Reaction tube
By independently aliquoting the lyophilized balls and buffers in the PCR reaction tube and using magnetic components to achieve rapid mixing, the problems of complex operation and insufficient component stability in the prior art are solved, and the operation is simplified and the test efficiency is improved.
Patent Information
- Application Number
- CN202421431488.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-21
- Publication Date
- 2025-05-09
- Estimated Expiration
- 2034-06-21
AI Technical Summary
The existing PCR reaction technology is complex in high-throughput testing, and the component configuration and assembly process are cumbersome, which can easily lead to contamination and errors. The aliquoting and sample addition of enzymes have high requirements for the operator's technical literacy and the enzyme cost is high.
A reaction tube was designed to independently aliquot lyophilized bulbs and buffers, and set up magnetic elements in the tube body, and use magnetic forces to quickly mix the components during the reaction process, simplifying the operation steps.
It realizes stable storage and rapid mixing of enzymes and buffers, simplifies PCR test operations, reduces operating errors and contamination risks, and improves the storage stability of reagents.
Smart Images
Figure CN222846712U_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of polymerase chain reaction, and in particular to a reaction tube. Background Art
[0002] In the field of biotechnology, polymerase chain reaction (PCR) has become a mature technology for amplifying specific nucleic acid sequences. This reaction process involves three main steps: denaturation, annealing, and extension, each of which requires a different reaction temperature. The temperature of denaturation is typically between 90 and 97°C; the temperature of annealing is selected based on the melting temperature of the primers used, typically between 50 and 65°C; the typical temperature of the extension reaction is 72°C. PCR reaction is a common technology for nucleic acid amplification and is widely used in the detection of pathogens, infectious diseases, and genetic diseases.
[0003] PCR reactions usually consist of the following components: reaction buffer (containing dNTPs, magnesium ions, buffer salts), enzymes (reverse transcriptase, Taq enzyme, UDG enzyme, RNase inhibitor, etc.), primer probes, sample solutions, etc.
[0004] In the actual application stage, operators are usually required to manually add some of the above components to the PCR tube quickly to complete the reaction. Especially in high-throughput testing, the component configuration and packaging process is a huge workload, the reaction system is also very easy to be contaminated, and errors are easy to occur between each batch and each tube of the reaction system. In addition, the packaging and addition of enzymes have certain requirements on the technical literacy of operators, otherwise it is easy to damage and contaminate the enzymes; and the cost of enzymes is also high.
[0005] Patent CN210458197U discloses a detection kit that can detect eight types of hand, foot and mouth disease viruses simultaneously through a single channel, and a freeze-dried reagent layer is provided in the kit. Among them, the freeze-dried reagent includes reverse transcriptase, DNA polymerase, PCR buffer, Mg2+, dNTP, specific primers and probes for hand, foot and mouth disease related viruses, and freeze-dried protective agents. However, this mixed pre-storage structure is difficult to ensure the stability of each component during long-term storage and transportation, and it also has higher requirements for pre-storage technology (such as freeze-drying technology).
[0006] Therefore, there is an urgent need for a PCR reaction scheme that can simplify the operator's manual operations and improve the storage stability of reagents. Utility Model Content
[0007] The utility model aims to provide a reaction tube, which partially solves or alleviates the above-mentioned deficiencies in the prior art. Through the independent packaging design, each key component (such as freeze-dried balls and buffer) can be stably stored, thereby simplifying the operation steps during the PCR test. For example, the PCR reaction can be quickly started by directly adding a sample liquid (such as nucleic acid) to a reaction tube pre-stored with freeze-dried balls and buffer.
[0008] In order to solve the above-mentioned technical problems, the utility model specifically adopts the following technical solutions:
[0009] A reaction tube, comprising:
[0010] A tube body, wherein the tube body is sequentially formed with a first accommodating space and a second accommodating space along the direction from the first end to the second end thereof, wherein a buffer solution is stored in the first accommodating space, and at least one magnetic element is further arranged in the first accommodating space, wherein the magnetic element can move within the tube body under the action of a magnetic force; and a sealing layer is further arranged in the first accommodating space, wherein the sealing layer is used to seal the buffer solution and limit the magnetic element in the buffer solution;
[0011] An opening is provided at the first end of the second containing space, and a first inner diameter at the first end of the opening is smaller than a second inner diameter at the second end of the opening. The second end of the opening is a sample addition port, and a freeze-dried ball is provided in the opening, and a diameter of the freeze-dried ball is larger than the first inner diameter.
[0012] In some embodiments, the diameter of the projection pattern of the magnetic element on the action surface is 30%-90% of the diameter of the tube body; wherein the action surface refers to a plane that is perpendicular or approximately perpendicular to the axis of the tube body.
