WO2016180333A1 - 一种可控制液体环流路径的核酸扩增反应管 - Google Patents

一种可控制液体环流路径的核酸扩增反应管 Download PDF

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WO2016180333A1
WO2016180333A1 PCT/CN2016/081649 CN2016081649W WO2016180333A1 WO 2016180333 A1 WO2016180333 A1 WO 2016180333A1 CN 2016081649 W CN2016081649 W CN 2016081649W WO 2016180333 A1 WO2016180333 A1 WO 2016180333A1
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Prior art keywords
nucleic acid
reaction tube
acid amplification
reaction
region
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PCT/CN2016/081649
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English (en)
French (fr)
Inventor
葛胜祥
张师音
徐飞海
王进
李金洁
张军
夏宁邵
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厦门大学
厦门万泰凯瑞生物技术有限公司
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Application filed by 厦门大学, 厦门万泰凯瑞生物技术有限公司 filed Critical 厦门大学
Priority to JP2018511317A priority Critical patent/JP6718956B2/ja
Priority to US15/573,381 priority patent/US10487301B2/en
Priority to ES16792183T priority patent/ES2798287T3/es
Priority to KR1020177035750A priority patent/KR102389800B1/ko
Priority to EP16792183.2A priority patent/EP3296386B1/en
Publication of WO2016180333A1 publication Critical patent/WO2016180333A1/zh

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    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
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    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/24—Apparatus for enzymology or microbiology tube or bottle type
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0663—Stretching or orienting elongated molecules or particles
    • 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
    • B01L2300/042—Caps; Plugs
    • 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
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • 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
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0442—Moving fluids with specific forces or mechanical means specific forces thermal energy, e.g. vaporisation, bubble jet
    • B01L2400/0445—Natural or forced convection

Definitions

  • the invention belongs to the field of molecular biology, in particular to the field of nucleic acid amplification.
  • the present invention relates to a nucleic acid amplification reaction tube, and more particularly to a nucleic acid amplification reaction tube that can control a liquid circulation path.
  • the present invention relates to a method of amplifying a nucleic acid comprising using a nucleic acid amplification reaction tube of the present invention which can control a liquid circulation path.
  • the present invention relates to a nucleic acid amplification reaction apparatus comprising the reaction tube.
  • the present invention relates to a kit comprising the reaction tube, and the use of the reaction tube for preparing a kit.
  • PCR Polymerase Chain Reaction
  • Each cycle includes three processes of denaturation, annealing and extension.
  • This process is called a low-temperature annealing reaction; after the annealing reaction, the temperature is rapidly increased to The extension reaction is carried out at about 72 ° C, and a single nucleotide is bound from the 3' end of the primer under the conditions of DNA polymerase and appropriate magnesium ion concentration to form a new DNA.
  • the original DNA double-stranded molecule formed two DNA molecules, which doubled in number.
  • the number of nucleic acid molecules of interest is doubled, and these newly formed double strands can be used as a template for the next cycle.
  • the number of nucleic acid molecules of interest is amplified to nearly 10 9 . Times. PCR is a method for obtaining a large amount of DNA fragments of interest in vitro, which facilitates further analysis and testing of nucleic acid molecules.
  • the reaction device of the mainstream PCR amplification technology generally uses a temperature-controlled metal block to heat a PCR reaction tube made of plastic, and after heating and cooling the metal block, the equilibrium temperature is reached, and the heat is transferred to the PCR reaction solution through the reaction tube.
  • the drawback of this device is that the reaction volume is large, that is, the system usually has a large volume and heat capacity, and conventional PCR completes 30 cycles. It takes 2-3 hours, most of which is consumed in the heating and cooling process, that is, the metal block reaches an equilibrium temperature and heat is transferred to the PCR reaction solution through the reaction tube, so that it is difficult to achieve rapid and efficient PCR.
  • RB-PCR Rayleigh-Benard PCR
  • Madhavi Krishnan et al. based on the principle of heat conduction and thermal convection, using two upper and lower constant temperature heat sources to establish a closed reaction with a bottom-up temperature gradient.
  • the lumen, and thus the spontaneous convection motion of the PCR reagent within the chamber repeatedly flows through different temperature regions to complete the amplification.
  • the method has the advantages of rapid amplification and simpler instrument than the conventional PCR instrument, but the amplification reagent must be filled with the entire closed cavity, which causes problems such as difficulty in loading, leakage, and pollution.
  • Taiwan University Chou et al. improved on the basis of RB-PCR technology, turning the closed reaction chamber into an open reaction tube with specific specifications, and using a single constant temperature heat source to heat the spontaneous recovery of the reagent in the bottom of the tube. Amplification.
  • the method solves the problem that RB-PCR is easy to leak and pollute.
