WO2020135873A1 - 一种可进行多重核酸扩增的反应管 - Google Patents

一种可进行多重核酸扩增的反应管 Download PDF

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WO2020135873A1
WO2020135873A1 PCT/CN2019/129968 CN2019129968W WO2020135873A1 WO 2020135873 A1 WO2020135873 A1 WO 2020135873A1 CN 2019129968 W CN2019129968 W CN 2019129968W WO 2020135873 A1 WO2020135873 A1 WO 2020135873A1
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Prior art keywords
reaction
nucleic acid
acid amplification
reaction tube
multiple nucleic
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PCT/CN2019/129968
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English (en)
French (fr)
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王升启
荣振
肖瑞
王封
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中国人民解放军军事科学院军事医学研究院
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Priority to DE112019006515.7T priority Critical patent/DE112019006515T5/de
Priority to US17/418,947 priority patent/US20220111379A1/en
Publication of WO2020135873A1 publication Critical patent/WO2020135873A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/043—Hinged closures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers

Definitions

  • the present disclosure relates to the fields of application technologies such as biological science research and medical testing, and in particular, to a reaction tube capable of performing multiple nucleic acid amplification.
  • Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) technology is a technique for rapid DNA amplification in vitro. Each cycle includes three processes: denaturation, annealing and extension.
  • This process is called low-temperature annealing reaction; after the annealing reaction is completed, the temperature should be rapidly increased to The extension reaction is carried out at about 72°C.
  • the extension reaction is carried out at about 72°C.
  • a single nucleotide is bound from the 3'end of the primer to form a new DNA.
  • the original DNA double-stranded molecule formed two DNA molecules, and the number doubled.
  • the number of target nucleic acid molecules is doubled, and these newly formed double strands can be used as a template for the next cycle. After 30-40 cycles, the number of target nucleic acid molecules is amplified by nearly 109 times .
  • PCR is also known as cell-free molecular cloning or specific DNA sequence in vitro primer-directed enzymatic amplification technology, which allows the target DNA to be rapidly amplified. It has the characteristics of strong specificity, high sensitivity, easy operation, time saving and high efficiency. It can be used not only for basic research such as gene isolation, cloning and nucleic acid sequence analysis, but also for diagnosis of diseases and other places containing DNA and RNA.
  • constant temperature amplification is also a new method of nucleic acid amplification, which has received more and more attention in recent years.
  • the objective of the present disclosure includes providing a reaction tube capable of performing multiple nucleic acid amplifications, so as to solve one of the technical problems existing in the nucleic acid amplification reaction tubes in the prior art.
  • the present disclosure provides a reaction tube capable of performing multiple nucleic acid amplification, including a base and multiple reaction lumens; the base is provided with a reference plane, and the openings of multiple reaction lumens are all provided on the reference.
  • the inner surfaces of the flat surfaces extend perpendicular to the reference plane toward the inside of the base.
  • a plurality of the reaction lumens are distributed annularly around the same axis.
  • the technical effect of the technical solution lies in that the multiple reaction lumens distributed in a ring shape are more compact on the one hand, and the layout is more regular on the other hand, which facilitates the operation of injecting and extracting test samples in the test. For example, eight, twelve, or sixteen reaction lumens are distributed annularly around the same central axis.
  • a plurality of the reaction lumens are distributed in a ring shape along a plurality of different radii.
  • this structure can divide multiple reaction lumens into a plurality of different radius but coaxial annular distribution, and set as many reaction lumens as possible in a small space.
  • the outer ring is provided with twelve reaction lumens
  • the inner ring is provided with six reaction lumens, evenly arranged around the same central axis.
  • a plurality of the reaction lumens located in the same ring shape are individually fixed to the base.
  • reaction lumens located in the same ring shape are integrally formed and fixed to the base.
  • a central lumen is further included; the central lumen is disposed on the reference plane and is located in the annular center of the plurality of reaction lumens.
  • the central lumen can not only reduce the weight of the reaction tube body, but also use the central lumen as the operating space, and perform the amplification reaction operation through the operating handle matched with the central lumen.
  • the central lumen penetrates the base.
  • the base is cylindrical, and its axis coincides with the axis of the central lumen.
  • the technical effect of this technical solution is that the cylindrical base is convenient for setting the screw cap, and the structure of the screw thread of the side wall is used to realize the uniform sealing of the screw cap on all the reaction lumens.
  • a blocking member or a screw cap is further included; the blocking member is provided at an opening of any of the reaction lumens; the screw cap is spirally provided at one end of the base , Blocking multiple openings of the reaction lumens.
  • the sealing piece is set at the opening of the sealing reaction cavity, preferably using a heat sealing film or heat sealing glue to achieve sealing
  • the screw cap is used to completely seal all the reaction cavities and temporarily seal the reaction
  • the nucleic acid sample solution in the lumen is protected from external dust, light and other factors, and can also prevent the nucleic acid sample solution from pouring out.
  • an injection hole may be provided on the screw cap.
  • reaction lumens are distributed in rows and columns on the reference plane.
  • the beneficial effects of the present disclosure include, for example:
  • the reaction tube for multiple nucleic acid amplification provided by the present disclosure is provided with multiple reaction lumens on the reference plane of the base, and nucleic acid samples and multiple PCR systems can be added for multiple PCR amplification, or the tube contains PCR freeze Dry system, according to the need to distribute a nucleic acid sample to different reaction lumens to achieve amplification, you can also amplify a variety of different nucleic acids at the same time, and stored in the same reaction environment or other reaction tests, greatly The efficiency of nucleic acid amplification is improved, and the uniformity of the conditions for nucleic acid amplification is ensured.
  • FIG. 1 is a perspective structural view of a first reaction tube capable of performing multiple nucleic acid amplification according to an embodiment of the present disclosure
  • Figure 2 is the front view of Figure 1;
  • Figure 3 is a top view of Figure 2;
  • FIG. 4 is a perspective structural view of a second reaction tube capable of performing multiple nucleic acid amplification according to an embodiment of the present disclosure
  • Figure 5 is a front view of Figure 4.
