CN115786097A

CN115786097A - Nucleic acid amplification reactor and application thereof

Info

Publication number: CN115786097A
Application number: CN202110968998.4A
Authority: CN
Inventors: 岂源
Original assignee: Beijing Qingfengtang Pharmaceutical Technology Co ltd
Current assignee: Beijing Qingfengtang Pharmaceutical Technology Co ltd
Priority date: 2021-07-30
Filing date: 2021-08-23
Publication date: 2023-03-14
Also published as: CN115703989A; CN215667945U; CN115703990A; CN215906212U; CN215667988U; CN215906211U; CN115704049A; CN215757272U; CN115703991A

Abstract

The invention discloses a nucleic acid amplification reactor, which comprises a sample adding part, a sample part and a reaction part, wherein before reaction, the sample part and the reaction part are in an independent sealing state, the sample adding part is of a piston structure, and a reaction system is arranged in the reaction part; 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; the sample adding part moves by external force to realize that the sample liquid passes through the micropores to enter the reaction part in a sealed state. The invention also discloses the application of the reactor, and the reactor is matched with an isothermal or variable temperature nucleic acid amplification instrument for color development detection. The reactor and the application thereof can solve the problem of uncovering pollution in the amplification method in the prior art, can directly react after the sample is added, do not need to uncover in the reaction process and need not to add liquid for many times, can realize the whole nucleic acid detection without a professional laboratory and have no aerosol pollution.

Description

Nucleic acid amplification reactor and application thereof

Technical Field

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

Background

Nucleic acid detection, which is a method having high sensitivity and specificity, is now 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 thermocycler device be necessary as a support, and a professional laboratory also be a necessary condition because of aerosol contamination problems. Among them, the PCR extension technology platform, particularly the quantitative PCR (qPCR) method, is the most widely used pathogen detection method, and is considered as 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 for use with point-of-use applications (point-of-use applications) can be challenging. This is because digital PCR requires expensive instrumentation (i.e., bio-rad droplet digital PCR), a full-scale laboratory environment, and trained technicians to perform the assay. These factors severely limit the accessibility and applications of digital PCR in resource-limited contexts.

To overcome these drawbacks, a large class of new isothermal nucleic acid amplification methods 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 Rongy chemical Co., ltd. Japan in 2000. It is characterized by that it designs 4 specific primers for 6 regions of target gene, and under the action of strand displacement DNA polymerase (Bst DNA polymerase), it can implement constant-temp. amplification at 60-65 deg.C for 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 applied to 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 centralized laboratories, for example, where on-site point-of-use testing of environmental water in resource-limited places is required.

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

The isothermal amplification instrument performs amplification reaction by using strand displacement DNA polymerase under the constant temperature condition, can realize 109-1010 times of amplification within 15-60 minutes, can generate a large amount of amplification products, namely magnesium pyrophosphate white precipitate, and can judge whether a target gene exists by observing the existence of the white precipitate by naked eyes.

Some studies have shown that a microreactor prepared from Polydimethylsiloxane (PDMS) is used for LAMP reaction, but PDMS is inherently porous and gas permeable, which is advantageous for cell culture and other work, but is not suitable for nucleic acid amplification applications because of evaporation of solution and generation of bubbles in the microreactor. In fact, the most common cause of aerosol contamination is the bursting of the bubbles during amplification, and such aerosols easily break through the sealing interface of the PDMS microreactor. In addition, PDMS is very hydrophobic, and PDMS-based microfabrication tends to have irregular geometries on a microscopic scale, which can lead to the generation of bubbles during sample loading and heating, which are likely to break through seals made to the sample inlet, sample outlet, or between different reaction cells. Although there have been some research efforts directed to the above disadvantages of PDMS, some material modifications have been made to the original PDMS with the intent of increasing its impermeability and regularity of geometry, but this has certainly increased the complexity and cost of preparation.

In practice, it is still an urgent need to efficiently perform simultaneous measurements of multiple targets with an inexpensive, simple device. There are efforts to miniaturize conventional PCR vials into a PDMS chip for multiplex amplification, but the chip fabrication still relies on precision micromachining. In recent years, on-chip droplet technology has also been increasingly used for nucleic acid amplification, but they all require complicated fluid control systems, often have bulky external devices and require external power supply, and thus such devices have not really been miniaturized as a whole.

