reaction result is shown in figure 7, the positive amplification result is purple red, the negative amplification result is faint yellow, and the result is consistent with the disclosure of CN 112442555A.

The above embodiments are merely preferred embodiments of the present invention, which is not intended to limit the present invention in any way. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention, unless the technical essence of the present invention departs from the content of the technical solution of the present invention.

Claims (22)

1. A method for detecting nucleic acid, comprising the steps of:

s1, preparing a detection reagent: selecting an amplification method according to the object to be detected and adaptively designing a detection reagent; after preparing a detection reagent, adding the detection reagent into a reactor in advance; the detection reagent comprises a reaction system and a sample preservation solution, and the reaction system and the sample preservation solution are respectively and independently arranged in the same reactor;

s2, taking a sample to be detected and adding the sample to be detected into a sample preservation solution to form a sample solution, wherein the sample is a sample source from which nucleic acid can be obtained;

s3, adding a sample liquid, pressing the sample liquid into a reaction system through an external force, wherein the addition amount of the sample liquid is less than 100 microliters;

s4, carrying out amplification reaction on the reaction system, reading the reaction result to complete detection, and judging the detection result through colorimetry or fluorescence;

wherein:

the amplification reaction is a temperature-variable amplification reaction or an isothermal amplification reaction;

the reaction system is 10-200 microliter, and at least comprises: reaction enzyme, primer, dNTP, buffer and dye;

adaptively designing a specific primer sequence according to the nucleic acid to be detected;

the sample is selected from: nasopharyngeal secretions, intestinal secretions, respiratory secretions, genital secretions, or blood samples.

2. The method of claim 1, wherein: when the nucleic acid to be detected is RNA, the reaction enzyme comprises RNA-reverse transcriptase; and when the isothermal amplification reaction is carried out, the reaction enzyme also comprises BST polymerase; or when the temperature-variable amplification reaction is carried out, the reaction enzyme also comprises Taq polymerase; or when the nucleic acid to be detected is DNA, the reaction enzyme of the isothermal amplification system comprises BST polymerase, and the reaction enzyme of the variable temperature amplification system comprises Taq polymerase.

3. The method of claim 1, wherein: the dye is a chromogenic dye or a fluorescent dye.

4. The method according to claim 3, wherein the chromogenic dye is selected from one or a combination of several of the following: HNB, neutral red dye, calcein, acid chrome blue K, ammonium molybdate or visible light dye; or the fluorescent dye is selected from one or a combination of several of the following: SYBR Green, SYBR Gold, SYBR-safe, evaGreen, RTGreen, SYTO-9, gel Green, gel Red, EB dyes or Gold View I dyes.

5. The method of claim 1, wherein: the sampling mode is swab sampling; after sampling, putting the swab with the nucleic acid into a reaction tube added with a sample preservation solution to obtain a sample solution.

6. The method of claim 1, wherein: the reaction system in S1 is in a freeze-dried state.

7. The method of claim 1, wherein: at least one component of the reaction enzyme, the primer and the dNTP in the reactor in the S1 and the other two components are independently and respectively added into the same cavity of the reactor through a sealing layer which can be melted when being heated.

8. The method of claim 7, wherein: the melting point of the isolation layer is 50-70 ℃, and the freezing point is 0-40 ℃.

9. The method of claim 7, wherein: s4, after the heating, the sealing layer is melted, and then the reaction system is in a reaction liquid state and is subjected to amplification reaction.

10. The method of claim 8, wherein: the sealing layer is selected from one or a mixture of several of the following: paraffin, silicone oil, or octadecane, and the density of the sealing layer is less than that of water.

11. The method of claim 1, wherein: the reaction system further comprises a lyoprotectant.

12. A reactor used for the method for detecting a nucleic acid according to any one of claims 1 to 11, characterized in that: the reactor comprises a sample adding part, a sample part and a reaction part which are connected in sequence, wherein the sample adding part, the sample part and the reaction part are movably connected to realize a sealing state; the connecting part of the reaction part and the sample part is provided with micropores, and the aperture of each micropore is not more than the capillary length of the sample liquid or the sample preservation liquid; under the sealed state of the reactor, the sample adding part moves towards the reaction part by external force to realize that the sample liquid passes through the micropores and is pressed into the reaction part under the sealed state.

13. The reactor of claim 12, wherein: the reaction part is internally provided with a plurality of reaction cavities, each reaction cavity is internally provided with an independent reaction system in advance, so that simultaneous detection of a plurality of detection items aiming at the same sample can be realized, the micropores are arranged at the cavity openings of the reaction cavities, and each reaction cavity is provided with one micropore.

14. The reactor according to claim 12 or 13, characterized in that: the reaction part is provided with a shunt plug, the shunt plug is positioned at the joint of the reaction part and the sample part, the reaction cavity is independently sealed through the shunt plug, and the shunt plug is arranged in the micropore.

15. The reactor of claim 14, wherein: one side of the shunting plug protrudes towards the reaction cavity to seal the cavity opening, and the micropores penetrate through the protrusion.

16. The reactor of claim 12, wherein: the reaction part is provided with an inner reaction tube and an outer sleeve which are sleeved, the inner reaction tube is clamped in the outer sleeve, and the outer sleeve is provided with a thread which is in coordination and screw connection with the sample part.

17. The reactor of claim 16, wherein: the inner reaction tube is provided with a plurality of reaction cavities.

18. The reactor of claim 13, wherein: the sample adding part comprises a concave plug and a sample adding cap which are sleeved, a hard protruding piece which is inserted into the concave plug and is sleeved is arranged in the sample adding cap, and the sample adding cap is also provided with a threaded connection section which is in coordination threaded connection with the sample part.

19. The reactor of claim 18, wherein: the sample adding part is also provided with a limiting part, and the limiting part is used for limiting the rotation of the sample adding part.

20. The reactor of claim 19, wherein: the locating part is a tearing ring, the tearing ring is respectively connected with the sample adding cap and the sample port, and the tearing ring is used for limiting the position between the sample adding part and the sample part.

21. The reactor of claim 12, wherein: the port of the sample part near the reaction part is closed, and is in an independent sealing state when no sample is added, and the port is closed through a sealing plug or a sealing film.

22. The reactor of claim 12, wherein: the sample part is a coaxial sleeve, wherein the inner pipe is a hollow pipe, the inner diameter of the hollow pipe is in sealed fit connection with the concave plug, and one end of the outer pipe is provided with a thread which is in coordination screw connection with the sample adding part; when the reactor is sealed, one section of the inner tube is positioned in the gap between the concave plug and the sample adding part; the sample preservation solution is pre-arranged in the cavity of the inner tube before reaction.

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