  • FIG. 6 is a top view of FIG. 5;
  • FIG. 7 is a perspective structural view of a third reaction tube capable of performing multiple nucleic acid amplification according to an embodiment of the present disclosure
  • Figure 8 is a top view of Figure 7;
  • FIG. 9 is a perspective structural view of a fourth reaction tube capable of performing multiple nucleic acid amplification according to an embodiment of the present disclosure.
  • FIG. 10 is a front view of FIG. 9;
  • FIG. 10 is a top view of FIG. 10;
  • Figure 13 is a graph of integrated PCR amplified agarose gel electrophoresis results.
  • Icons 1-base; 2-reference plane; 3-reaction lumen; 4-central lumen.
  • connection should be understood in a broad sense, for example, it can be a fixed connection or a removable Connected, or connected integrally; either mechanically or electrically; directly connected, or indirectly connected through an intermediary, or internally connected between two components.
  • connection should be understood in a broad sense, for example, it can be a fixed connection or a removable Connected, or connected integrally; either mechanically or electrically; directly connected, or indirectly connected through an intermediary, or internally connected between two components.
  • the reaction tube capable of performing multiple nucleic acid amplification includes a base 1 and a plurality of reaction lumens 3; the base 1 is provided with a reference plane 2, and the openings of the plurality of reaction lumens 3 are all set on the reference plane 2, The inner cavities extend perpendicular to the reference plane 2 toward the inside of the base 1.
  • the above technical solution of the reaction tube capable of performing multiple nucleic acid amplification can better solve the problems that the existing nucleic acid amplification reaction tube in the prior art does not focus and unify the operation of the multiple nucleic acid amplification, the reaction conditions are inconsistent, cross interference and expansion.
  • multiple reaction lumens 3 are set on the reference plane 2 of the base 1, nucleic acid samples and multiple PCR systems can be added for multiple PCR amplification, or the lumina contains a PCR freeze-drying system, as needed Distribute a nucleic acid sample to different reaction lumens 3 to achieve amplification, or amplify multiple different nucleic acids at the same time, and store in the same reaction environment or perform other reaction tests, greatly improving nucleic acid expansion Increase efficiency and ensure the uniformity of the conditions for nucleic acid amplification.
  • FIG. 1 is a perspective structural view of the first type of reaction tube capable of performing multiple nucleic acid amplification provided by an embodiment of the present disclosure
  • FIG. 2 is a diagram 1 is a front view
  • FIG. 3 is a top view in FIG. 2
  • FIG. 4 is a perspective structural view of a second reaction tube capable of performing multiple nucleic acid amplification provided by an embodiment of the present disclosure
  • FIG. 5 is a main view in FIG. 4 View
  • Figure 6 is a top view in Figure 5.
  • FIGS. 1 is a perspective structural view of the first type of reaction tube capable of performing multiple nucleic acid amplification provided by an embodiment of the present disclosure
  • FIG. 2 is a diagram 1 is a front view
  • FIG. 3 is a top view in FIG. 2
  • FIG. 4 is a perspective structural view of a second reaction tube capable of performing multiple nucleic acid amplification provided by an embodiment of the present disclosure
  • FIG. 5 is a main view in FIG. 4 View
  • Figure 6 is a top view
  • a reaction tube capable of performing multiple nucleic acid amplification includes a base 1 and a plurality of reaction lumens 3 provided on the base 1; the base 1 is provided with a reference plane 2 and a plurality of reaction lumens The openings of 3 are all set on the reference plane 2, and the inner cavities thereof extend perpendicular to the reference plane 2 into the base 1.
  • a plurality of reaction lumens 3 are distributed in an annular shape around the same axis along the same radius.
  • the plurality of annularly distributed reaction lumens 3 has a compact structure on the one hand, and a more regular layout on the other hand, which facilitates the operations of injecting and extracting the test sample in the test.
  • eight, twelve, or sixteen reaction lumens 3 are distributed annularly around the same central axis along the same radius.
  • the number of optional reaction lumens 3 may be eight, ten, twelve, sixteen or twenty-four.
  • the material of the optional reaction lumen 3 may be polypropylene plastic.
  • FIG. 7 is a perspective structural view of a third reaction tube capable of performing multiple nucleic acid amplification according to an embodiment of the present disclosure
  • FIG. 8 is a top view of FIG. 7.
  • multiple reaction lumens 3 are distributed along a plurality of rings of different radii, wherein the number of optional different radii may be 2, 3 Or four.
  • the reaction tubes of this structure divide multiple reaction lumens 3 into a plurality of different radius but coaxial annular distribution, and set as many reaction lumens 3 as possible in a small space to enable simultaneous detection of more samples .
  • reaction lumens 3 are provided on the outer ring of the same radius R, and six reaction lumens 3 are provided on the inner ring of the same radius r, evenly arranged around the same central axis, R>r.
  • the material of the optional reaction lumen 3 may be polypropylene plastic.
  • reaction lumens 3 located in the same ring shape are individually fixed to the base 1.
  • the independently arranged reaction chambers 3 save the material of the entire reaction tube and reduce the weight of the reaction tube.
  • multiple reaction lumens 3 are independently set, it is easy to distinguish and arrange in appearance, which facilitates the operation of the test and improves the detection efficiency.
  • a plurality of reaction lumens 3 in the same ring shape are integrally formed and fixed to the base 1. At this time, all the reaction lumens 3 are integrally formed on the base 1, which is beneficial to uniformly heating all the reaction lumens 3 to achieve the effect of constant temperature, and the entire reaction tube has a more compact structure and a more complete appearance.
  • FIGS. 1, 3, 4, 6, 7, and 8 further includes a central lumen 4; the central lumen 4 is disposed on the reference plane 2 and is located in multiple reaction lumens The ring center of 3.
  • the central lumen 4 can not only reduce the weight of the reaction tube body, but also use the central lumen 4 as an operation space to perform an amplification reaction operation through an operation handle matched with the central lumen 4.
  • the shape of the central lumen 4 is not limited, and the optional central lumen 4 may be a cylinder, a rectangular parallelepiped, a polyhedron, or a vertebral body.
  • the central lumen 4 penetrates the base 1, further reducing the quality of the reaction tube, and providing sufficient space for the test operation.
  • the reaction tube provided by the present disclosure that can perform multiple nucleic acid amplification further includes a blocking cap provided on the base 1, and the blocking cap can achieve uniform blocking of all reaction lumens 3 .