[0013] In some embodiments, the magnetic element is a magnetic iron block.
[0014] In some embodiments, the magnetic element is designed as a hollow structure, wherein a plurality of mesh structures penetrating the magnetic element are distributed on the magnetic element.
[0015] In some embodiments, at least one of the mesh structures is modified with a binding group, and the binding group is used to bind to the enzyme in the lyophilized ball.
[0016] In some embodiments, the lyophilized pellet contains only enzyme and excipient, and does not contain buffer. The enzyme can be in the form of a single enzyme or a complex connected with magnetic microspheres, specifically, the magnetic microspheres are surface modified (such as carboxyl) and then coupled with the enzyme.
[0017] In some embodiments, the diameter of the lyophilized ball is about 2-5 mm, and the diameter of the lyophilized ball is larger than the diameter of the tube.
[0018] In some embodiments, the tube is a capillary tube with a diameter of about 1-4 mm.
[0019] In some embodiments, the sealing layer is paraffin.
[0020] In some embodiments, the inner diameter of the opening gradually increases from the first end to the second end thereof.
[0021] Beneficial technical effects:
[0022] The utility model provides a PCR reaction tube that can independently package enzymes and buffers, and its packaging scheme and packaging structure design can ensure the stability of each component during the storage process. At the same time, the design of the magnetic element can promote the rapid mixing of the components in the tube during the reaction process, saving reaction time. Furthermore, the utility model also proposes a magnetic element with a grid design for the above-mentioned packaging scheme, wherein the hollow design facilitates the passage of liquid on the one hand, and at the same time, it is adsorbed and combined with magnetic beads modified with binding groups (such as carboxyl groups), which is also conducive to achieving local enrichment of enzymes, thereby helping to improve the efficiency of PCR reactions. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the utility model or the prior art, the drawings required for use in the embodiments or the prior art description are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the utility model. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without paying creative labor.
[0024] Figure 1 It is a schematic diagram of a first cross-sectional structure of a reaction tube in an exemplary embodiment of the utility model;
[0025] Figure 2a It is a structural schematic diagram of a magnetic element in an exemplary embodiment of the utility model;
[0026] Figure 2b It is a structural schematic diagram of a magnetic element in another exemplary embodiment of the utility model;
[0027] Figure 3 It is a perspective structural schematic diagram of a reaction tube in an exemplary embodiment of the utility model;
[0028] Figure 4It is a schematic diagram of the structure of a reaction tube in an exemplary embodiment of the present invention.
[0029] Summary of reference numerals:
[0030] 10 is a tube body, 11 is a first accommodating space, 12 is a second accommodating space, 13 is a magnetic element, 14 is a sealing layer, 20 is an opening, and 21 is a freeze-dried ball. DETAILED DESCRIPTION
[0031] In order to make the purpose, technical solution and advantages of the embodiment of the utility model clearer, the technical solution in the embodiment of the utility model will be clearly and completely described below in conjunction with the drawings in the embodiment of the utility model. Obviously, the described embodiment is a part of the embodiment of the utility model, not all of the embodiments. Based on the embodiment of the utility model, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.
[0032] Herein, the suffixes such as "module", "component" or "unit" used to represent elements are only used to facilitate the description of the present invention, and have no specific meanings. Therefore, "module", "component" or "unit" can be used in a mixed manner.
[0033] In this document, the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.
[0034] In this document, unless otherwise clearly specified or limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this utility model can be understood according to specific circumstances.
[0035] Herein "and / or" includes any and all combinations of one or more of the associated listed items.
[0036] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.
[0037] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.
[0038] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.
[0039] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.
[0040] See also Figure 1-Figure 4 As shown, the utility model provides a reaction tube, comprising:
[0041] A tube body 10, wherein the tube body 10 is sequentially formed with a first accommodating space 11 and a second accommodating space 12 along the direction from the first end to the second end thereof, wherein a buffer solution is stored in the first accommodating space 11, and at least one magnetic element 13 is further provided in the first accommodating space 11, wherein the magnetic element 13 (also referred to as a movable block) can move within the tube body under the action of a magnetic force; a sealing layer 14 is further provided in the first accommodating space, wherein the sealing layer 14 is used to seal the buffer solution and limit the magnetic element in the buffer solution;
[0042] The first end of the second accommodation space 12 is provided with an opening 20, and the first inner diameter at the first end of the opening 20 is smaller than the second inner diameter at the second end of the opening 20. A freeze-dried ball 21 is provided in the opening, and the diameter of the freeze-dried ball 21 is larger than the first inner diameter. The first end of the tube body 10 is the bottom of the tube, and the second end is the opening direction.