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 of the amplification process is also needed, 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 a reactor which can directly complete reaction once after a sample is directly added is not adopted, the traditional eight-connected tube or EP tube is still adopted, and the important elbow that the nucleic acid detection cannot be well applied to POCT and the development and application of pathogenic microorganisms is stopped.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: aiming at the technical problems in the prior art, the invention provides a nucleic acid amplification reactor and application thereof, which can simultaneously carry out a plurality of joint detections on the same sample, can realize that the detection conditions in the whole nucleic acid detection process are basically unlimited and have no aerosol pollution

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

a nucleic acid amplification reactor comprises a sample adding part, a sample part and a reaction part, wherein before reaction, the sample part and the reaction part are in an independent sealing state, the sample adding part is of a piston structure, and a reaction system is arranged in the reaction part; the connecting part of the reaction part and the sample part is provided with micropores, the pore diameter of each micropore is not more than the capillary length of the sample liquid or the sample preservation liquid, in other words, the surface tension of the liquid at each micropore is more than the gravity thereof; under the sealed state of the reactor, the sample adding part moves by external force to realize that the sample liquid under the sealed state passes through the micropores and enters the reaction part.

The technical proposal is further improved as follows:

one embodiment of the present invention is: the sample adding part, the sample part and the reaction part are sequentially connected, the sample adding part and the sample part are movably connected to realize a sealing state, and a sample preservation solution is pre-filled in the sample part; 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.

Preferably, at least one reaction chamber is arranged in the reaction part, an independent reaction system is preset in the reaction chamber, simultaneous detection of multiple detection items of the same sample can be realized, the micropores are arranged at the orifice of the reaction chamber, each reaction chamber corresponds to one micropore, and liquid cannot flow into the reaction chamber from the sample part under the condition of no external force. The reaction system is independently and respectively embedded in the same cavity of the reactor through preheating meltable packing materials.

Preferably, the reaction part is equipped with the reposition of redundant personnel stopper, the reposition of redundant personnel stopper adopts flexible material to make, the reposition of redundant personnel stopper is located the junction of reaction part and sample portion, the reaction chamber is independently sealed through the reposition of redundant personnel stopper, the reposition of redundant personnel stopper is located to the micropore.

Preferably, one side of the shunting plug protrudes towards the reaction cavity to seal the cavity opening, the micropores penetrate through the protrusion, and one side of the shunting plug facing the sample part is a smooth surface.

Preferably, the pore diameter of the micropores is 0.3 to 0.6mm.

Preferably, 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 screw connection with the sample part. The reaction part is arranged into an inner pipe and an outer pipe so as to facilitate the processing of the reaction cavity during production, control the precision and the tightness and achieve the corresponding production cost.

Preferably, the reaction chambers are closed at the ports close to the sample part, each reaction chamber is in an independent sealing state before reaction, and the ports are closed through a sealing plug or a sealing film. Because a sealing film or a sealing plug is provided, the reaction chamber can be kept in a sealed state even in the case where the threaded joint of the reaction part and the sample part is unscrewed.

Preferably, the sample adding part comprises a sleeved concave plug and a sample adding cap, a hard protruding part inserted into the concave plug and sleeved in the sample adding cap is arranged in the sample adding cap, the sample adding cap is further provided with a threaded connection section in coordination and screw connection with the sample part, so that the sample adding part and the sample part can be hermetically connected, and sample liquid can enter the reaction part by pressurizing the sample part through unidirectional rotation.

Preferably, 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.

Preferably, the sample part is a coaxial sleeve, wherein the inner tube is a hollow tube, the inner diameter of the hollow tube is in sealed 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. The inner and outer tubes of the sample section may be integral and divided into inner and outer tubes to reduce machining costs, control accuracy and sealing.

Preferably, one end of the outer tube of the sample part, which is close to the reaction part, is provided with a thread which is in coordination threaded connection with the reaction part, and the reaction part is not communicated with the outer tube.

Preferably, the sample part is further provided with a sealing member disposed between the reaction part and the sample part, the sealing member being used for sealing the sample preservation solution, so that the lumen of the sample part can be in a sealed state even when the threaded joint of the reaction part and the sample part is unscrewed.