  • an effective annealing reaction may occur from a single-stranded template and a primer passing through the A1 region which is not higher than the annealing temperature required for the template or the amplicon; Below the single-chain template and primers passing through the A2 region of the A1 region, an effective annealing reaction cannot occur, resulting in a low overall annealing efficiency.
  • the temperature of the upper end of the reaction tube is lower than the annealing temperature of the primer by the control of the temperature field and the flow field, so as to ensure that the primer can be annealed as much as possible. .
  • the specificity of annealing is lowered, and it is easy to form a non-specific pairing between the primers or between the primers or between the primers and the template (/amplicon), and then an extension reaction occurs. A non-specific amplification product is formed.
  • the earlier, higher-proportion reaction tubes in which non-specific amplification occurs are less efficient than the later, lower-producing reaction tubes in which non-specific amplification occurs, and are lower than those in which non-specific amplification occurs. tube.
  • the earlier, higher-proportionation reaction tubes in which non-specific amplification occurs have a higher proportion of non-specific products in the tube product composition, and the non-specific amplification occurs later and the proportion of the reaction tubes is lower. There is a lower proportion of non-specific products in the composition of the tube, and no non-specific amplification occurs, and the products in the tube are correct amplification products.
  • the object of the present invention is to provide a novel nucleic acid amplification reaction tube and a nucleic acid amplification method, which can solve the problem of low amplification efficiency and poor specificity in the existing convection PCR technology. Problems such as large differences between tubes and inaccurate quantitative methods.
  • a first aspect of the present invention provides a nucleic acid amplification reaction tube comprising a tube body closed at one end, the tube body comprising a liquid storage region and a nucleic acid amplification region located below the liquid storage region, wherein the nucleic acid is expanded Inserts are suspended in the extension zone.
  • the physical barrier of the insert causes the reagent in the reaction tube to form a circulation path around the upper and lower sides of the insert under the action of external force or internal force.
  • the tabs are disposed along a central axis of the tubular body, and both sides of the insert are coupled to the inner wall of the nucleic acid amplification region.
  • the insert divides the nucleic acid amplification region into a first region and a second region along a central axis direction of the tube body, and the first region and the second region are in communication at an upper portion and a lower portion of the nucleic acid amplification region.
  • the distance between the lower end of the insert and the bottom of the tubular body is greater than 0 mm (for example, 1 mm or more) and less than 1/2 of the height of the nucleic acid amplification region; more preferably, the lower end of the insert and the bottom of the tubular body The distance between them is greater than 0 mm (eg, greater than or equal to 1 mm) and less than 1/3 of the height of the nucleic acid amplification region; further preferably, the distance between the lower end of the tab and the bottom of the tubular body is greater than 0 mm (eg, greater than or equal to 1 mm) and less than Equal to 4mm.
  • the distance between the upper end of the insert and the upper end of the nucleic acid amplification region is greater than 0 mm (for example, 1 mm or more) and less than 1/2 of the height of the nucleic acid amplification region; more preferably, the upper end of the insert and the nucleic acid are expanded.
  • the distance between the upper ends of the increasing regions is greater than 0 mm (eg, greater than or equal to 1 mm) and less than 1/3 of the height of the nucleic acid amplification region; further preferably, the distance between the upper end of the insert and the upper end of the nucleic acid amplification region is greater than 0 mm (eg, Greater than or equal to 1 mm) and less than or equal to 3 mm.
  • the bottom of the tubular body is closed at one end by a bottom plug that cooperates with the tubular body.
  • the tubular body and the bottom plug are tightly connected to each other by a rotating thread structure, or an annular bayonet structure, or a bump locking structure, and other sealing connections in the prior art may also be adopted.
  • the tubular body further includes a tube cover that cooperates therewith.
  • the tube body and the tube cover are tightly connected to each other by a rotating thread structure, or an annular bayonet structure, or a bump locking structure.
  • a rotating thread structure or an annular bayonet structure, or a bump locking structure.
  • other sealing methods in the prior art may also be adopted.
  • the ratio of the height/inner diameter of the nucleic acid amplification region is from 3 to 12, more preferably, The height/inner diameter ratio of the nucleic acid amplification region is 6-9.
  • the nucleic acid amplification region has a volume of 30 to 200 ⁇ l, and more preferably, the nucleic acid amplification region has a volume of 40 to 150 ⁇ l.
  • the tube body and the insert are made of a heat resistant material selected from, for example, glass, polycarbonate, polypropylene, polyether sulfone, and polysulfone.
  • the inner wall of the tube body may be passivated by bovine serum albumin (BSA) or a silylating agent or the like to reduce adsorption of certain components in the nucleic acid and the reagent.
  • BSA bovine serum albumin
  • the lumen of the nucleic acid amplification region is a columnar hollow structure having an equal inner and lower inner diameter, and may also be a narrow and narrow tapered hollow structure or a multilayer trapezoidal hollow structure in cross section, and the nucleic acid is expanded.
