CN215906212U

CN215906212U - Nucleic acid amplification reactor

Info

Publication number: CN215906212U
Application number: CN202121991523.9U
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: 2022-02-25
Anticipated expiration: 2031-08-23
Also published as: CN115704049A; CN115703990A; CN215906211U; CN215757272U; CN215667945U; CN115786097A; CN215667988U; CN115703989A; CN115703991A

Abstract

The utility model discloses a nucleic acid amplification reactor, which comprises a sample part, a sample adding part and a reaction part which are connected in sequence, wherein the sample adding part is positioned between the sample part and the reaction part, 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. The reactor can solve the problem of uncovering pollution in the amplification method in the prior art, can directly react after a sample is added, does not need to uncover in the reaction process and does not need to add liquid for many times, and can realize the whole nucleic acid detection without a professional laboratory and aerosol pollution.

Description

Nucleic acid amplification reactor

Technical Field

The utility model relates to the technical field of nucleic acid detection (DNA or RNA) consumables, in particular to a nucleic acid amplification 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 biology technique for amplifying and amplifying a specific DNA fragment, which can be regarded as special DNA replication in vitro, and the biggest characteristic of PCR is that a trace amount of DNA can be greatly increased. However, PCR, a classical nucleic acid detection method, its inherent denaturation-renaturation-extension cycle, requires that it necessarily requires thermocycler equipment as a support, and professional laboratories also serve as a necessary condition because of aerosol contamination issues. 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 no external quantification standards need to be compared 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 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 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 min⁹～10¹⁰The double nucleic acid amplification has the characteristics of simple operation, strong specificity, easy detection of 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 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 utilizes the strand displacement type DNA polymerase to carry out amplification reaction under the constant temperature condition, can realize amplification of 109-1010 times within 15-60 minutes, can generate a large amount of amplification products, namely magnesium pyrophosphate white precipitate, and can judge whether the 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 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 a reactor which can directly complete reaction at one time after directly adding a sample is not available, and the traditional eight-connected tube or EP tube is still adopted, which is an important toggle that the nucleic acid detection cannot be well applied to POCT and the development and application of pathogenic microorganisms.

SUMMERY OF THE UTILITY MODEL

The technical problem to be solved by the utility model is as follows: aiming at the technical problems in the prior art, the utility model provides a nucleic acid amplification reactor 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 has no aerosol pollution

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

a nucleic acid amplification reactor comprises a sample part, a sample adding part and a reaction part which are connected in sequence, wherein the sample adding part is positioned between the sample part and the reaction part, before reaction, the sample part and the reaction part are in an independent sealing state, the sample adding part is in 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.

The further improvement of the technical scheme is as follows:

in the above embodiment, preferably, 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 to realize a sealed 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.

In the above aspect, preferably, the sample addition part is provided with a sealing member, and the sealing member moves in the connection cavity of the sample addition part by an external force to mix the sample liquid and the reaction liquid in a sealed state.

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

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 1 mm.

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.

In the above scheme, preferably, the reaction system is pre-embedded in the reaction chamber through an isolation layer, and the isolation layer is a material which is solid at normal temperature and is molten into liquid after being heated. The temperature of the molten packing material is matched with the heating reaction temperature, and the amplification reaction system is not influenced

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

(1) the nucleic acid amplification reactor 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 application of the nucleic acid amplification reactor, the pore diameter of the micropores in the reactor is set, so that the sample liquid or the sample preservation liquid is ensured not to enter the reaction cavity in the processes of storage, transportation, addition of a sample to be detected and the like, and the sample liquid can smoothly enter the reaction cavity for sufficient reaction under the condition of applying external force.

(3) The utility model provides a nucleic acid amplification reactor, packing material melting temperature matches heating reaction temperature, and can not exert an influence to the amplification reaction system, and the seal melts after the heating, has guaranteed that reaction system intensive mixing under the condition of not uncapping, and the packing material after the melting is because density floats on reaction system for a short time, has advanced one step and has become the seal on the reaction system, provides dual assurance to the abundant going on of reaction.

(4) 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 utility model 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 the present invention.

The reference numbers in the figures illustrate:

1. a sample section; 11. a seal member; 12. a sample chamber; 2. a sample addition part; 21. a moving member; 22. a communicating position; 3. a reaction section; 31. a reaction chamber; 32. 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 utility model.

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.

FIG. 1 shows an embodiment of a nucleic acid amplification reaction vessel according to 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 11, and the sealing member 11 is moved in a connection chamber of the sample addition part 2 by an external force to mix a sample solution and a reaction solution 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 12 corresponds to one micropore, each reaction chamber 31 corresponds to one micropore, and the diameter of the micropore is 0.3mm to 1mm, preferably 0.5 mm. One sealing member 11 can simultaneously block the minute hole of the sample portion 1 and the minute hole of the reaction chamber 31. The reaction part 3 is provided with at least one reaction site, the sealing elements 11 are arranged in pairs, and a communication site 22 is formed between the two sealing elements 11.