  • the side wall of the base 1 is provided with a connector, which can be detachably connected to the blocking cover (not shown in the figure).
  • the base 1 is provided with elastic protrusions configured as connecting parts, and the blocking cover is provided with grooves, so as to realize the detachable connection of the connecting part and the blocking cover; or on the base 1
  • the threaded structure is configured as a connecting piece, and the blocking cover is provided with a structure matching with the above threading structure, so as to realize the detachable connection of the connecting piece and the blocking cover.
  • the base 1 is provided with a connecting member, which can be hinged with the blocking cover (not shown in the figure).
  • the base 1 is cylindrical, and its axis coincides with the axis of the central lumen 4.
  • the cylindrical base 1 is conveniently provided with a screw cap configured as a plugging cap, and a structure that cooperates with the screw thread of its side wall is used to realize a uniform plugging of all reaction tubes 3 by the screw cap.
  • the blocking member is provided at the opening of any reaction lumen 3, and the screw cap is spirally provided at one end of the base 1 to block the openings of the multiple reaction lumen 3.
  • the sealing member may use a heat-sealing film or a heat-sealing glue to seal any single reaction lumen 3, and the screw cap is used to seal all the reaction lumen 3 as a whole, and the nucleic acid sample solution in the reaction lumen 3 is temporarily sealed.
  • an injection hole can be provided on the screw cap.
  • FIG. 9 is a perspective structural view of a fourth reaction tube capable of performing multiple nucleic acid amplification according to an embodiment of the present disclosure
  • FIG. 10 is a front view in FIG. 9
  • FIG. 11 is a top view in FIG.
  • a plurality of reaction lumens 3 are distributed in a matrix on the reference plane 2.
  • the structure of the reaction lumens 3 distributed in rows and columns is more regular and the positioning is more accurate.
  • the plurality of reaction lumens 3 may be individually fixed to the base 1.
  • the independently set reaction chambers 3 save the material of the entire reaction tube and reduce the weight of the reaction tube. At the same time, since multiple reaction lumens 3 are independently set, it is easy to distinguish and arrange in appearance, which facilitates the operation of the test and improves the detection efficiency.
  • all the reaction lumens 3 may be integrally formed on the base 1.
  • the integrally formed reaction lumens 3 are beneficial for uniform heating of all reaction lumens 3 to achieve a constant temperature effect, and the entire reaction tube has a more compact structure and a more complete appearance.
  • a blocking cap provided on the base 1 is also included, and the blocking cap can achieve uniform blocking of all reaction lumens 3.
  • the side wall of the base 1 is provided with a connector, which can be detachably connected to the blocking cover (not shown in the figure).
  • the base 1 is provided with elastic protrusions configured as connecting parts, and the blocking cover is provided with grooves, so as to realize the detachable connection of the connecting part and the blocking cover; or on the base 1
  • a connecting piece is provided, and the connecting piece can be hinged with the blocking cover (not shown in the figure).
  • a blocking member (not marked) is also included.
  • the blocking member is disposed at the opening of any reaction cavity 3, and the blocking cover is hingedly disposed at one end of the base 1, to block the openings of the multiple reaction lumens 3.
  • the sealing member may use a heat sealing film or a heat sealing glue to seal any single reaction lumen 3, and the blocking cap may block all the reaction lumen 3 as a whole, and temporarily store the nucleic acid sample in the reaction lumen 3
  • the solution can be protected from external dust, light and other factors, and can also prevent the nucleic acid sample solution from pouring out.
  • an injection hole can be provided on the screw cap.
  • a reaction tube capable of performing multiple nucleic acid amplification includes a base 1 and a plurality of reaction lumens 3 provided on the base 1; the base 1 is provided with a reference plane 2, and a plurality of reaction tubes The openings of the cavity 3 are all set on the reference plane 2, and the inner cavities thereof extend perpendicular to the reference plane 2 toward the inside of the base 1.
  • a plurality of reaction lumens 3 are distributed in a ring shape along the same radius around the same axis, and are individually fixed to the base 1.
  • the reaction lumen 3 includes a lumen body and a conical bottom narrowing from top to bottom connected to the lumen body.
  • the reaction tube capable of performing multiple nucleic acid amplification further includes a central lumen 4 penetrating through the base 1; the central lumen 4 is disposed on the reference plane 2 and is located in the annular center of the multiple reaction lumens 3.
  • the reaction tube capable of performing multiple nucleic acid amplification further includes a blocking cap (not shown in the figure) provided on the base 1.
  • the base 1 is cylindrical, and its axis coincides with the axis of the central lumen 4.
  • the base 1 is provided with a screw structure configured as a connector, and the blocking cover is provided with a structure that cooperates with the above screw structure, so as to achieve all reactions Uniform plugging of the lumen 3.
  • Figure 12 is a graph of the results of integrated and multiplex tube PCR amplification agarose gel electrophoresis. As shown in Figure 12, where,
  • M 1 DNA2000, 50ng; 2 DNA1000, 50ng; 3 DNA750, 150ng; 4 DNA500, 50ng; 5 DNA250, 50ng; 6 DNA100, 50ng;
  • Figure 13 is a graph of integrated PCR amplified agarose gel electrophoresis results. As shown in Figure 13, where,
  • M 1 DNA2000, 50ng; 2 DNA1000, 50ng; 3 DNA750, 150ng; 4 DNA500, 50ng; 5 DNA250, 50ng; 6 DNA100, 50ng;
  • Example 1 Qualitative and semi-quantitative detection of integrated tube PCR amplification of four mosquito-mediated viruses
  • Select mosquito-borne viruses West Nile virus, Oriental equine encephalitis virus, Venezuelan equine encephalitis virus and forest encephalitis virus, use their gene coding regions as amplification target regions, and design specific primers. The sequences are shown in Table 1. Specific primer sequences for mosquito vector viruses.
  • PCR reaction total volume of 50 ⁇ L 5 ⁇ PCR buffer 10 ⁇ L, 25 ⁇ enzyme 2 ⁇ L, West Nile virus, Oriental equine encephalitis virus, Venezuelan equine encephalitis virus and forest brain Inflammation virus upstream and downstream primers 0.3 ⁇ mol/L, template 6 ⁇ L, water to a final volume of 50 ⁇ L;
  • PCR reaction total reaction volume is 25 ⁇ L, 5 ⁇ PCR buffer 5 ⁇ L, 25 ⁇ enzyme 1 ⁇ L, upstream and downstream primers each 0.3 ⁇ mol/L, template 6 ⁇ L, make up water to final volume 25 ⁇ L.