A second embodiment of the reactor of the present application is: the application of sample portion includes application of sample stopper and application of sample lid, application of sample lid is equipped with the inner chamber, application of sample stopper is located application of sample lid's inner chamber, just application of sample stopper has resistance unidirectional motion in the inner chamber, application of sample stopper constitutes piston structure with application of sample lid's inner chamber, and under the reactor encapsulated situation, through application of sample stopper to sample portion direction motion messenger's sample liquid gets into the reaction chamber.

Preferably, the reaction part and the sample part are integrally processed, and the reaction part and the sample part are separated by a shunt plug.

A third embodiment of the reactor of the present application is: sample portion, application of sample portion, reaction part connect in proper order, and sample portion application of sample portion is located between sample portion and the reaction part, application of sample portion is equipped with the sealing member, the sealing member realizes the mixture of sample liquid and reaction liquid under encapsulated situation through the motion of external force in the connection chamber of application of sample portion.

In the above scheme, preferably, the sample part is provided with at least one sample cavity, the reaction part is provided with at least one reaction cavity, and the sample cavity is not directly communicated with the reaction cavity.

In the above-mentioned embodiment, preferably, micropores are provided between the sample part and the sample adding part, and between the reaction part and the sample adding part, each sample chamber corresponds to one micropore, and each reaction chamber corresponds to one micropore.

In the above solution, preferably, the sample adding part is provided with a moving part, the moving part is arranged in the connecting cavity of the sample adding part, the moving part drives the sealing part to move in the connecting cavity, and the sealing part is arranged on the moving part.

In the above-mentioned embodiment, preferably, the sample addition part is provided with at least one communication position, the sealing members are arranged in pairs, one communication position is formed between two sealing members, and one communication position corresponds to one or more reaction chambers and one sample chamber.

In the above scheme, preferably, the cross-sectional area of the moving member is smaller than the diameter of the sample addition part connection cavity, and the sealing member is in interference connection with the inner wall of the sample addition part, so that each connection position is independently sealed.

In the above aspect, preferably, the sealing member is a rubber member.

In the above aspect, preferably, the diameter of the micro-hole is 0.3mm to 1mm.

In the above scheme, preferably, the reactor is further provided with a sealing cover, and the liquid inlets of the sample chamber and the reaction chamber are provided with sealing covers.

The application also discloses application of the reactor, and the reactor is matched with constant-temperature or variable-temperature amplification equipment for use and is used for realizing color development detection of nucleic acid constant-temperature amplification or variable-temperature amplification.

Compared with the prior art, the nucleic acid amplification reactor and the application thereof provided by the invention have the following advantages:

(1) The nucleic acid amplification reactor and the application thereof can pre-add a reaction system in the amplification operation, the pre-added reaction system not only avoids the limitation of a field configuration reaction system on the environment, simplifies the system configuration steps before detection, but also can ensure the rapid and simple use of the detection. The subsequent amplification operation only needs to add a sample to be detected, the sample can be directly reacted after reaching the amplification reaction condition (such as temperature), liquid is not needed to be added again, so that the sample to be detected is added into the reactor only by opening the cover once in the detection process, the sample is not in contact with other components in the reaction system, the reaction is directly carried out by full contact in the reaction process, the cover is not needed again, the whole nucleic acid detection process can be realized, the detection condition is 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 amplification reactor and the application thereof can simultaneously detect a plurality of nucleic acids to be detected in a sample to be detected by pre-burying different reaction systems in one tube of multi-reaction cavity, thereby not only improving the detection efficiency, but also jointly considering the detection results of related nucleic acids, and providing a more accurate gene level suggestion for clinical judgment.

(3) The application discloses nucleic acid amplification reactor and application thereof, the special design of application of sample portion can control the volume of adding that sample liquid got into the reaction chamber, can not cause mutual pollution when guaranteeing to react fully in the reaction chamber.

(4) The application discloses nucleic acid amplification reactor and application thereof, micropore's aperture setting in the reactor has guaranteed promptly that storage transportation, the sample that awaits measuring add in-process sample liquid or sample save the liquid and do not get into the reaction intracavity, has also guaranteed under the condition of exerting external force, and the sample liquid can get into the reaction intracavity smoothly fully to react.

(5) The application discloses nucleic acid amplification reactor and application thereof, detection reactor overall structure is simple, low in production cost, and the material environment is friendly, and cylindrical body suitability is strong, can be used to each type of firing equipment, also is convenient for exert external force and guarantees that the reaction is abundant.