  • the transcription, RNA transcription, and acquisition of real-time detection signals are located in this region.
  • the nucleic acid amplification region can be provided with a visible volumetric scale.
  • Another aspect of the present invention provides a nucleic acid amplification reaction apparatus comprising the reaction tube according to any one of the first aspects of the present invention and one or more temperature control devices capable of providing or removing heat, the temperature control device setting Outside or inside the reaction tube.
  • a still further aspect of the invention provides a kit comprising the reaction tube of any of the first aspects of the invention.
  • a still further aspect of the present invention provides a method of amplifying a nucleic acid of interest in a sample, comprising using the reaction tube according to any one of the first aspects of the present invention or the nucleic acid amplification reaction device according to any one of the present invention,
  • the nucleic acid is DNA or RNA
  • the amplification is a PCR reaction or a reverse transcription reaction.
  • the method comprises the steps of:
  • RNA reverse transcription and/or DNA amplification reaction using a temperature control device to supply or remove heat to a specific portion of the reaction tube, Complete RNA reverse transcription and/or DNA amplification reaction;
  • the step of detecting the amplification product is also included while the nucleic acid amplification is being performed or after the amplification is completed.
  • the present invention also provides the use of the reaction tube according to any one of the first aspects of the present invention or the nucleic acid amplification reaction device of any of the present invention for nucleic acid amplification.
  • the invention also provides the use of a reaction tube according to any of the first aspects of the invention for the preparation of a kit for nucleic acid amplification.
  • the reagent in the tube can only pass from below and above the insert in the movement driven by the external force or the internal force, and passes through one or more temperatures disposed outside or inside the reaction tube.
  • the control device heats a specific region of the reaction tube so that a denaturing reaction occurs when the circulation passes through a certain region below the insert, and an annealing reaction may occur when the circulation passes through the region above the insert.
  • Both the chain template and the primer can undergo an annealing reaction through the effectively annealed region, and no ineffective loop is caused by the random circulation path (ie, when the circulation moves to the lower side, it does not pass through the temperature region satisfying the denaturing condition, and/or) When the circulation moves to the top, it does not pass through the temperature region that satisfies the annealing condition, thereby ensuring the uniformity of amplification between the tubes per unit time.
  • Figure 1a illustrates the circulation trajectory in the natural convection state
  • Figure 1b illustrates the liquid circulation trajectory controlled by the reaction tube of the present invention
  • Figure 2a is a front elevational view of a reaction tube that controls the spontaneous circulation path of the liquid
  • Figure 2b is a side view of a reaction tube that controls the spontaneous circulation path of the liquid
  • Figure 2c is a front exploded view of a reaction tube that controls the spontaneous circulation path of the liquid
  • Figure 2d is a top plan view of a reaction tube that controls the spontaneous circulation path of the liquid
  • Figure 3 is a view showing a device for heating and detecting fluorescence of a reaction tube capable of controlling a spontaneous circulation path of a liquid;
  • Figure 4 is a graph showing the results of agarose gel electrophoresis detection of a product obtained by amplifying a DNA template using the reaction tube of the present invention
  • Figure 5 shows the results of agarose gel electrophoresis detection of the product after amplification of the RNA template by using the reaction tube of the present invention
  • Figure 6a shows the results of agarose gel electrophoresis after nucleic acid amplification using a reaction tube that controls the spontaneous circulation path of the liquid;
  • Figure 6b shows the results of agarose gel electrophoresis after nucleic acid amplification using a loop-free control function reaction tube
  • Figure 7a shows the results of agarose gel electrophoresis after nucleic acid amplification using a reaction tube that controls the spontaneous circulation path of the liquid at different amplification times
  • Figure 7b shows the results of agarose gel electrophoresis after nucleic acid amplification using a loop-free control function reaction tube at different amplification times
  • Figure 8a shows the results of nucleic acid amplification and simultaneous real-time fluorescence detection using a reaction tube that controls the spontaneous circulation path of the liquid;
  • Figure 8b shows the results of nucleic acid amplification using a loop-free control function reaction tube and simultaneous real-time fluorescence detection.
  • amplification is to be understood broadly and includes the process of obtaining DNA from RNA or DNA, and includes, but is not limited to, PCR reactions, reverse transcription reactions, and various modifications thereof (eg, real-time PCR reactions). ).
  • nucleic acid as used herein includes ribonucleic acid (RNA) and deoxyribonucleic acid (RNA).
  • Figure 1a shows the trajectory of the circulation inside the reaction tube in the natural convection state. Convection can be formed by forming a certain temperature difference in a specific section in the reaction tube. Therefore, in the space of the reaction tube, the circulation trajectory is not single, but is characterized by multiple levels and multiple directions.