  • PCR reaction system a total of 4 groups are: 1 West Nile virus; 2 Oriental equine encephalitis virus; 3 Venezuelan equine encephalitis virus; 4 Forest encephalitis virus group; including: the total reaction volume of the PCR reaction is 15 ⁇ L, 5 ⁇ PCR buffer 3 ⁇ L, 25 ⁇ enzyme 0.6 ⁇ L, upstream and downstream primers 0.3 ⁇ mol/L each, template 6 ⁇ L, make up water to final volume 15 ⁇ L.
  • the total reaction volume of the PCR reaction in each well is 15 ⁇ L, 5 ⁇ PCR buffer 3 ⁇ L, 25 ⁇ enzyme 0.6 ⁇ L, upstream and downstream primers each 0.3 ⁇ mol/L, template 6 ⁇ L, and make up water to a final volume of 15 ⁇ L.
  • Reaction conditions 50 °C, 2 min; 94 °C, 2 min; 94 °C, 15 s, 58 °C, 45 s, 35 cycles in total.
  • the reaction tube capable of performing multiple nucleic acid amplification can add a nucleic acid sample and multiple PCR systems for multiple PCR amplification, or contain a PCR freeze-drying system in the lumen, and allocate a nucleic acid sample to different Amplification can be achieved in the reaction lumen, and multiple different nucleic acids can be amplified at the same time, and stored in the same reaction environment or other reaction tests, which greatly improves the efficiency of nucleic acid amplification and ensures the nucleic acid amplification Conditional unity.

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Abstract

一种可进行多重核酸扩增的反应管，涉及生物科学研究与医学检验等应用技术领域。该可进行多重核酸扩增的反应管包括基座（1）和多个反应管腔（3）；基座（1）设置一个基准平面（2），多个反应管腔（3）的开口均设置于基准平面（2），其内腔均垂直于基准平面（2）向基座（1）内部延伸。可进行多重核酸扩增的反应管，在基座（1）的基准平面（2）上设置多个反应管腔（3），可加入核酸样本及多种PCR体系进行多重PCR扩增，或者管腔（3）内含PCR冻干体系，根据需要将一种核酸样本分配到不同的反应管腔（3）中实现扩增，也可以同时对多种不同的核酸进行扩增，并于同样的反应环境中保存或者进行其他反应试验，大大提高了核酸扩增效率，并保证了核酸扩增的条件统一性。

Description

一种可进行多重核酸扩增的反应管
相关申请的交叉引用
本申请要求于2018年12月29日提交中国专利局的申请号为201811647409.7、名称为“一种可进行多重核酸扩增的反应管”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物科学研究与医学检验等应用技术领域，具体而言，涉及一种可进行多重核酸扩增的反应管。
背景技术
聚合酶链式反应(Polymerase Chain Reaction，PCR)技术，是一种体外快速扩增DNA的技术，每个循环包括变性、退火和延伸三个过程。首先，在大约95℃的高温下加热双链DNA样品，双链间的氢键会断裂，使得DNA热分解成两条互补的单链DNA分子，这一过程称为高温解链反应；然后，温度迅速降到大约50-65℃的范围内，在这个温度下单链DNA与引物按碱基互补配对原则结合，这一过程称为低温退火反应；退火反应结束后，温度要迅速升高到72℃左右进行延伸反应，在DNA聚合酶以及适当镁离子浓度的条件下，从引物的3’端开始结合单核苷酸，从而形成一条新的DNA。经过一个这样的过程，原来的一个DNA双链分子就形成了两个DNA分子，数量增加了一倍。每经过一个循环，目的核酸分子的数目扩增一倍，并且这些新形成的双链又可 以作为下次循环的模板，经过30-40个循环，目的核酸分子数目扩增到原来的近109倍。
所以，PCR又被称为无细胞分子克隆或特异性DNA序列体外引物定向酶促扩增技术，使目标DNA得以迅速扩增，具有特异性强、灵敏度高、操作简便、省时高效等特点，它不仅可用于基因分离、克隆和核酸序列分析等基础研究，还可用于疾病的诊断等任何含有DNA、RNA的地方。
另外，恒温扩增也属于核酸扩增的新方法，近年来越来越受到广泛关注。
然而不管是PCR扩增技术还是恒温扩增技术，在对多重核酸同时扩增反应时，仍存在很多缺陷。
公开内容
本公开的目的包括提供一种可进行多重核酸扩增的反应管，以解决现有技术中的核酸扩增反应管存在的技术问题之一。