[0043] When the reaction tube is activated, corresponding reagents or sample solutions (such as nucleic acids) can be added into it through the opening to dissolve the freeze-dried balls, so that the reagents, freeze-dried balls, and buffer can be mixed with each other to form a PCR reaction solution.
[0044] The sealing layer is preferably paraffin, which can be dissolved at high temperature to cut off the sealing effect on the buffer solution and achieve liquid mixing.
[0045] In some embodiments, one end of the reaction tube is also configured with an electromagnetic driving module, which is used to provide a magnetic force with a specific direction and a specific size to the magnetic element, so that the magnetic element can move back and forth in different liquid areas in the tube body 10 under the action of the magnetic force to mix the liquid.
[0046] In some embodiments, see Figure 2a As shown, the diameter of the projection figure of the at least one magnetic element 13 on the active surface is approximately 30%-80% of the diameter D2 of the tube body 10; wherein the active surface refers to a plane perpendicular or approximately perpendicular to the axis of the tube body 10 (or, the active surface refers to a plane parallel or approximately parallel to the cross-section of the tube body).
[0047] The projection pattern refers to the orthographic projection of the magnetic element on the action surface.
[0048] For example, in some embodiments, the magnetic element 13 may be a magnetic iron ball, which may be a solid iron ball or a hollow iron ball, and the diameter D1 of the magnetic iron ball is 30%-90% of the diameter D2 of the tube body.
[0049] Alternatively, in some embodiments, the magnetic element 13 may also be a columnar magnetic iron block.
[0050] For another example, in some embodiments, a plurality of small-sized magnetic elements (or magnetic particles) may be provided in the tube body, and the plurality of magnetic elements can be combined with each other under the action of corresponding magnetic forces to form a hollow structure. Specifically, the plurality of magnetic elements are connected to each other under the action of magnetic forces, and the contact surfaces between at least two adjacent magnetic elements are not completely fitted to form pores (also referred to as: mesh structures or hollow structures), and the pores can accommodate liquids. In other words, a plurality of small-sized magnetic elements can form a grid-like structure / mesh-like structure under the action of magnetic adsorption.
[0051] Preferably, the plurality of magnetic elements may have different diameters and sizes to form an irregular hollow structure, and in particular, to form a plurality of pore spaces of different sizes on the hollow structure.
[0052] Alternatively, in other embodiments, the magnetic element is designed as a hollow structure, wherein a plurality of mesh structures are distributed on the magnetic element and penetrate the magnetic element; when the freeze-dried balls and the buffer solution in the reaction tube are subjected to polymerase chain reaction, at least one of the mesh structures can be used to accommodate the corresponding mixed liquid and form a local ultra-high concentration reaction area.
[0053] Specifically, the magnetic element is designed as a hollow structure, wherein at least one first mesh structure formed by an inward depression and a second mesh structure penetrating the magnetic element are distributed on the magnetic element; when the freeze-dried balls and the buffer solution in the reaction tube are subjected to polymerase chain reaction, at least one mesh structure can be used to accommodate the corresponding mixed liquid and form a local ultra-high concentration reaction area.
[0054] The second mesh structure provided through the magnetic element can also reduce the resistance borne by the magnetic element during the reciprocating movement to a certain extent.
[0055] In some embodiments, at least one of the mesh structures is modified with a binding group, and the binding group is used to bind to the enzyme in the lyophilized ball.
[0056] For example, in some embodiments, the modification group on the surface of the magnetic iron ball can be a carboxyl group or other groups that can bind to the surface of a protein.
[0057] PCR involves three main steps: denaturation, annealing, and extension; the denaturation step does not require the participation of enzymes, only the extension step requires the participation of enzymes. The use of magnetic elements modified with binding groups can control the enzyme to react only in the extension step, forming a local high concentration, and the extension temperature is 60-72°C, which enhances the optimal activity half-life of the enzyme.
[0058] In this embodiment, an optional scheme is to modify the binding group on the magnetic element, that is, to be able to fix the enzyme through the chemical bond between the binding group and the enzyme, and form a local enrichment area of the enzyme (that is, an ultra-high concentration reaction area) on the magnetic element (such as in the pores of the grid structure).
[0059] Alternatively, in other embodiments, the lyophilized pellet contains only enzymes and excipients, but does not contain PCR buffer, thereby simplifying the lyophilization process and maintaining the performance of the enzyme for a longer period of time after lyophilization.
[0060] For example, in some embodiments, a lyophilized ball may include a plurality of lyophilized particles composed of magnetic particles and enzymes, and the plurality of lyophilized particles may be combined into a large lyophilized ball through a lyophilization process.