  • a template or an amplicon passing through the D1 region which is not lower than the denaturation temperature required for the template or the amplicon can be effectively denatured; and the position is higher than D1
  • the template or amplicon passing through the D2 region of the region cannot be subjected to an effective denaturation reaction, resulting in a low overall denaturation efficiency; (2) a single passage through the A1 region which is not higher than the annealing temperature required for the template or the amplicon.
  • Chain template and primers are likely to have an effective annealing reaction; however, from the single-chain template and primers passing through the A2 region of the A1 region, an effective annealing reaction cannot occur, resulting in low overall annealing efficiency; (3) When the single-stranded template and/or primer pass through a low temperature region in the circulation, the specificity of annealing is reduced, and it is easy to form non-specificity inside the primer or between the primers or between the primer and the template (/amplicon).
  • the circulation trajectory shown in Fig. 1b is a unidirectional, relatively concentrated, regular circulation trajectory formed by controlling the circulation path through physical obstruction on the basis of natural convection.
  • the reaction tube capable of controlling the liquid circulation path due to the physical barrier of the insert 2 in the reaction tube, when the circulation moves to the lower end of the reaction tube, it can only pass under the insert 2, and the area is in the temperature control device.
  • the present invention first provides a preferred embodiment of a nucleic acid amplification reaction tube with a controllable liquid circulation path, including a tube 1 closed at one end.
  • the tube body 1 includes a liquid storage area 4 and a nucleic acid amplification area 3 located below the liquid storage area; the nucleic acid amplification area 3 is provided with inserts 2 suspended vertically, and when the reagent is injected into the reaction tube, the insertion is performed.
  • the physical barrier of the sheet 2 causes the reagent in the reaction tube to form a circulation path around the upper and lower sides of the insert under the action of an external force or an internal force.
  • the tab 2 is disposed along the central axis of the tubular body 1, and both sides a and b of the insert are connected to the inner wall of the nucleic acid amplification region 3; further preferably, both sides a and b of the insert and the nucleic acid
  • the inner wall of the amplification zone 3 is hermetically connected.
  • the insert 2 divides the nucleic acid amplification region 3 into a first region 3-1 and a second region 3-2 along the central axis direction of the tube 1, and the first region 3-1 and the second region 3-2 are amplified in nucleic acid.
  • the upper portion 3-A and the lower portion 3-B of the zone 3 are in communication.
  • the distance between the lower end d of the tab 2 and the bottom of the tubular body 1 is greater than 0 mm (eg, 1 mm or more) and smaller than the nucleic acid amplification region 3 More preferably, the distance between the lower end d of the tab 2 and the bottom of the tubular body 1 is greater than 0 mm (for example, 1 mm or more) and less than 1/3 of the height of the nucleic acid amplification region 3; further preferably The distance between the lower end d of the tab 2 and the bottom of the tubular body 1 is greater than 0 mm (for example, greater than or equal to 1 mm) and less than or equal to 4 mm.
  • the distance between the upper end c of the insert 2 and the upper end of the nucleic acid amplification region 3 is greater than 0 mm (eg, 1 mm or more) and is less than nucleic acid amplification. 1/2 of the height of the region 3; more preferably, the distance between the upper end c of the insert 2 and the upper end of the nucleic acid amplification region 3 is greater than 0 mm (for example, 1 mm or more) and less than 1/3 of the height of the nucleic acid amplification region 3. Further preferably, the distance between the upper end c of the tab 2 and the upper end of the nucleic acid amplification region 3 is greater than 0 mm (for example, 1 mm or more) and less than or equal to 3 mm.
  • the bottom of the tubular body 1 is closed at one end by a bottom plug 1-1 that cooperates with the tubular body 1.
  • the tubular body and the bottom plug may be closely connected to each other by a rotating thread structure, or an annular bayonet structure, or a bump locking structure, and other sealing methods in the prior art may also be adopted.
  • the tubular body 1 further comprises a tube cover that cooperates therewith.
  • the tube body 1 and the tube cover are tightly connected to each other by a rotating thread structure, or an annular bayonet structure, or a bump locking structure.
  • a rotating thread structure or an annular bayonet structure, or a bump locking structure.
  • other sealing methods in the prior art may also be adopted.
  • the ratio of the height/inner diameter of the nucleic acid amplification region 3 is 3 to 12, and more preferably, the ratio of the height/inner diameter of the nucleic acid amplification region 3 is 6 to 9, for example, 7 to 8.
  • the inner diameter of the nucleic acid amplification region 3 is 10 mm or less, for example, 5 mm or less, and the inner diameter of the nucleic acid amplification region 3 is smaller than the inner diameter of the liquid storage region 4.