为了实现上述目的中的至少一个目的，本公开采用以下技术方案：
本公开提供的一种可进行多重核酸扩增的反应管，包括基座和多个反应管腔；所述基座设置一个基准平面，多个所述反应管腔的开口均设置于所述基准平面，其内腔均垂直于所述基准平面向所述基座内部延伸。
在上述技术方案的基础上，进一步，多个所述反应管腔绕同一轴线呈环状分布。
——该技术方案的技术效果在于：环状分布的多个反应管腔一方面结构比较紧凑，另一方面布局更加规整，方便了检测试验中注入和提取检验样本的操作。例如，八个、十二个或者十六个反应管腔绕同一中心轴呈环状分布。
在上述任一技术方案的基础上，进一步，多个所述反应管腔沿多个不同半 径的环状分布。
——该技术方案的技术效果在于：该结构可将多个反应管腔分多个不同半径但同轴的环状分布，在较小的空间内设置尽可能多个反应管腔。例如，外圈设置十二个反应管腔，内圈设置六个反应管腔，绕相同的中心轴均匀布置。
在上述任一技术方案的基础上，进一步，位于同一环形的多个所述反应管腔各自单独固定于所述基座。
——该技术方案的技术效果在于：独立设置的各个反应管腔节省了整个反应管的材料，减轻了反应管的重量。同时，由于多个反应管腔独立设置，在外形上容易区分和编排，方便了试验的操作，提高了检测效率。
或者，位于同一环形的多个所述反应管腔一体成型固定于所述基座。
——该技术方案的技术效果在于：将全部反应管腔一体成型设置在基座上，利于对所有反应管腔统一加热实现恒温的效果，并且整个反应管结构更紧凑，外形更完整。
在上述任一技术方案的基础上，进一步，还包括中心管腔；所述中心管腔设置于所述基准平面，位于多个所述反应管腔的环状中心。
——该技术方案的技术效果在于：中心管腔不仅能够降低反应管体的重量，并能够利用中心管腔作为操作空间，通过与中心管腔配合的操作手柄，进行扩增反应操作。
在上述任一技术方案的基础上，进一步，所述中心管腔贯穿所述基座。
——该技术方案的技术效果在于：贯穿基座的中心管腔进一步降低了反应管的质量，也为试验操作提供了足够的空间。
在上述任一技术方案的基础上，进一步，所述基座呈圆柱状，其轴线与所述中心管腔的轴线重合。
——该技术方案的技术效果在于：圆柱状的基座方便设置旋盖，利用与其侧壁的螺纹配合的结构，实现旋盖对所有反应管腔的统一封堵。
在上述任一技术方案的基础上，进一步，还包括封堵件或者旋盖；所述封堵件设置在任一所述反应管腔的开口；所述旋盖螺旋设置在所述基座的一端，封堵多个所述反应管腔的开口。
——该技术方案的技术效果在于：封堵件设置在封堵反应管腔的开口，优选采用热封膜或者热封胶实现密封，旋盖对所有反应管腔实现整体封堵，暂时封存反应管腔内的核酸样本溶液，防止受到外界灰尘、光照等因素的影响，也可防止核酸样本溶液倾倒流出。另外，为了方便注入核酸样本溶液，可在旋盖上设置注入孔。
在上述任一技术方案的基础上，进一步，多个所述反应管腔在所述基准平面上呈行列式分布。
——该技术方案的技术效果在于：呈行列式分布的反应管腔结构更为规整，定位更加准确。此时，可采用热封膜或者热封胶密封反应管腔。
本公开具有的有益效果例如包括：
本公开提供的可进行多重核酸扩增的反应管，在基座的基准平面上设置多个反应管腔，可加入核酸样本及多种PCR体系进行多重PCR扩增，或者管腔内含PCR冻干体系，根据需要将一种核酸样本分配到不同的反应管腔中实现扩增，也可以同时对多种不同的核酸进行扩增，并于同样的反应环境中保存或者进行其他反应试验，大大提高了核酸扩增效率，并保证了核酸扩增的条件统一性。
本公开的附加技术特征及其优点将在下面的描述内容中阐述地更加明显，或通过本公开的具体实践可以了解到。
附图说明
为了更清楚地说明本公开具体实施方式的技术方案，下面将对具体实施方式描述中所需要使用的附图作简单地介绍。显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开实施例提供的第一种可进行多重核酸扩增的反应管的外形立体结构图；
图2为图1中的主视图；
图3为图2中的俯视图；
图4为本公开实施例提供的第二种可进行多重核酸扩增的反应管的外形立体结构图；
图5为图4中的主视图；
图6为图5中的俯视图；
图7为本公开实施例提供的第三种可进行多重核酸扩增的反应管的外形立体结构图；
图8为图7中的俯视图；
图9为本公开实施例提供的第四种可进行多重核酸扩增的反应管的外形立体结构图；
图10为图9中的主视图；
图11为图10中的俯视图；
图12为集成与多重管式PCR扩增琼脂糖凝胶电泳结果图；
图13为集成PCR扩增琼脂糖凝胶电泳结果图。
图标：1-基座；2-基准平面；3-反应管腔；4-中心管腔。
具体实施方式
下面将结合附图对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
在本公开的描述中，需要说明的是，术语“中心”、“上”、“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本公开和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本公开的限制。此外，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性。
在本公开的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本公开中的具体含义。
一、现有技术说明：
不管是PCR扩增技术还是恒温扩增技术，在对多重核酸同时扩增反应时，由于反应装置的设计缺陷，难免存在反应环境条件和操作时间上的不一致，出现先后反应的扩增效果差别较大的情况。而传统PCR管进行多重PCR时扩增体系易出现交叉干扰，影响扩增效果。
二、本公开技术方案概述：
本公开提供的可进行多重核酸扩增的反应管，包括基座1和多个反应管腔3；基座1设置一个基准平面2，多个反应管腔3的开口均设置于基准平面2，其内腔均垂直于基准平面2向基座1内部延伸。
