A multiplex reaction detection unit
Patent Information
- Application Number
- CN202521876544.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2026-09-22
- Estimated Expiration
- 2035-09-02
AI Technical Summary
[0004]但是，“单管封闭”模式也存在很多缺陷和不足，例如：1.引物间干扰，每增加一个靶标，就需要一对新引物，引物之间容易形成二聚体或非特异性扩增；2.扩增效率差异，不同靶标的扩增效率差异大，导致“强者愈强，弱者愈弱”，难以实现均衡扩增；3.荧光信号重叠，荧光通道有限，多个探针信号可能串扰，影响结果判读；受限于引物二聚体、荧光通道数、扩增效率差异，单管重数通常≤20重；4.反应条件不兼容，不同靶标可能要求不同的退火温度、Mg2+浓度等，统一条件难度大
[0026]本申请的多重反应检测单元，仅需要通过一个液体注入口即可实现对所有反应管加样，操作简单方便；每个反应管都是独立反应，避免了交叉污染，确保了检测的有效性和准确性；并且，随着反应管数量增加，可以实现更多重检测，具有可扩展更多重检测的优势；总之，本申请的多重反应检测单元，同时兼顾“单管封闭”模式操作简单方便的优点，和可扩展更多重检测的优势，为即时检验提供了一种更优的多重、高通量检测方式。
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Figure CN224784168U_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of point-of-care testing technology, and in particular to a multiplex reaction detection unit. Background Technology
[0002] Point-of-care testing (POCT) is a testing method that uses portable analytical instruments and reagents to quickly obtain test results at the sampling site.
[0003] To simplify operation and reduce contamination risks, current POCT products generally adopt a "single-tube closed" mode, performing multiple reactions within a single tube. Specifically, pre-packaged lyophilized reaction beads contain optimized primer / probe pairs, anti-inhibitory enzymes, internal controls, and other reaction reagents; then, the cartridge is sealed, leaving only the sample injection port. During use, simply inject the sample to be tested through the sample injection port. Its advantages are: extremely simple operation: one-step sample addition, tube closure complete; extremely low contamination risk: no opening step; low consumable cost: single-tube reaction, consuming only a tiny amount of reagent per reaction.
[0004] However, the "single-tube closed" mode also has many defects and shortcomings, such as: 1. Primer interference: each additional target requires a new pair of primers, and primers are prone to dimerization or non-specific amplification; 2. Differences in amplification efficiency: different targets have large differences in amplification efficiency, leading to "the strong getting stronger and the weak getting weaker," making it difficult to achieve balanced amplification; 3. Fluorescence signal overlap: with limited fluorescence channels, multiple probe signals may crosstalk, affecting result interpretation; due to limitations in primer dimerization, the number of fluorescence channels, and differences in amplification efficiency, the single-tube weight is usually ≤20; 4. Incompatible reaction conditions: different targets may require different annealing temperatures and Mg... 2+ 5. There is a conflict between sensitivity and specificity; improving multiplicity often sacrifices sensitivity or specificity, especially in samples with low template amounts.
[0005] In addition, a few high-end technologies have broken through the detection throughput limit of the "single tube closed" mode and can achieve >100-fold detection through physical separation, such as microfluidic chips, droplet PCR, and CRISPR arrays; however, these technologies require precision instruments, are expensive, and have complex microfabrication and high cost of disposable consumables.
[0006] Therefore, how to balance the advantages of the "single-tube closed" mode in terms of simplicity and convenience with the advantages of high-end technologies such as microfluidic chips in terms of scalability and multiple detection remains a key research focus and challenge in the field of POCT technology. Summary of the Invention
[0007] The purpose of this application is to provide an improved multiple reaction detection unit.