  • a region having a larger inner diameter above the tubular body 1 serves as the liquid storage region 4, because the inner diameter of the nucleic acid amplification region 3 is relatively small, it is inconvenient to insert the pipetting tip into the bottom, and the liquid cannot flow to the bottom, so that the liquid does not flow to the bottom.
  • the reaction reagent may be temporarily stored in the liquid storage zone 4, and then the reaction reagent of the upper liquid storage zone 4 is introduced into the nucleic acid amplification zone 3 below by centrifugation, shaking or other means, and the amplification reaction is completed in the nucleic acid amplification zone 3. Or fluorescent signal acquisition, etc.
  • the liquid storage zone 4 has a larger diameter with respect to the nucleic acid amplification zone 3, and is easier to grip and take, thereby providing the operator with great convenience in the dosing.
  • the volume of the nucleic acid amplification region 3 is 30 to 200 ⁇ l, and more preferably, the volume of the nucleic acid amplification region 3 is 40 to 150 ⁇ l.
  • the lumen of the nucleic acid amplification region 3 is a tapered hollow structure or a multilayer trapezoidal hollow structure having a narrow and narrow cross section, and nucleic acid amplification, RNA transcription, and real-time detection signal collection are located in this region. .
  • the inner and outer narrow inner diameter of the nucleic acid amplification region 3 of the present invention has the advantage that when the reagent is convected due to the established upper and lower temperature gradient, the wider inner diameter region above the reaction tube increases the path of the reagent in the temperature region. That is equivalent to increasing the time of the "extension" step in the PCR reaction, which is advantageous for the amplification of long fragments.
  • the inner cavity of the nucleic acid amplification region 3 may also be a columnar hollow structure having the same inner and lower inner diameters.
  • the tubular body 1 and the insert 2 are made of a heat resistant material, and the heat resistant material is exemplified.
  • a heat resistant material such as selected from the group consisting of glass, polycarbonate (PC), polypropylene (PE), polyethersulfone (PES), and polysulfone (PSF).
  • the inner wall of the tube body 1 may be passivated by bovine serum albumin (BSA) or a silylating agent or the like to reduce adsorption of certain components in the nucleic acid and the reagent.
  • BSA bovine serum albumin
  • the above reaction tube may contain: sample nucleic acid to be tested, DNA polymerase, adenine triphosphate deoxynucleotide, cytosine deoxynucleotide triphosphate, deoxynucleotide triphosphate deoxynucleotide, thymidine triphosphate deoxygenation Nucleotide, reaction buffer, divalent magnesium ion, non-primary component PCR additives (eg, betaine, bovine serum albumin, DMSO, etc.) and at least two oligonucleotide primers specifically complementary to the nucleic acid sequence to be detected And a fluorescent dye or a specific fluorescent probe or the like optionally bound to double-stranded DNA. Thereafter, the low-density nonvolatile matter (such as paraffin oil or various low-melting waxes) is applied to the surface of the reagent or the reaction tube is closed with a cap to prevent evaporation.
  • the low-density nonvolatile matter such as par
  • the present invention also provides a nucleic acid amplification reaction device comprising the reaction tube of any one of the present invention and one or more temperature control devices capable of providing or removing heat, wherein the temperature control device is disposed in the reaction tube External or internal.
  • the function of the temperature control device is to establish a temperature gradient and a density gradient of the reagent in the reaction tube based on the Rayleigh-Benard principle, thereby driving the spontaneous circulation of the reagent in the reaction tube; controlling the reaction tube and the reagent in the tube The temperature of the part; and further, through the spontaneous circulation of the reagent and temperature control, a nucleic acid amplification reaction such as a polymerase chain reaction is realized.
  • Temperature control devices capable of establishing temperature gradients and density gradients of reagents in the reaction tubes are well known in the art, for example, reference is made to the invention patents CN103173434A, CN1571849A, CN101983236A.
  • the device includes an upper heating module 4 and a lower heating module 5 for respectively supplying or removing heat to the bottom and upper portions of the reaction tube, and by such temperature control,
  • the above-mentioned denaturing temperature can be provided to the bottom of the reaction tube which can realize high-efficiency PCR amplification by controlling the liquid circulation path, so as to ensure that the reagent flowing through the insert can be efficiently degenerated; and the upper part of the reaction tube is provided.
  • Annealing temperature to ensure efficient annealing of the reagent flowing over the insert.
  • a module 6 with a low thermal conductivity is constructed for wrapping
  • the indirect heating zone of the reaction tube can avoid the interference of the region due to exposure to the outside air, and can avoid the difference of the temperature field distribution in the reaction tube in different pores due to the large difference in the heat dissipation capacity between the middle and the outer portion of the multi-flux module. .
  • the device further comprises a real-time fluorescent signal detection module.
  • the module is an organic whole composed of the excitation light source 7, the filter 8 and the sensitometric detecting device 9, and can complete the rapid equalization scanning detection of multiple specimens in a millisecond time.