上述可进行多重核酸扩增的反应管的技术方案，能够较好地解决现有技术中的核酸扩增反应管存在的对多重核酸扩增时操作不集中统一、反应条件不一致、交叉干扰且扩增效率低下等问题：在基座1的基准平面2上设置多个反应管腔3，可加入核酸样本及多种PCR体系进行多重PCR扩增，或者管腔内含PCR冻干体系，根据需要将一种核酸样本分配到不同的反应管腔3中实现扩增，也可以同时对多种不同的核酸进行扩增，并于同样的反应环境中保存或者进行其他反应试验，大大提高了核酸扩增效率，并保证了核酸扩增的条件统一性。
三、本公开技术方案具体实施方式：
针对上述现有技术方案存在的技术问题，下面结合具体的实施方式对本公开的技术方案做进一步的解释说明：
本实施例提供了一种可进行多重核酸扩增的反应管，其中：图1为本公开实施例提供的第一种可进行多重核酸扩增的反应管的外形立体结构图；图2为图1中的主视图；图3为图2中的俯视图；图4为本公开实施例提供的第二种可进行多重核酸扩增的反应管的外形立体结构图；图5为图4中的主视图；图6为图5中的俯视图。如图1～6所示，可进行多重核酸扩增的反应管包括基座1和设置于基座1上的多个反应管腔3；基座1设置一个基准平面2，多个反应管腔3的开口均设置于基准平面2，其内腔均垂直于基准平面2向基座1内部延伸。
在上述实施例的基础上，进一步地，如图1、3、4、6所示，多个反应管 腔3沿同一半径绕同一轴线呈环状分布。此时，环状分布的多个反应管腔3一方面结构比较紧凑，另一方面布局更加规整，方便了检测试验中注入和提取检验样本的操作。例如，八个、十二个或者十六个反应管腔3沿同一半径绕同一中心轴呈环状分布。可选的反应管腔3的个数可以为八个、十个、十二个、十六个或二十四个。可选的反应管腔3的材料可以为聚丙烯塑料。
图7为本公开实施例提供的第三种可进行多重核酸扩增的反应管的外形立体结构图；图8为图7中的俯视图。在上述实施例的基础上，如图7、8所示，进一步地，多个反应管腔3沿多个不同半径的环状分布，其中，可选的不同半径的数量可以为2个、3个或4个。该结构的反应管，将多个反应管腔3分多个不同半径但同轴的环状分布，在较小的空间内设置尽可能多个反应管腔3，能够实现更多样本的同时检测。例如，位于同一半径R的外圈设置十二个反应管腔3，位于同一半径r的内圈设置六个反应管腔3，绕相同的中心轴均匀布置，R＞r。可选的反应管腔3的材料可以为聚丙烯塑料。
在上述实施例的基础上，如图1、2所示，进一步地，位于同一环形的多个反应管腔3各自单独固定于基座1。在该结构中，独立设置的各个反应管腔3节省了整个反应管的材料，减轻了反应管的重量。同时，由于多个反应管腔3独立设置，在外形上容易区分和编排，方便了试验的操作，提高了检测效率。
或者，如图4、5、7所示，位于同一环形的多个反应管腔3一体成型固定于基座1。此时，将全部反应管腔3一体成型设置在基座1上，利于对所有反应管腔3统一加热实现恒温的效果，并且整个反应管结构更紧凑，外形更完整。
在上述实施例的基础上，如图1、3、4、6、7、8所示，进一步地，还包括中心管腔4；中心管腔4设置于基准平面2，位于多个反应管腔3的环状中心。在该结构中，中心管腔4不仅能够降低反应管体的重量，并能够利用中心 管腔4作为操作空间，通过与中心管腔4配合的操作手柄，进行扩增反应操作。对中心管腔4的形状不做限定，可选的中心管腔4可以为圆柱体、长方体、多面体或椎体。
在上述实施例的基础上，如图1、3、7、8所示，进一步地，中心管腔4贯穿基座1，进一步降低了反应管的质量，也为试验操作提供了足够的空间。
在上述实施例的基础上，本公开提供的可进行多重核酸扩增的反应管还包括设置于基座1上的封堵盖，该封堵盖可实现对所有反应管腔3的统一封堵。如图1～8所示，在一个实施方式中，基座1侧壁设置有连接件，该连接件可与封堵盖(图中未显示)可拆卸连接。连接件的形式有很多，例如基座1上设置具有弹性的凸起配置成连接件，封堵盖内设置有凹槽，从而实现连接件与封堵盖的可拆卸连接；或者基座1上设置有螺纹结构配置成连接件，封堵盖内设置有与上述螺纹结构配合的结构，从而实现连接件与封堵盖的可拆卸连接。在另一个实施方式中，基座1上设置有连接件，该连接件可与封堵盖(图中未显示)铰接。
进一步地，基座1呈圆柱状，其轴线与中心管腔4的轴线重合。在该结构中，圆柱状的基座1方便设置旋盖配置成封堵盖，利用与其侧壁的螺纹配合的结构，实现旋盖对所有反应管腔3的统一封堵。
在上述实施例的基础上，进一步地，还包括封堵件(未标注)或者旋盖(未标注)。其中，封堵件设置在任一反应管腔3的开口，旋盖螺旋设置在基座1的一端，封堵多个反应管腔3的开口。此时，封堵件可采用热封膜或者热封胶实现任一单个反应管腔3的密封，旋盖对所有反应管腔3实现整体封堵，暂时封存反应管腔3内的核酸样本溶液，防止受到外界灰尘、光照等因素的影响，也可防止核酸样本溶液倾倒流出。另外，为了方便注入核酸样本溶液，可在旋 盖上设置注入孔。
图9为本公开实施例提供的第四种可进行多重核酸扩增的反应管的外形立体结构图；图10为图9中的主视图；图11为图10中的俯视图。在上述实施例的基础上，如图9～11所示，进一步地，多个反应管腔3在基准平面2上呈行列式分布。呈行列式分布的反应管腔3结构更为规整，定位更加准确。
可选地，多个反应管腔3可各自单独固定于基座1。独立设置的各个反应管腔3节省了整个反应管的材料，减轻了反应管的重量。同时，由于多个反应管腔3独立设置，在外形上容易区分和编排，方便了试验的操作，提高了检测效率。
可选地，全部反应管腔3可一体成型设置在基座1上。一体成型的各个反应管腔3利于对所有反应管腔3统一加热实现恒温的效果，并且整个反应管结构更紧凑，外形更完整。
可选地，还包括设置于基座1上的封堵盖，该封堵盖可实现对所有反应管腔3的统一封堵。在一个实施方式中，基座1侧壁设置有连接件，该连接件可与封堵盖(图中未显示)可拆卸连接。连接件的形式有很多，例如基座1上设置具有弹性的凸起配置成连接件，封堵盖内设置有凹槽，从而实现连接件与封堵盖的可拆卸连接；或者基座1上设置有连接件，该连接件可与封堵盖(图中未显示)铰接。