(6) The application discloses nucleic acid amplification reactor and application thereof, packing material melts temperature matching heating reaction temperature, and can not exert an influence to the amplification reaction system, and the seal melts after the heating, has guaranteed that the reaction system intensive mixing under the condition of not uncapping, and the packing material after melting floats on the reaction system because density is little, has further gone into the seal on the reaction system, provides dual assurance to the abundant going on of reaction.

(7) The nucleic acid amplification reactor and the application thereof can be realized by matching simple heating equipment (even a vacuum cup) with the reactor of the invention aiming at public health events, do not need professional operation, 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 particularly in relatively laggard areas.

Drawings

Fig. 1 is a schematic structural view of embodiment 1 of the present invention.

Fig. 2 is a schematic diagram of an explosive structure of embodiment 1 of the present invention.

Fig. 3 is a schematic structural diagram of embodiment 2 of the present invention.

Fig. 4 is a schematic structural view of embodiment 3 of the present invention.

The reference numbers in the figures illustrate:

1. a sample section; 11. an outer tube; 12. an inner tube; 13. a seal member; 14. a sample chamber; 2. a sample addition part; 21. a concave plug; 22. a sample adding cap; 23. tearing the pull ring; 24. a moving member; 25. a communication position; 26. a sample adding plug; 27. a sample application cover; 3. a reaction section; 31. an outer sleeve; 32. an inner reaction tube; 33. a reaction chamber; 34. a shunt plug; 341. a boss member; 342. micropores; 4. and (7) sealing the cover.

Detailed Description

The present invention will be described more fully hereinafter 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.

Example 1

FIGS. 1 and 2 show one embodiment of a nucleic acid amplification reactor of the present invention,

the reactor comprises a sample adding part 2, a sample part 1 and a reaction part 3 which are connected in sequence, wherein the sample adding part 2 is movably connected with the sample part 1 and the sample part 1 is movably connected with the reaction part 3 to realize a sealing state, the sample adding part 2 is of a piston structure, a sample preserving fluid is pre-filled in the sample part 1, and a reaction system is filled in the reaction part 3; a micropore 342 is arranged at the joint of the reaction part 3 and the sample part 1, and the aperture of the micropore 342 is not more than the capillary length of the sample liquid or the sample preservation liquid; the surface tension of the liquid at the micropores is greater than its gravity. In this embodiment, the pore diameter of the micropores is 0.3 to 0.6mm. In the sealed state of the reactor, the sample addition part 2 is moved toward the reaction part 3 by an external force, and the sample liquid is pressed into the reaction part 3 through the micro hole 342 in the sealed state. In order to be conveniently placed in the isothermal amplification instrument, the whole reactor adopts a circular tube type structure, and particularly, the whole body is preferably made of a transparent plastic material.

In this embodiment, a plurality of reaction chambers 33 are disposed in the reaction portion 3, an independent reaction system is preset in the reaction chamber 33, simultaneous detection of a plurality of detection items for the same sample can be achieved, and the end portion of each reaction chamber 33 near the joint is provided with a micropore 342, so that liquid cannot flow into the reaction chamber 33 from the sample portion 1 without external force.

In this embodiment, the reaction portion 3 is provided with an inner reaction tube 32 and an outer sleeve 31 which are sleeved, the inner reaction tube 32 is tightly clamped in the outer sleeve 31, one end of the outer sleeve 31 is provided with an internal thread which is in coordination threaded connection with the sample portion 1, the reaction portion 3 is provided with a shunt plug 34, the shunt plug 34 is made of a flexible material, and the transparent rubber material is adopted in this embodiment. The shunting plug 34 is located at the connection position of the reaction chamber 33 and the sample part 1, one section of the upper part of the reaction part 3 is an internal thread section, the position of the reaction chamber 33 is not located at the connection position of the internal thread, namely, the shunting plug 34 divides the connection position of the internal thread and the reaction chamber 33, one surface of the shunting plug 34 protrudes towards the interior of the reaction chamber 33 to seal a chamber opening, each protrusion is provided with a micropore 342, the micropore 342 penetrates through the protrusion, and one surface of the shunting plug 34 facing the sample part 1 is a smooth surface. The reaction chamber 33 is sealed independently by a shunt plug 34. Before the reaction, a reaction system is pre-buried in each reaction cavity 33, and the reaction system is sealed and buried through a sealing material, for example, the reaction system is sealed and stored by a material which is not compatible with water and can change form along with temperature change. Each reaction chamber 33 may be provided with a different reaction system for detecting different items. Since the reactor is small and compact as a whole, it is preferable to provide 4 reaction chambers 33.