  • the present invention is not limited to the reaction tube and the detecting device described in Figs. 2 and 3, and the change in the heating mode and the change in the shape of the container are within the scope of the present invention.
  • Fig. 4 is a graph showing the results of agarose gel electrophoresis detection of a DNA template after amplification of a nucleic acid amplification reaction tube which can control a spontaneous circulation path of a liquid of the present invention.
  • the reaction tube contains: DNA template to be detected, DNA polymerase, adenine triphosphate deoxynucleotide, cytosine deoxynucleotide triphosphate, adenosine triphosphate deoxynucleotide, thymidine triphosphate deoxygenation Nucleotide, reaction buffer, divalent magnesium ion, non-primary component PCR additives (eg, betaine, bovine serum albumin, DMSO, etc.) and at least two oligonucleotide primers specifically complementary to the nucleic acid sequence to be detected .
  • non-primary component PCR additives eg, betaine, bovine serum albumin, DMSO, etc.
  • the low-density non-volatile substance (such as paraffin oil or various low-melting waxes) is used to cover the surface of the reagent or the tube is closed with a cap to prevent evaporation.
  • the reaction tube was placed in a heating device, the heating module at the bottom of the reaction tube was set to 95 ° C, the heating module at the upper portion of the reaction tube was set to 60 ° C, and the reaction time was set to 30 minutes.
  • the reagent in the reaction tube will continuously flow under the driving of the temperature difference, and under the physical barrier of the insert in the reaction tube, only passes from the bottom and the top of the insert, and a denaturation reaction occurs when flowing through the bottom of the insert, flowing through At the top of the insert, an annealing reaction occurs and the extension reaction is carried out within the polymerase activity temperature range.
  • 5 ⁇ l of the product was taken from the tube for analysis by agarose gel electrophoresis. Lanes 1 and 2 are positive samples, and lanes 3 and 4 are negative controls (DEPC water). As can be seen from the results, the present invention can achieve amplification of DNA templates.
  • Fig. 5 shows the results of agarose gel electrophoresis detection of an RNA template after amplification of a nucleic acid amplification reaction tube which can control the spontaneous circulation path of the liquid of the present invention.
  • the reaction tube also contains reverse transcriptase for cDNA synthesis using RNA as a template.
  • the temperature of the reaction module The setting is also different: the heating module at the bottom of the reaction tube is first set to 60 ° C, after 20 minutes, and then heated to 95 ° C for 30 minutes; the heating module located in the upper part of the reaction tube is still set to a constant temperature of 60 ° C, 50 minutes .
  • the present invention also enables amplification of the RNA template.
  • FIG. 6 illustrates that a nucleic acid amplification reaction tube that can control a spontaneous circulation path of a liquid according to the present invention can improve the consistency and specificity of amplification between different tubes compared to previous convection PCR methods.
  • an extraction template for a 4-tube repeated cytomegalovirus (CMV) positive sample (CMV DNA concentration is 10) 3 copies/tube) and 4 tubes of CMV-negative HBV-positive samples were extracted (HBV DNA concentration was 10 6 copies/tube) for amplification.
  • CMV cytomegalovirus
  • Fig. 6a The results of a reaction tube amplification using a spontaneous circulation path for controlling a liquid of the present invention are shown in Fig. 6a, and the results of the amplification of the reaction tube using the loopless control function are shown in Fig. 6b.
  • Lanes 1-4 are repeated amplification results for 4 tubes of the same CMV nucleic acid positive sample, and lanes 5-8 are controls for CMV nucleic acid negative HBV nucleic acid positive samples.
  • the results showed that the parallel detection of the four groups of positive samples using the present invention, the consistency of the end point detection results (Fig. 6a, lanes 1-4) was significantly better than the results of the amplification using the loopless control function tube (Fig. 6b, Lanes 1-4).
  • the reaction tube of the present invention for controlling the spontaneous circulation path of a liquid can improve the consistency of amplification between different tubes.
  • the present invention was used to perform parallel amplification of four groups of negative samples, and in the endpoint detection results (Fig. 6a, lanes 5-8), non-specific amplification (such as primer dimer) was less than that of the loop-free control function tube ( Figure 6b, lanes 5-8) have been greatly improved, indicating that a reaction tube of the present invention that controls the spontaneous circulation path of the liquid enhances the specificity of amplification.
  • Figure 7 illustrates that a nucleic acid amplification reaction tube that can control a spontaneous spontaneous circulation path of the present invention can increase the rate of amplification relative to previous convection PCR methods.
  • three sets of CMV DNA with a concentration of 10 3 copies/ml were amplified in a reaction tube for controlling the spontaneous circulation path of the liquid and the reaction tube for the non-circulation control function of the present invention (four replicates per group).