In this embodiment, the reactor is a cuboid, the reaction chamber 31 and the sample chamber 12 are circular holes, which are respectively provided with a plurality of circular holes, and the connection between the reaction chamber 31 and the sample chamber 12 and the micro-holes is conical. The sample adding part 2 is provided with a circular connecting cavity. In practical application, according to requirements, one sample cavity 12 can correspond to one or more reaction cavities 31, different reaction liquids are buried in each reaction cavity 31, the communication positions 22 are correspondingly arranged, and if the sample cavity 12 and the reaction cavities 31 are arranged in a one-to-one manner, the two sealing members 11 are arranged at corresponding positions of the sample cavity 12 and the reaction cavity 31; if one sample chamber 12 corresponds to three reaction chambers 31, the area enclosed by the two sealing members 11 is three reaction chambers 31.

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 21, the moving member 21 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 11 is arranged on the moving pin, and the moving pin can move in the connecting cavity.

In this embodiment, the sealing member 11 is in interference connection with the inner wall of the sample adding part 2, and the sealing member 11 is made of high temperature resistant rubber, and is made into a non-transparent color, so that the communicating part 22 can be observed conveniently. The sealing elements 11 are sleeved and fixed on the moving pins and arranged at intervals. The thickness of the sealing member 11 is larger than the diameter of the micro-hole so as to block the micro-holes 32 at both sides of the sealing member 11.

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. When the reaction is carried out, the convex pressing piece pushes the moving pin to move in the connecting cavity, and the sealing piece 11 is driven to leave the micro hole. The 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 11 during moving. The convex pressing piece can be arranged independently or on the centrifugal vibration machine.

In this embodiment, the reactor is further provided with a sealing cover 4, the sample chamber 12 and the reaction chamber 31 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 utility model comprises the following steps when in detection:

1) firstly, determining that the sealing element 11 blocks micropores on two sides, namely the sample cavity 12, the reaction cavity 31 and the connecting cavity are not communicated, sealing the reaction liquid in the reaction cavity 31 in advance, and sealing the reaction cavity 31 through the sealing cover 4;

2) after the sample liquid is injected into the sample cavity 12, the sample cavity 12 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 11 to move, so that the sample cavity 12 is communicated with the reaction cavity 31 through the communication position 22, and the sample liquid and the reaction liquid cannot flow into the micropores;

4) the sample liquid passes through the micropores and the reaction liquid passes through the micropores through centrifugal vibration of the centrifugal vibration machine, 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 a template nucleic acid strand in a nucleic acid amplification reaction.

The reaction solution contains one or more of a primer for nucleic acid to be detected, a DNA polymerase and a reaction buffer. Each reaction liquid may be injected into one reaction chamber 31 or may be mixed and injected into one reaction chamber. One communication position 22 can correspond to a plurality of reaction chambers, and only one sample chamber 12 needs to be filled with a sample.

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 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 part, a sample adding part and a reaction part which are connected in sequence, wherein the sample adding part is positioned between the sample part and the reaction part, before reaction, the sample part and the reaction part are in an independent sealing state, the sample adding part is in 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 reactor according to claim 1, wherein the sample addition part is provided with a sealing member which achieves 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 part.

3. The nucleic acid amplification reactor according to claim 2, 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.

4. The nucleic acid amplification reaction vessel according to claim 3, wherein a microwell is provided between the sample part and the sample application part and between the reaction part and the sample application part, and one microwell is provided for each sample chamber and one microwell is provided for each reaction chamber.

5. The nucleic acid amplification reactor of claim 4, wherein the sample addition part is provided with a moving member, the moving member is disposed in the connection cavity of the sample addition part, the moving member drives the sealing member to move in the connection cavity, and the sealing member is disposed on the moving member.

6. The nucleic acid amplification reaction vessel according to claim 5, 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.

7. The nucleic acid amplification reactor according to claim 5, wherein the moving member has a cross-sectional area smaller than a diameter of the sample addition part connection chamber, and the sealing member is in interference contact with an inner wall of the sample addition part.

8. The nucleic acid amplification reactor according to claim 4, wherein the diameter of the microwell is 0.3mm to 1 mm.

9. The nucleic acid amplification reactor of claim 3, wherein the reaction system is pre-embedded in the reaction chamber through an isolation layer, and the isolation layer is a material which is solid at normal temperature and melted into liquid after being heated.