In this example, the reaction system was sealed in the reaction chamber with paraffin. The melted paraffin floats on the liquid in the reaction chamber 33 to play a sealing role. In addition, after the reaction is finished, the paraffin is in a solid state above the liquid, and plays a role in sealing the liquid after the reaction. The melting temperature of the packing material is matched with the heating reaction temperature, the amplification reaction system cannot be influenced, the sealing layer is melted after heating, the reaction system is fully mixed under the condition that the cover is not opened, the melted packing material floats on the reaction system due to the fact that the density of the melted packing material is small, the sealing layer on the reaction system is formed, and double guarantee is provided for full reaction.

In this embodiment, the outer sleeve 31 is a hollow tube, the inner reaction tube 32 is clamped in the outer sleeve 31, the inner reaction tube 32 has one or more ribs for positioning, the outer sleeve 31 has corresponding grooves, and the inner reaction tube 32 and the outer sleeve 31 can be integrally formed or can be separately formed. In practice, because the reactor is small, the outer tube 31 and the inner tube 32 are generally manufactured as two parts and then assembled for ease of handling and ease of operation for embedding the reaction system. In other embodiments, the reaction tube may be a unitary body.

In this embodiment, the outer surface of the outer sleeve 31, i.e. the outer surface of the reaction part 3, is provided with at least one rib or groove for positioning the reactor when being placed in the loading cavity 11 of the isothermal amplification apparatus, and the loading cavity 11 is provided with a groove or rib matching with the groove or rib.

In this embodiment, the sample adding part 2 includes a sleeved concave plug 21 and a sleeved sample adding cap 22, one section of the sample adding cap 22 connected with the sample part 1 is provided with an external thread connecting section, a rigid protruding part 341 inserted into the concave plug 21 and sleeved is arranged in the sample adding cap, the protruding part 341 adopts a cylindrical shape or a cylindrical shape, the protruding part 341 and the external thread connecting section adopt a concentric ring structure, and an annular cavity is arranged between the protruding part 341 and the external thread connecting section. The concave plug 21 is made of transparent rubber materials, the concave plug 21 is of a piston structure with a cylindrical cavity arranged inside, a circular ring extending outwards is arranged at the cavity opening of the top of the piston, the outer diameter of the circular ring is the same as that of the annular cavity, the diameter of the piston head at the other end is slightly larger than that of the inner cavity of the sample part 1, and the function of the piston head in the inner cavity of the sample part 1 is achieved. When the concave plug 21 is sleeved on the sample adding cap 22, the head part of the piston protrudes out of the sample adding cap 22, so that the sample adding part 2 and the sample part 1 can be hermetically connected, and the sample liquid can enter the reaction part 3 by pressurizing the sample adding part 2 to the sample part 1 through unidirectional rotation.

In this embodiment, the sample portion 1 is a coaxial sleeve, the inner tube 12 and the outer tube 11 are both hollow tubes, one end of the outer tube 11 is provided with an internal thread, and the other end of the outer tube is provided with an internal thread, and the diameter of the section provided with the external thread is slightly smaller than the diameter of the middle section of the outer tube 11 and the section provided with the internal thread. The inner tube 12 has the same diameter as the small tube diameter of the outer tube 11. The sample part 1 is further provided with a sealing member 13, the sample preservation solution is sealed in the cavity of the inner tube 12 through the sealing member 13 and the sample adding part 2 before reaction, and the sealing member 13 is removed when the sample to be measured is put in.

In this embodiment, the external thread of the sample-adding cap 22 of the sample-adding part 2 and the internal thread of the outer tube 11 are screwed in the cavity between the outer tube 11 and the inner tube 12, and the concave plug 21 is located in the inner tube 12 to seal the inner tube 12. The internal thread of the outer sleeve 31 of the reaction part 3 is in threaded connection with the external thread of the outer sleeve 11, and the end part of the inner tube 12 at the joint extends outwards to form a limiting ring, so that the position of the inner tube 12 in the outer sleeve 11 is limited, and the sample preserving fluid or the sample fluid can be prevented from entering the outer sleeve 11 into the compartment of the inner tube 12.