  • the sample and one template were negative controls for DEPC water), and the amplification time of the three groups was set to 15 minutes, 20 minutes, and 25 minutes, respectively.
  • the results of a reaction tube amplification using a novel control of the spontaneous circulation path of the present invention are shown in Fig.
  • Fig. 7a and the results of the amplification of the reaction tube using the loopless control function are shown in Fig. 7b.
  • the results showed that with the amplification of the present invention, a weak band can be observed in the positive tube after 20 minutes of amplification, and a strong band can be observed in the positive tube after 25 minutes of amplification; The reaction tube needs to be amplified for 25 minutes, and a weak band can be observed in the positive reaction tube.
  • This result demonstrates that a nucleic acid amplification reaction tube that can control a spontaneous spontaneous circulation path of the present invention can increase the efficiency of amplification relative to previous convection PCR methods.
  • Figure 8 illustrates that a nucleic acid amplification reaction tube that can control a spontaneous circulation path of a liquid to which the present invention is applied can improve the accuracy of quantitative detection relative to previous convection PCR methods.
  • the reaction tube and the reaction tube without loop control apparatus of the present invention respectively In one control liquid circulation path by the same spontaneous heating, to a concentration of 10 6 copies / tube, 10 5 copies / tube of the human cytomegalovirus (CMV) DNA and negative samples (DEPC water) were amplified and assayed for real-time detection using a taqman hydrolysis probe.
  • CMV human cytomegalovirus
  • DEPC water negative samples
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method described in FMAusubel et al., Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; enzymes are used according to the conditions recommended by the product manufacturer.
  • the invention is described by way of example, and is not intended to limit the scope of the invention.
  • Example 1 A reaction tube for controlling a spontaneous circulation path of a liquid
  • a nucleic acid amplification reaction tube of the present invention for controlling a liquid circulation path includes a tube body 1 closed at one end, the tube body 1 including a liquid storage region 4 And a nucleic acid amplification region 3 located below the liquid storage region, wherein the nucleic acid amplification region 3 is provided with a top and bottom suspended insert 2, and when the reagent is injected into the reaction tube, the physical barrier of the insert 2 is such that the reagent in the reaction tube is Under the action of external force or internal force, a circulation path around the top and bottom of the insert is formed.
  • the tab 2 is disposed along the central axis of the tubular body 1, and both sides a and b of the insert are connected to the inner wall of the nucleic acid amplification region 3.
  • the insert 2 divides the nucleic acid amplification region 3 into a first region 3-1 and a second region 3-2 along the central axis direction of the tube 1, and the first region 3-1 and the second region 3-2 are amplified in nucleic acid.
  • the upper portion 3-A and the lower portion 3-B of the zone 3 are in communication.
  • the distance between the lower end d of the tab 2 and the bottom of the tubular body 1 is greater than 0 mm (eg, 1 mm or more) and is less than the height of the nucleic acid amplification region. 1/2, for example less than 1/3, for example less than or equal to 4 mm.
  • the distance between the upper end c of the insert 2 and the upper end of the nucleic acid amplification region 3 is greater than 0 mm (eg, 1 mm or more) and is less than nucleic acid amplification. 1/2 of the height of the zone 3, for example less than 1/3, for example less than or equal to 3 mm.
  • the bottom of the tubular body 1 is closed at one end by a bottom plug 1-1 that cooperates with the tubular body 1.
  • the tube body and the bottom plug may be tightly connected to each other by a rotating thread structure, or an annular bayonet structure, or a bump lock structure, and other closed connection manners in the prior art may also be adopted.
  • the ratio of the height/inner diameter of the nucleic acid amplification region 3 is 3 to 12, and more preferably 6 to 9.
  • the volume of the nucleic acid amplification region 3 is 30 to 200 ⁇ l, and more preferably 40 to 150 ⁇ l.
  • the inner cavity of the nucleic acid amplification region 3 may be a tapered hollow structure or a multi-layered trapezoidal hollow structure having a narrow and narrow cross section, or may be a columnar hollow structure having substantially the same inner and lower inner diameters. Amplification of nucleic acids, transcription of RNA, and acquisition of real-time detection signals are located in this region.
  • the tube body 1 and the insert 2 may be made of a heat resistant material selected from, for example, glass, polycarbonate (PC), polypropylene (PE), polyethersulfone (PES), and Polysulfone (PSF).
  • PC polycarbonate
  • PE polypropylene
  • PES polyethersulfone
  • PSF Polysulfone
  • the inner wall of the tube body 1 may be passivated by bovine serum albumin (BSA) or a silylating agent or the like to reduce adsorption of certain components in the nucleic acid and the reagent.