[0008] The following technical solution is adopted in this application:
[0009] This application discloses a multiple reaction detection unit, including a reaction tube assembly and a flow channel sealing plate. The reaction tube assembly includes several independent reaction tubes, each of which is divided into upper and lower cavities by solid paraffin. The lower cavity of the reaction tube is pre-sealed with reaction reagents by solid paraffin, and the upper cavity is used to add the test solution. The injection volume of each reaction tube depends on the shape of the solid paraffin and the corresponding upper cavity of the reaction tube. The flow channel sealing plate includes a liquid inlet, a liquid outlet, and a reaction tube sealing cap. The number and position of the reaction tube sealing caps correspond to the reaction tubes of the reaction tube assembly. The reaction tube sealing caps have a reaction tube injection port and a reaction tube outlet for adding the test solution to the upper cavity of the corresponding reaction tube. The reaction tube injection port of the first reaction tube arranged in sequence is connected to the liquid injection port of the flow channel sealing plate, the reaction tube outlet of the first reaction tube is connected to the reaction tube injection port of the second reaction tube, the reaction tube outlet of the second reaction tube is connected to the reaction tube injection port of the third reaction tube, and so on, until the reaction tube outlet of the last reaction tube is connected to the liquid outlet of the flow channel sealing plate.
[0010] It should be noted that the multiplex reaction detection unit of this application, for example, can perform 4-5 real-time fluorescent PCR amplifications in each reaction tube during multiplex PCR amplification. Taking a conventional eight-tube pack as an example, one multiplex reaction detection unit can simultaneously perform 32-40 real-time fluorescent PCR amplifications, offering the advantage of scalability to perform more multiplex detections. Simultaneously, the multiplex reaction detection unit of this application uses the same liquid inlet for sample addition to each reaction tube, offering the advantages of a simple and convenient "single-tube closed" operation. Furthermore, the sample volume added to each reaction tube can be determined based on the position and shape of the solid paraffin, thus allowing for different sample volumes to be designed in different reaction tubes to meet different experimental design requirements. Additionally, after sample addition to each reaction tube, the solid paraffin can be melted by heating. The test solution on top of the paraffin will mix with the reaction reagent below, and the liquid paraffin acts as a sealant for the reaction system, eliminating the need for a heat-sealed cap.
[0011] In one implementation of this application, the flow channel sealing plate includes a microchannel plate and a sealing plate; the bottom of the microchannel plate is designed with a reaction tube sealing cap, and through holes are opened on both sides of the reaction tube sealing cap to serve as the reaction tube injection port and the reaction tube outlet port, respectively; a groove is opened on the top of the microchannel plate; the sealing plate is sealed on the top of the microchannel plate, so that the top groove forms a relatively sealed connecting pipe, and a through hole is opened at the injection position to form a liquid injection port, and a through hole is opened at the liquid discharge position to form a liquid outlet.
[0012] In one implementation of this application, the microfluidic plate and the sealing plate are fixed by double-sided tape or soft glue and screws.
[0013] In one implementation of this application, the microfluidic plate and sealing plate are made of polycarbonate (PC) or acrylonitrile-butadiene-styrene copolymer (ABS); the reaction tube assembly is made of polypropylene (PP). In this application, the materials PC, ABS, and PP are all medical-grade.
[0014] In one implementation of this application, the upper surface of the solid paraffin, i.e. the surface corresponding to the upper cavity of the reaction tube, has a hydrophilic treatment layer.
[0015] In one implementation of this application, the hydrophilic treatment layer is a hydrophilic structure formed by surface roughening.
[0016] In one implementation of this application, solid paraffin wax is thermoplastically formed into L-shapes, U-shapes, cylindrical shapes, or inverted triangles.
[0017] It should be noted that the shape of solid paraffin directly affects the amount of sample added to the test solution. The required shape can be selected according to the needs, and is not limited to L-shaped, U-shaped, cylindrical or inverted triangle.
[0018] In one implementation of this application, the reaction reagent is a PCR liquid mix or its in-situ lyophilized powder.
[0019] It should be noted that the multiple reaction detection unit of this application is not limited to PCR amplification, but can also be applied to other point-of-care tests, such as rolling circle amplification, etc., without specific limitations.
[0020] In one implementation of this application, the reaction tube assembly is an eight-tube bundle.
[0021] It should be noted that the eight-tube configuration is only one specific design of the number of reaction tubes in this application. More reaction tubes can be designed according to requirements or equipment conditions, and it is not limited to the eight-tube configuration.