In this embodiment, the sample adding part 2 is further provided with a tearing ring 23, and the tearing ring 23 is used for limiting the rotation of the sample adding part 2. The tearing ring 23 is located between the outer tube 11 of the sample part 1 and the sample adding cap 22, and is connected in a point-breaking manner, and the tearing ring 23 extends outwards to be provided with an operation section convenient for tearing. The outer circumference of one end of the sample-adding cap 22 is provided with a plurality of ribs for facilitating the rotary pressurizing operation. The outer circumferences of the sample part 1 and the reaction part 3 are also provided with a plurality of friction ribs for facilitating the operation during rotation.

Before the rapid detection equipment is used for detection, the reaction systems are respectively pre-embedded in the reaction cavities 33, and the sample preservation solution is packaged in the cavity of the inner tube 12 in the sample part 1. Sampling a to-be-detected sample of the swab, dipping secretion or saliva, opening the reaction part and the sample part, removing a sealing piece, loading a swab section with the sample into the sample part 1 of the reactor, screwing the reaction part 3 and the sample adding part 2, tearing off the tearing pull ring 23, rotating the sample adding part 2 to pressurize sample liquid, and forcing the sample liquid to flow into the reaction cavity 33 through the micropores 342; then the reactor is put into a heating device for heating.

Example 2

FIG. 3 shows a second embodiment of the nucleic acid amplification reaction vessel of the present invention, which is different from example 1 mainly in the structure of the sample addition part 2, and further, the reaction part 3 and the sample part 1 are formed in an integrated structure.

In this embodiment, the sample adding part 2 comprises a sample adding plug 26 and a sample adding cover 27, the sample adding cover 26 is provided with an inner cavity, the sample adding plug 27 is positioned in the inner cavity of the sample adding cover 26, and the sample adding plug 26 has resistance unidirectional motion in the inner cavity. The sample adding plug 26 is provided with a plug in the inner cavity of the sample adding cover from top to bottom, so that the sample adding plug 26 can not move out of the sample adding cover 27. The sample adding plug 26 and the inner cavity of the sample adding cover 27 form a piston structure, a notch communicated with the inner cavity is formed above the sample adding cover 26, and a tool can be conveniently adopted to apply force to the sample adding plug 26. A vent hole is arranged below the sample adding cover, the vent hole is communicated with the sample part 1, and when the sample adding plug 26 moves towards the sample part 1, the sample liquid is pressurized through the vent hole, so that the sample liquid flows towards the reaction cavity 33. In this embodiment, the main body of the sample adding plug 26 is made of a hard transparent material, and sealing rubber rings are arranged at the lower end and the middle part.

In this embodiment, the reaction part 3 and the sample part 1 are integrally structured, and the sample part 1 and the sample addition cap 27 are screwed together.

Example 3

FIG. 4 shows a third embodiment of the nucleic acid amplification reaction vessel of the present invention, in this embodiment, a sample part 1, a sample addition part 2, and a reaction part 3 of the reaction vessel are connected in order, the sample addition part 2 is located between the sample part 1 and the reaction part 3, the sample addition part 2 is provided with a sealing member 13, and the sealing member 13 is moved in a connection chamber of the sample addition part 2 by an external force to mix a sample liquid and a reaction liquid in a sealed state.

In this embodiment, micropores are provided between the sample part 1 and the sample addition part 2, and between the reaction part 3 and the sample addition part 2, each sample chamber 14 corresponds to one micropore, each reaction chamber 33 corresponds to one micropore, and the diameter of the micropore is 0.3mm to 1mm, preferably 0.5mm. One sealing member 13 may simultaneously block the minute holes of the sample portion 1 and the minute holes of the reaction chamber 33. The reaction part 3 is provided with at least one reaction position, the sealing elements 13 are arranged in pairs, and a communication position 25 is formed between the two sealing elements 13.

In this embodiment, the reactor is a cuboid, the reaction chamber 33 and the sample chamber 14 are circular holes, which are respectively provided with a plurality of circular holes, and the connection positions of the reaction chamber 33 and the sample chamber 14 with the micropores are conical. The sample adding part 2 is provided with a circular connecting cavity. In practical application, according to requirements, one sample cavity 14 can correspond to one or more reaction cavities 33, different reaction liquids are buried in each reaction cavity 33, the communication positions 25 are correspondingly arranged, and if the sample cavity 14 and the reaction cavities 33 are arranged in a one-to-one manner, the two sealing members 13 are arranged at corresponding positions of the sample cavity 14 and the reaction cavity 33; if one sample chamber 14 corresponds to three reaction chambers 33, the area enclosed by the two sealing members 13 is three reaction chambers 33.