[0061] For example, in some other embodiments, a plurality of freeze-dried particles can be directly stored at the opening. Correspondingly, a grid plate can be provided at the opening, and a plurality of holes (whose inner diameter is smaller than the diameter of the freeze-dried particles) can be provided on the grid plate to isolate and store the freeze-dried particles at the opening before they are reconstituted.
[0062] Subsequently, when the freeze-dried particles (or freeze-dried balls) are redissolved and enter the mixed liquid, under the dual effects of magnetic attraction, enzymes and binding groups, the redissolved freeze-dried particles (or freeze-dried balls) will be preferentially adsorbed on the magnetic element to form a local enrichment area of the enzyme thereon, thereby improving the efficiency of the PCR reaction.
[0063] In some embodiments, the diameter of the lyophilized spheres is 2-5 mm.
[0064] In some embodiments, the tube is a capillary tube.
[0065] In some embodiments, the sealing layer is paraffin.
[0066] In some embodiments, the inner diameter of the opening gradually increases from the first end to the second end thereof.
[0067] In some embodiments, the lyophilized pellets include enzymes and trehalose. Preferably, the components of the lyophilized pellets are enzymes without glycerol, and trehalose is used as an excipient. In this embodiment, the lyophilized pellets do not contain a buffer (i.e., the enzyme and the buffer are packaged separately), so there is no need to consider the effect of the increased salt ion concentration of the buffer during the lyophilization process, and the lyophilization process is simpler.
[0068] In some embodiments, the components of the buffer are conventional PCR components, such as one or more of the following: Tri-HCl, KCl, MgCl2.
[0069] In some embodiments, the invention further includes: a tube cover disposed at the second end of the opening.
[0070] Alternatively, in other embodiments, a sealing film is provided at the tube opening to seal the tube body.
[0071] It is worth noting that the tube design and magnetic element used in the utility model cooperate with each other to achieve stable packaging of freeze-dried balls (mainly enzymes) and buffer solutions, and at the same time ensure that the two can be quickly and evenly mixed from independent packaging states during the reaction process. This independent packaging scheme for enzymes and buffer solutions has little effect of aerosolization during the enzyme re-dissolution process and is not prone to generate bubbles.
[0072] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.
[0073] The embodiments of the utility model are described above in conjunction with the accompanying drawings, but the utility model is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the utility model, ordinary technicians in this field can also make many forms without departing from the scope of protection of the utility model and the claims, which all fall within the protection of the utility model.
Claims
1. A reaction tube, characterized in that: include: A tube body (10), wherein the tube body (10) is sequentially formed with a first accommodating space (11) and a second accommodating space (12) along the direction from the first end to the second end thereof, wherein a buffer solution is stored in the first accommodating space (11), and at least one magnetic element (13) is also arranged in the first accommodating space (11), wherein the magnetic element (13) can move in the tube body under the action of a magnetic force; and a sealing layer (14) is also arranged in the first accommodating space, wherein the sealing layer (14) is used to seal the buffer solution and limit the magnetic element in the buffer solution; The first end of the second accommodating space (12) is provided with an opening (20), and the first inner diameter at the first end of the opening (20) is smaller than the second inner diameter at the second end of the opening (20), the second end of the opening is a sample addition port, and a freeze-dried ball (21) is provided in the opening, and the diameter of the freeze-dried ball (21) is larger than the first inner diameter.
2. The reaction tube according to claim 1, characterized in that: The diameter of the projection figure of the magnetic element (13) on the action surface is 30%-90% of the diameter of the tube body (10); wherein the action surface refers to a plane perpendicular or approximately perpendicular to the axis of the tube body (10).
3. The reaction tube according to claim 2, characterized in that: The magnetic element is a magnetic iron block.
4. The reaction tube according to claim 2, characterized in that: The magnetic element is designed as a hollow structure, wherein a plurality of mesh structures penetrating the magnetic element are distributed on the magnetic element; when the freeze-dried balls and the buffer solution in the reaction tube are subjected to polymerase chain reaction, at least one of the mesh structures can be used to accommodate the corresponding mixed liquid and form a local ultra-high concentration reaction zone.
5. The reaction tube according to claim 4, characterized in that: At least one of the mesh structures is modified with a binding group, and the binding group is used to bind to the enzyme in the freeze-dried ball.
6. The reaction tube according to claim 4, characterized in that: The freeze-dried ball comprises magnetic particles, and the periphery of the magnetic particles is covered with enzymes.
7. The reaction tube according to claim 1, characterized in that: The diameter of the freeze-dried balls is 2-5 mm.
8. The reaction tube according to claim 1, characterized in that: The tube body is a capillary tube.
9. The reaction tube according to claim 1, characterized in that: The sealing layer is paraffin.
10. The reaction tube according to any one of claims 1 to 9, characterized in that: The inner diameter of the opening gradually increases from the first end to the second end.