[0022] In one implementation of this application, the reaction tube assembly and the flow channel sealing plate are detachably and fixedly connected. After the two are fixed, only the liquid injection port and the liquid outlet are left for adding the test solution to the reaction tube.
[0023] In one implementation of this application, the injection port and outlet of the reaction tube extend into the interior of the reaction tube. After extension, the distance between the pipe openings of the injection port and outlet and the predetermined liquid surface after the reaction tube is filled with liquid is greater than 1 mm. In a further improvement, the distance between the pipe openings of the injection port and outlet and the predetermined liquid surface after the reaction tube is filled with liquid is greater than 2 mm, preferably greater than 3 mm.
[0024] It should be noted that the injection port and outlet pipes of the reaction tube extend into the interior of the reaction tube. The length of the pipes is related to the highest point of the shaped paraffin, i.e., the liquid level after the reaction tube is filled with liquid. The distance between the two must be maintained at 1-3mm. This is because the internal diameter of the injection port and outlet pipes is only 0.4mm. When a certain air pressure is applied to the injection port, there will be a water pressure, which will force water into the tube. When the entire reaction tube is filled, it will flow out from the outlet. The air pressure will form a pressure column, forcing the excess liquid with a height of 1-3mm to flow to the next reaction tube, while the liquid left in the shaped paraffin groove remains.
[0025] The beneficial effects of this application are as follows:
[0026] The multiplex reaction detection unit of this application only requires one liquid injection port to add samples to all reaction tubes, making operation simple and convenient. Each reaction tube reacts independently, avoiding cross-contamination and ensuring the effectiveness and accuracy of the detection. Furthermore, as the number of reaction tubes increases, more multiplex detections can be achieved, offering the advantage of scalability for multiplex detections. In summary, the multiplex reaction detection unit of this application combines the advantages of simple and convenient operation in the "single-tube closed" mode with the advantage of scalability for multiplex detections, providing a superior multiplex, high-throughput detection method for point-of-care testing. Attached Figure Description
[0027] Figure 1 This is an exploded structural diagram of the multiple reaction detection unit in the embodiments of this application;
[0028] Figure 2 This is an exploded structural diagram of the multiple reaction detection unit from another perspective in the embodiments of this application;
[0029] Figure 3 This is a schematic diagram of the internal structure of the assembled multiple reaction detection unit in the embodiments of this application. Detailed Implementation
[0030] The present application will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.
[0031] Example
[0032] The multiple reaction detection unit in this example, such as Figures 1 to 3As shown, the assembly includes a reaction tube assembly 1 and a flow channel sealing plate 2. The reaction tube assembly 1 includes several independent reaction tubes 11, specifically eight reaction tubes in this example, i.e., an eight-tube assembly. Each reaction tube is divided into upper and lower cavities by solid paraffin. The lower cavity of the reaction tube is pre-sealed with the reaction reagent by solid paraffin, and the upper cavity is used to add the test solution. The injection volume of each reaction tube depends on the shape of the solid paraffin and its corresponding upper cavity. In this example, the solid paraffin is L-shaped. The flow channel sealing plate 2 includes a liquid inlet 221, a liquid outlet 222, and a reaction tube sealing cap 213. The number and position of the reaction tube sealing caps 213 are specified. Corresponding to reaction tube 11 of reaction tube assembly 1, reaction tube sealing cap 213 has a reaction tube injection port 214 and a reaction tube outlet 215 for adding the test solution to the upper cavity of the corresponding reaction tube. The reaction tube injection port of the first reaction tube arranged in sequence is connected to the liquid injection port 221 of the flow channel sealing plate 2, the reaction tube outlet of the first reaction tube is connected to the reaction tube injection port of the second reaction tube, the reaction tube outlet of the second reaction tube is connected to the reaction tube injection port of the third reaction tube, and so on, until the reaction tube outlet of the last reaction tube is connected to the liquid outlet 222 of the flow channel sealing plate 2. The reaction tube assembly 1 and the flow channel sealing plate 2 are detachably and fixedly connected. After they are fixed, only the liquid injection port 221 and the liquid outlet 222 are left open for adding the test solution to the reaction tube.