In this embodiment, the material of the reactor may be glass or plastic.

In this embodiment, the sample adding part 2 is provided with a moving member 24, the moving member 24 in this embodiment is a moving pin, the diameter of the moving pin is smaller than that of the connecting cavity of the sample adding part 2, the sealing member 13 is arranged on the moving pin, and the moving pin can move in the connecting cavity.

In this embodiment, the sealing member 13 is in interference connection with the inner wall of the sample adding part 2, and the sealing member 13 is made of high temperature resistant rubber, and is made into a non-transparent color, so that the communicating part 25 can be observed conveniently. The sealing elements 13 are sleeved and fixed on the moving pins and arranged at intervals. The thickness of the sealing element 13 is larger than the diameter of the micro-hole, so that the micro-hole on both sides of the sealing element 13 can be sealed.

In this embodiment, the reactor is provided with corresponding raised pressers, the diameter of which is slightly smaller than the diameter at the port of the moving pin. During the reaction, the convex pressing piece pushes the moving pin to move in the connecting cavity and drive the sealing piece 13 to leave the micro hole. And two ends of the connecting cavity are provided with limiting steps or the protruding pressing piece is provided with limiting steps for positioning the position of the sealing piece 13 during movement. The convex pressing pieces can be independently arranged or arranged on the centrifugal vibration machine.

In this embodiment, the reactor is further provided with a sealing cover 4, the sample chamber 14 and the reaction chamber 33 are provided with liquid inlets for introducing liquid from the outside to the inside thereof, and the liquid inlets are provided with the sealing cover 4 to prevent the sample liquid or the reaction liquid from leaking.

The nucleic acid amplification reactor of the invention comprises the following steps when in detection:

1) Firstly, determining that the sealing element 13 blocks micropores on two sides, namely the sample cavity 14, the reaction cavity 33 and the connecting cavity are not communicated, sealing the reaction liquid in the reaction cavity 33 in advance, and sealing the reaction cavity 33 through the sealing cover 4;

2) After the sample liquid is injected into the sample cavity 14, the sample cavity 14 is sealed by the sealing cover 4;

3) The reactor is placed on a centrifugal vibrator, the convex pressing piece pushes the moving pin to move to drive the sealing piece 13 to move, so that the sample cavity 14 is communicated with the reaction cavity 33 through the communication position 25, and the sample liquid and the reaction liquid cannot flow into the micropores;

4) And the sample liquid passes through the micropores and the reaction liquid passes through the micropores through the centrifugal vibration of the centrifugal vibrator, and the sample liquid and the reaction liquid are mixed and then react.

In this example, the sample solution contains DNA, genomic RNA, mRNA, etc., which are used as template nucleic acid strands in the nucleic acid amplification reaction.

The reaction solution contains one or more of a primer for detecting nucleic acid, DNA polymerase and reaction buffer.

The reaction solution may be injected into one reaction chamber 33 for each reaction solution or may be mixed and injected into one reaction chamber. One communication site 25 can correspond to a plurality of reaction chambers, and only one sample chamber 14 needs to be filled with a sample.

The reactor of example 3 above, used with an isothermal amplification apparatus: and (3) putting the reactor into an isothermal amplification instrument or heating equipment (a water bath kettle, a metal bath or a common PCR instrument), heating, and then sealing the interlayer to melt the reaction system and mix the reaction system with the sample liquid to form reaction liquid. The temperature in the reactor is maintained to the amplification temperature, and the reactor is oscillated to fully mix the reaction solution for amplification reaction. To ensure the reaction is complete, the reactor may be oscillated or rotated repeatedly. And judging the detection result through colorimetry.

The reactor of the embodiment of 3 is used in combination with a temperature-variable amplification device, and different reaction systems are adopted, and the detection result is judged by color development.

The above-described embodiments are merely preferred examples 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 spirit of the present invention should fall within the protection scope of the technical scheme of the present invention, unless the technical spirit of the present invention departs from the content of the technical scheme of the present invention.