可选地，还包括封堵件(未标注)。其中，封堵件设置在任一反应管腔3的开口，封堵盖铰接设置在基座1的一端，封堵多个反应管腔3的开口。此时，封堵件可采用热封膜或者热封胶实现任一单个反应管腔3的密封，封堵盖对所有反应管腔3实现整体封堵，暂时封存反应管腔3内的核酸样本溶液，防止受到外界灰尘、光照等因素的影响，也可防止核酸样本溶液倾倒流出。另外，为 了方便注入核酸样本溶液，可在旋盖上设置注入孔。
如图1和图2所示，可进行多重核酸扩增的反应管包括基座1和设置于基座1上的多个反应管腔3；基座1设置一个基准平面2，多个反应管腔3的开口均设置于基准平面2，其内腔均垂直于基准平面2向基座1内部延伸。多个反应管腔3沿同一半径绕同一轴线呈环状分布，且各自单独固定于基座1。其中，反应管腔3包括管腔主体和与管腔主体连接的由上至下收窄的锥形底。该可进行多重核酸扩增的反应管还包括贯穿基座1的中心管腔4；中心管腔4设置于基准平面2，位于多个反应管腔3的环状中心。该可进行多重核酸扩增的反应管还包括设置于基座1上的封堵盖(图中未标注)。基座1呈圆柱状，其轴线与中心管腔4的轴线重合，基座1上设置有螺纹结构配置成连接件，封堵盖内设置有与上述螺纹结构配合的结构，从而实现对所有反应管腔3的统一封堵。
下面，通过多重管式PCR扩增试验进行说明：
图12为集成与多重管式PCR扩增琼脂糖凝胶电泳结果图。如图12所示，其中，
M：①DNA2000，50ng；②DNA1000，50ng；③DNA750，150ng；④DNA500，50ng；⑤DNA250，50ng；⑥DNA100，50ng；
1：四个孔的阴性对照；
2：集成PCR管式扩增西尼罗河病毒电泳结果图；
3：集成PCR管式扩增东方马脑炎病毒电泳结果图；
4：集成PCR管式扩增委内瑞拉马脑炎病毒电泳结果图；
5：集成PCR管式扩增森林脑炎病毒电泳结果图；
6：8联排管式双重扩增西尼罗河病毒、东马脑炎病毒电泳结果图(条带从上至下)；
7：8联排管式双重扩增西尼罗河病毒、委内瑞拉马脑炎病毒电泳结果图(条带从上至下)；
8：8联排管式双重扩增西尼罗河病毒、森林脑炎病毒电泳结果图(条带从上至下)；
9：8联排管式双重扩增东方马脑炎病毒、委内瑞拉马脑炎病毒电泳结果图(条带从上至下)；
10：8联排管式双重扩增东方马脑炎病毒、森林脑炎病毒电泳结果图(条带从上至下)；
11：8联排管式双重扩增委内瑞拉马脑炎病毒、森林脑炎病毒电泳结果图(条带从上至下)；
12：8联排管式3重扩增西尼罗河病毒、东方马脑炎病毒、委内瑞拉马脑炎病毒电泳结果图(条带从上至下)；
13：8联排管式3重扩增西尼罗河病毒、东方马脑炎病毒、森林脑炎病毒电泳结果图(条带从上至下)；
14：8联排管式3重扩增西尼罗河病毒、委内瑞拉马脑炎病毒、森林脑炎病毒电泳结果图(条带从上至下)；
15：8联排管式3重扩增东方马脑炎病毒、委内瑞拉马脑炎病毒、森林脑炎病毒电泳结果图(条带从上至下)；
16：8联排管式4重扩增西尼罗河病毒、东方马脑炎病毒、委内瑞拉马脑炎病毒及森林脑炎病毒电泳结果图(条带从上至下)。
图13为集成PCR扩增琼脂糖凝胶电泳结果图。如图13所示，其中，
M：①DNA2000，50ng；②DNA1000，50ng；③DNA750，150ng；④DNA500，50ng；⑤DNA250，50ng；⑥DNA100，50ng；
2-8：集成管式自动加样PCR扩增森林脑炎病毒电泳结果图；
9-16：集成管式手动加样PCR扩增森林脑炎病毒电泳结果图；
1、9：阴性对照
实施例1、四种蚊媒类病毒集成管式PCR扩增定性、半定量检测
1、四种蚊媒类病毒特异性引物设计
选取蚊媒类病毒：西尼罗河病毒、东方马脑炎病毒、委内瑞拉马脑炎病毒及森林脑炎病毒，以它们的基因编码区为扩增靶区域，设计特异性引物，序列见表1.四种蚊媒类病毒特异性引物序列。
表1.四种蚊媒类病毒特异性引物序列
Figure PCTCN2019129968-appb-000001
2、PCR体系配制
(1)配制4重PCR反应体系，包括：PCR反应总反应体积为50μL，5×PCR缓冲液10μL，25×酶2μL，西尼罗河病毒、东方马脑炎病毒、委内瑞拉马脑炎病毒及森林脑炎病毒上、下游引物各0.3μmol/L，模板6μL，补水至终体积50μL；
(2)配制3重PCR反应体系，共4组分别为：①西尼罗河病毒、东方马脑炎病毒、委内瑞拉马脑炎病毒组；②西尼罗河病毒、东方马脑炎病毒、森林脑炎病毒组；③西尼罗河病毒、委内瑞拉马脑炎病毒、森林脑炎病毒组；④东方马脑炎病毒、委内瑞拉马脑炎病毒及森林脑炎病毒组。包括：PCR反应总反应体积为25μL，5×PCR缓冲液5μL，25×酶1μL，上、下游引物各0.3μmol/L，模板6μL，补水至终体积25μL。
(3)配制2重PCR反应体系，共6组分别为：①西尼罗河病毒、东方马脑炎病毒组；②西尼罗河病毒、委内瑞拉马脑炎病毒组；③西尼罗河病毒、森林脑炎病毒组；④东方马脑炎病毒、委内瑞拉马脑炎病毒组；⑤东方马脑炎病毒、森林脑炎病毒组；⑥委内瑞拉马脑炎病毒、森林脑炎病毒组；包括：PCR反应总反应体积为25μL，5×PCR缓冲液5μL，25×酶1μL，上、下游引物各0.3μmol/L，模板6μL，补水至终体积25μL。
(4)配制单重PCR反应体系，共4组分别为：①西尼罗河病毒；②东方马脑炎病毒；③委内瑞拉马脑炎病毒；④森林脑炎病毒组；包括：PCR反应总反应体积为15μL，5×PCR缓冲液3μL，25×酶0.6μL，上、下游引物各0.3μmol/L，模板6μL，补水至终体积15μL。
3、PCR扩增
(1)
Figure PCTCN2019129968-appb-000002
96-Well Thermal Cycler PCR仪扩增
将以上2、3、4重体系分别加入Axgen 8联排PCR管中，反应条件：50℃， 2min；94℃，2min；94℃，15s，58℃，45s，共35个循环；
(2)集成管式PCR仪扩增
将以上单重体系由管顶部分别加入8孔集成管中，1、3、5、7号孔中分别加入①西尼罗河病毒；②东方马脑炎病毒；③委内瑞拉马脑炎病毒；④森林脑炎病毒单重反应体系，2、4、6、8号小孔中加入阴性对照体系，反应条件：50℃，2min；94℃，2min；94℃，15s，58℃45s，共35个循环。