[0033] In this example, the flow channel sealing plate 2 includes a micro-flow channel plate 21 and a sealing plate 22, such as Figure 2 As shown, the bottom of the microfluidic plate 21 is designed with a reaction tube sealing cap 213. The reaction tube sealing cap 213 has through holes on both sides, which serve as the reaction tube injection port 214 and the reaction tube outlet 215, respectively. The top of the microfluidic plate 21 has a groove. The sealing plate 22 is sealed on the top of the microfluidic plate 21, so that the top groove forms a relatively sealed connecting pipe. A through hole is opened at the injection position to form a liquid injection port 221, and a through hole is opened at the liquid discharge position to form a liquid outlet 222.
[0034] In this example, the microfluidic plate 21 and the sealing plate 22 are fixed with double-sided tape or soft glue and screws. The microfluidic plate 21 and the sealing plate 22 are made of medical-grade PC (polycarbonate) / medical-grade ABS (acrylonitrile-butadiene-styrene copolymer) material, and the reaction tube assembly 1 is made of medical-grade PP (polypropylene) material.
[0035] In this example, the upper surface of the solid paraffin, corresponding to the upper cavity of the reaction tube, has a hydrophilic treatment layer. This hydrophilic treatment layer is a hydrophilic structure formed by surface roughening.
[0036] In this example, the injection port 214 and the outlet port 215 of the reaction tube extend into the reaction tube 11. After extension, the distance between the pipe openings of the injection port 214 and the outlet port 215 and the pre-set liquid level after the reaction tube is filled with liquid is greater than 1-3 mm; in this example, it is designed to be greater than 3 mm. The connecting pipe 216 between two adjacent reaction tubes is located at the top of the injection port 214 and the outlet port 215 of the reaction tube, such as... Figure 3 As shown.
[0037] Taking real-time fluorescence PCR amplification as an example, a quantitative amount of liquid PCR liquid mix or in-situ lyophilized powder is placed at the bottom of each reaction tube, followed by a quantitative amount of solid paraffin. The paraffin is then molded into an L-shape using its thermoplasticity. After molding, a hydrophilic treatment is performed. The L-shaped paraffin allows for a relatively quantitative retention of the nucleic acid test solution after liquid injection. The reaction tubes are then fitted with a forced sequential injection tube; the solution will only proceed to the next reaction tube after the previous one has been completely filled, and so on, until all eight reaction tubes are filled with the nucleic acid test solution. Then, pressure is applied (positive or negative) to push away excess liquid. The multiplex reaction unit is heated to 55-65℃, causing the paraffin to melt. The nucleic acid test solution on top of the paraffin mixes with the PCR liquid mix or lyophilized powder below, forming a complete reaction system. The molten paraffin seals the PCR reaction system, allowing subsequent PCR reactions to proceed. Due to the liquid paraffin seal, a heat-sealed reaction system is not required. Furthermore, the direction of liquid injection of the test solution can be either from the left or right; no specific limitation is made here.
[0038] The multiple reaction detection unit in this example has the following advantages compared to existing point-of-care testing methods:
[0039] 1. High-repetition detection: The theoretical repetition rate is limited only by the number of chambers. For example, when performing multiplex PCR amplification, each reaction tube can perform 4-5 repetitions of real-time fluorescent PCR amplification, and eight reaction tubes can perform 32-40 repetitions of real-time fluorescent PCR amplification simultaneously, which has the advantage of being scalable to detect more repetitions.
[0040] 2. Each chamber has an independent reaction system to avoid primer cross-inhibition.
[0041] 3. One test solution is equivalent to being evenly distributed into multiple reaction tubes. Different amounts of test solution can also be designed to meet different experimental design requirements.
[0042] 4. The micropores are self-sealing, eliminating the need for a heat-sealing cap; the molten liquid paraffin acts as a seal, allowing the reaction system to proceed in a relatively closed environment.