Claims

1. A nucleic acid amplification reactor is characterized by comprising a sample adding part, a sample part and a reaction part, wherein before reaction, the sample part and the reaction part are in an independent sealing state, the sample adding part is of a piston structure, and a reaction system is arranged in the reaction part; 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 by external force to realize that the sample liquid under the sealed state passes through the micropores and enters the reaction part.

2. The nucleic acid amplification reaction vessel of claim 1, wherein the sample addition part, the sample part, and the reaction part are connected in sequence, the sample addition part and the sample part are movably connected, and a sample preservation solution is pre-loaded in the sample part; 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.

3. The nucleic acid amplification reactor of claim 2, wherein at least one reaction chamber is provided in the reaction part, an independent reaction system is preset in each reaction chamber, the micropores are arranged at the orifice of the reaction chamber, and each reaction chamber corresponds to one micropore.

4. The nucleic acid amplification reactor according to claim 3, wherein the reaction part is provided with a shunt plug made of a flexible material, the shunt plug is located at a 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.

5. The nucleic acid amplification reactor according to claim 4, wherein the pore size of the microwell is 0.3 to 0.6mm.

6. The nucleic acid amplification reactor of claim 3, wherein the sample loading part comprises a concave plug and a cap, the concave plug and the cap are sleeved with each other, a rigid protrusion is inserted into the cap and sleeved with the rigid protrusion, and the cap is further provided with a threaded connection section which is in threaded connection with the sample part.

7. The nucleic acid amplification reactor of claim 3, wherein the sample part is a coaxial sleeve, the inner tube is a hollow tube, the inner diameter of the hollow 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 addition 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.

8. The nucleic acid amplification reactor of claim 3, wherein the reaction chambers are closed at ports near the sample section, each reaction chamber is independently sealed before reaction, and the ports are closed by a sealing plug or a sealing film.

9. The nucleic acid amplification reactor according to claim 3, wherein the reaction part comprises an inner reaction tube and an outer sleeve, the inner reaction tube and the outer sleeve are sleeved with each other, the inner reaction tube is clamped in the outer sleeve, and the outer sleeve is provided with threads for coordination screw connection with the sample part.

10. The nucleic acid amplification reactor of claim 3, wherein the sample addition part comprises a sample addition plug and a sample addition cover, the sample addition cover is provided with an inner cavity, the sample addition plug is positioned in the inner cavity of the sample addition cover, the sample addition plug has resistance and one-way movement in the inner cavity, the sample addition plug and the inner cavity of the sample addition cover form a piston structure, and the sample solution enters the reaction cavity by the movement of the sample addition plug towards the direction of the sample part in a sealed state of the reactor.

11. The nucleic acid amplification reactor according to claim 1, wherein the sample section, the sample addition section, and the reaction section are connected in this order, the sample addition section is located between the sample section and the reaction section, and the sample addition section is provided with a sealing member that realizes mixing of the sample liquid and the reaction liquid in a sealed state by movement of an external force in the connection chamber of the sample addition section.

12. The nucleic acid amplification reactor according to claim 11, wherein the sample section is provided with at least one sample chamber, the reaction section is provided with at least one reaction chamber, and the sample chamber and the reaction chamber are not directly communicated with each other.

13. The nucleic acid amplification reaction vessel of claim 12, wherein a microwell is provided between the sample part and the sample addition part, and between the reaction part and the sample addition part, and one microwell is provided for each sample chamber, and one microwell is provided for each reaction chamber.

14. The nucleic acid amplification reactor of claim 13, wherein the sample addition part is provided with a moving member, the moving member is disposed in the connection chamber of the sample addition part, the moving member moves the sealing member in the connection chamber, and the sealing member is disposed on the moving member.

15. The nucleic acid amplification reaction vessel according to claim 14, wherein the sample addition part has at least one communication site, the sealing members are arranged in pairs, and a communication site is formed between the two sealing members, one communication site corresponding to one or more reaction chambers and one sample chamber.

16. The nucleic acid amplification reactor of claim 14, wherein the moving member has a cross-sectional area smaller than a diameter of the sample application part connection chamber, and the sealing member is in interference fit with an inner wall of the sample application part.

17. Use of a reactor according to any one of claims 1 to 16 in combination with a isothermal or temperature-variable amplification device for performing nucleic acid detection by color development during isothermal or temperature-variable amplification.