4、定性半定量检测结果
参考康为DM2000 DNA Marker用于琼脂糖凝胶电泳实验检测说明，使用
Figure PCTCN2019129968-appb-000003
Gel Image System ID分析软件，4种病毒集成管式扩增效果优于3、4重Axgen 8联排PCR管式反应。半定量结果如表2.不同扩增体系PCR扩增产物总量表所示，定性结果图12所示。
表2.不同扩增体系PCR扩增产物总量表
Figure PCTCN2019129968-appb-000004
a:未检测到目的产物条带。
实施例2、集成管式PCR扩增稳定性定性、半定量检测
1、PCR体系配制
配制PCR反应体系，每孔PCR反应总反应体积为15μL，5×PCR缓冲液3μL，25×酶0.6μL，上、下游引物各0.3μmol/L，模板6μL，补水至终体积15μL。
2、PCR扩增
将以上配制体系由管顶部分别加入8孔集成管中，1号小孔中加入阴性对照体系，2-8号孔中加入森林脑炎病毒反应体系。
反应条件：50℃，2min；94℃，2min；94℃，15s，58℃45s，共35个循环。
3、定性半定量检测结果
参考康为DM2000 DNA Marker用于琼脂糖凝胶电泳实验检测说明，使用
Figure PCTCN2019129968-appb-000005
Gel Image System ID分析软件，结果表明：自动与手动加样，PCR扩增效果较稳定均一，自动加样与手动加样效果相当，且较均一稳定。半定量结果如表3.集成PCR扩增琼脂糖凝胶电泳结果图所示，定性结果图13所示。
表3.集成PCR扩增琼脂糖凝胶电泳结果图
Figure PCTCN2019129968-appb-000006
a:未检测到目的产物条带。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
此外，本领域的技术人员能够理解，尽管上述的一些实施例包括其它实施例中所包括的某些特征而不是其它特征，但是不同实施例的特征的组合意味着处于本公开的范围之内并且形成不同的实施例。例如，在上面的权利要求书中，所要求保护的实施例的任意之一都可以以任意的组合方式来使用。另外，公开于该背景技术部分的信息仅仅旨在加深对本公开的总体背景技术的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域技术人员所公知的现有技术。
工业实用性
本公开提供的可进行多重核酸扩增的反应管，可加入核酸样本及多种PCR体系进行多重PCR扩增，或者管腔内含PCR冻干体系，根据需要将一种核酸样本分配到不同的反应管腔中实现扩增，也可以同时对多种不同的核酸进行扩增，并于同样的反应环境中保存或者进行其他反应试验，大大提高了核酸扩增效率，并保证了核酸扩增的条件统一性。

Claims (19)

  1. 一种可进行多重核酸扩增的反应管，其特征在于，包括基座和多个反应管腔；所述基座设置一个基准平面，多个所述反应管腔的开口均设置于所述基准平面，其内腔均垂直于所述基准平面向所述基座内部延伸。
  2. 根据权利要求1所述的可进行多重核酸扩增的反应管，其特征在于，多个所述反应管腔绕同一轴线呈环状分布。
  3. 根据权利要求2所述的可进行多重核酸扩增的反应管，其特征在于，多个所述反应管腔沿同一半径绕同一轴线呈环状分布。
  4. 根据权利要求2所述的可进行多重核酸扩增的反应管，其特征在于，多个所述反应管腔沿多个不同半径绕同一轴线呈环状分布。
  5. 根据权利要求3或4所述的可进行多重核酸扩增的反应管，其特征在于，位于同一环形的多个所述反应管腔各自单独固定于所述基座。
  6. 根据权利要求3或4所述的可进行多重核酸扩增的反应管，其特征在于，位于同一环形的多个所述反应管腔一体成型固定于所述基座。
  7. 根据权利要求1-6任一项所述的可进行多重核酸扩增的反应管，其特征在于，所述反应管腔包括管腔主体和与所述管腔主体连接的由上至下收窄的锥形底。
  8. 根据权利要求1-7任一项所述的可进行多重核酸扩增的反应管，其特征在于，所述反应管腔的材料包括聚丙烯塑料。
  9. 根据权利要求1-8任一项所述的可进行多重核酸扩增的反应管，其特征在于，还包括中心管腔；所述中心管腔设置于所述基准平面，位于多个所述反应管腔的环状中心。
  10. 根据权利要求9所述的可进行多重核酸扩增的反应管，其特征在 于，所述中心管腔贯穿所述基座。
  11. 根据权利要求10所述的可进行多重核酸扩增的反应管，其特征在于，所述基座呈圆柱状，其轴线与所述中心管腔的轴线重合。
  12. 根据权利要求1-10任一项所述的可进行多重核酸扩增的反应管，其特征在于，还包括设置于基座上的封堵盖，所述封堵盖用于对反应管腔的开口进行封堵。
  13. 根据权利要求12所述的可进行多重核酸扩增的反应管，其特征在于，所述封堵盖上还包括注入孔，用于向所述反应管腔内注入反应液。
  14. 根据权利要求12或13所述的可进行多重核酸扩增的反应管，其特征在于，所述封堵盖与基座可拆卸连接。
  15. 根据权利要求12或13所述的可进行多重核酸扩增的反应管，其特征在于，所述封堵盖与基座铰接。
  16. 根据权利要求1-15任一项所述的可进行多重核酸扩增的反应管，其特征在于，还包括设置于所述反应管腔的开口处的封堵件。
  17. 根据权利要求16所述的的可进行多重核酸扩增的反应管，其特征在于，所述封堵件为热封膜或者热封胶。
  18. 根据权利要求12-17任一项所述的可进行多重核酸扩增的反应管，其特征在于，所述封堵件设置在任一所述反应管腔的开口；
    所述旋盖螺旋设置在所述基座的一端，封堵多个所述反应管腔的开口。
  19. 根据权利要求1所述的可进行多重核酸扩增的反应管，其特征在于，多个所述反应管腔在所述基准平面上呈行列式分布。
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