[0043] 5. The detection sensitivity is higher than that of ultra-multiplex detection. Since each reaction tube only needs to perform 4-5 detections, or even fewer, the mutual influence between the detection targets is smaller. Therefore, it can detect each target more sensitively. In contrast, ultra-multiplex detection has more primers, probes and detection targets in a single reaction system, making it difficult to guarantee good sensitivity for all detection targets.
[0044] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.
Claims
1. A multiple reaction detection unit, characterized in that: It includes a reaction tube assembly (1) and a flow channel sealing plate (2); The reaction tube assembly (1) includes several independent reaction tubes (11). Each reaction tube is divided into upper and lower cavities by solid paraffin. The lower cavity of the reaction tube is pre-sealed with reaction reagents by solid paraffin, and the upper cavity is used to add the solution to be tested. The amount of liquid injected into each reaction tube depends on the shape of the solid paraffin and the corresponding upper cavity of the reaction tube. The flow channel sealing plate (2) includes a liquid inlet (221), a liquid outlet (222), and a reaction tube sealing cap (213). The number and position of the reaction tube sealing caps (213) correspond to the reaction tubes (11) of the reaction tube assembly (1). The reaction tube sealing caps (213) are provided with a reaction tube injection port (214) and a reaction tube outlet (215) for adding the test solution to the upper cavity of the corresponding reaction tube. The reaction tube injection port of the first reaction tube arranged in sequence is connected to the liquid inlet (221) of the flow channel sealing plate (2), the reaction tube outlet of the first reaction tube is connected to the reaction tube injection port of the second reaction tube, the reaction tube outlet of the second reaction tube is connected to the reaction tube injection port of the third reaction tube, and so on. The reaction tube outlet of the last reaction tube is connected to the liquid outlet (222) of the flow channel sealing plate (2).
2. The multiple reaction detection unit according to claim 1, characterized in that: The flow channel sealing plate (2) includes a microflow channel plate (21) and a sealing plate (22); The bottom of the microfluidic plate (21) is designed with a reaction tube sealing cap (213), and through holes are opened on both sides of the reaction tube sealing cap (213) to serve as the reaction tube injection port (214) and the reaction tube outlet port (215) respectively; a groove is opened on the top of the microfluidic plate (21); The sealing plate (22) covers the top of the microchannel plate (21), so that the top groove forms a relatively sealed connecting pipe, and a through hole is opened at the liquid injection position to form a liquid injection port (221), and a through hole is opened at the liquid discharge position to form a liquid outlet (222).
3. The multiple reaction detection unit according to claim 2, characterized in that: The microchannel plate (21) and the sealing plate (22) are fixed by double-sided tape or soft glue and screws.
4. The multiple reaction detection unit according to claim 1, characterized in that: The upper surface of the solid paraffin, that is, the surface corresponding to the upper cavity of the reaction tube, has a hydrophilic treatment layer.
5. The multiple reaction detection unit according to claim 4, characterized in that: The hydrophilic treatment layer is a hydrophilic structure formed by surface roughening.
6. The multiple reaction detection unit according to any one of claims 1-5, characterized in that: The solid paraffin wax is thermoplastically formed into L-shaped, U-shaped, cylindrical, or inverted triangular shapes.
7. The multiple reaction detection unit according to any one of claims 1-5, characterized in that: The reaction reagent is PCR liquid mix or its in-situ lyophilized powder.
8. The multiple reaction detection unit according to any one of claims 1-5, characterized in that: The reaction tube assembly (1) is an eight-tube assembly.
9. The multiple reaction detection unit according to any one of claims 1-5, characterized in that: The injection port (214) and outlet port (215) of the reaction tube extend into the interior of the reaction tube. After extension, the distance between the pipe openings of the injection port (214) and outlet port (215) and the preset liquid surface after the reaction tube is filled with liquid is greater than 1 mm.
10. The multiple reaction detection unit according to claim 9, characterized in that: The distance between the inlet (214) and outlet (215) of the reaction tube and the liquid surface after the reaction tube is filled with liquid is greater than 3 mm.