  • BSA bovine serum albumin
  • the reaction tube contains: sample nucleic acid to be tested, DNA polymerase, adenine triphosphate deoxynucleotide, cytosine deoxynucleotide triphosphate, pyrithione triphosphate deoxynucleotide, thymidine triphosphate deoxynucleoside Acid, reaction buffer, divalent magnesium ion, non-primary component PCR additives (eg, betaine, bovine serum albumin, DMSO, etc.) and at least two oligonucleotide primers that are specifically complementary to the nucleic acid sequence to be detected, and A fluorescent dye or a specific fluorescent probe or the like which optionally binds to double-stranded DNA.
  • a low-density nonvolatile substance such as paraffin oil or various low-melting waxes or the tube is closed with a cap to prevent evaporation.
  • Example 2 DNA-template-based amplification and detection using a nucleic acid amplification reaction tube of Example 1 for controlling a liquid circulation path
  • Instrument consumables self-made nucleic acid amplification instrument (see application CN201110456811.9); a nucleic acid amplification reaction tube, gel electrophoresis apparatus, gel imager (Bio-Rad) for controlling the liquid circulation path of the first embodiment
  • JxbUL54F1 GTGCCTTGACACTGTAC (SEQ ID NO. 1)
  • JxbUL54R11 CGACAAGTACTTTGAGCAGG (SEQ ID NO. 2)
  • Detection template 1 CMV virus DNA extract at a concentration of 10 3 copies/mL
  • amplification reagent 3.2 mM dNTP, 4 ⁇ L 10 ⁇ Fast Buffer I (Mg 2+ plus), 1 U SpeedSTAR HS DNA polymerase, 0.4 ⁇ L 10 ⁇ M JxbUL54F1, 0.4 ⁇ L 10 ⁇ M JxbUL54R11, 5 ⁇ l detection template, total volume 40 ⁇ l, The remaining volume is made up with DEPC water.
  • nucleic acid amplification a. Injecting the prepared amplification reagent into a nucleic acid amplification reaction tube of a controllable liquid circulation path of the present invention, adding 10 ⁇ l of paraffin oil dropwise, and by centrifugation, vibration or other means, The amplification reagent is filled in the reaction vessel; b. The bottom temperature of the self-made nucleic acid amplification instrument is set to 95 ° C, the upper temperature is 60 ° C, and the amplification time is set to 30 minutes. The reaction tube containing the nucleic acid amplification reagent is inserted into the nucleic acid amplification instrument, the program is turned on, and the reaction tube is taken out after the end of the program operation.
  • Example 3 Using RNA as a template for amplification and detection by using a nucleic acid amplification reaction tube capable of controlling a liquid circulation path of Example 1.
  • Chemical reagents SpeedSTAR HS DNA polymerase (TaKaRa), reverse transcriptase MMLV (Transgen), 10 ⁇ Fast Buffer I (Mg 2+ plus) (TaKaRa), dNTP (TaKaRa), DEPC water, paraffin oil, 6 ⁇ DNA Sample buffer (including Sybr Green)
  • Instrument consumables self-made nucleic acid amplification instrument (see application CN201110456811.9); a nucleic acid amplification reaction tube, gel electrophoresis apparatus, gel imager (Bio-Rad) for controlling the liquid circulation path of the first embodiment
  • CA16-WJ-F6-1 CAAGTAYTACCYACRGCTGCCAA (SEQ ID NO. 3)
  • CA16-WJ-R6-1 CAACACACAYCTMGTCTCAATGAG (SEQ ID NO.4)
  • Detection template 1 Coxsackievirus A16 (CA16 virus) RNA extract at a concentration of 10 3 copies/mL
  • (2) Nucleic acid amplification a. Injecting the prepared amplification reagent into a nucleic acid amplification reaction tube of a controllable liquid circulation path of the present invention, adding 10 ⁇ l of paraffin oil dropwise, and by centrifugation, vibration or other means, The amplification reagent is filled in the reaction vessel; b. The heating module program at the bottom of the instrument is 60 ° C for 20 minutes, 95 ° C for 30 minutes; the upper heating module of the instrument is set to a constant temperature of 60 ° C for 50 minutes. Insert the reaction tube containing the nucleic acid amplification reagent into the instrument, open the program, and take out the reaction tube after the program is finished.
  • Example 4 Comparison of homogeneity and specificity of amplification of reaction tubes with and without circulation control function
  • Instrument consumables self-made nucleic acid amplification instrument; a nucleic acid amplification reaction tube for controlling the liquid circulation path of Embodiment 1, a reaction tube without circulation control function (see application 201110360350.5), a gel electrophoresis apparatus, a gel imager ( Bio-Rad)
  • JxbUL54F1 GTGCCTTGACACTGTAC (SEQ ID NO. 1)
  • JxbUL54R11 CGACAAGTACTTTGAGCAGG (SEQ ID NO. 2)
  • Detection template 1 CMV virus DNA extract at a concentration of 10 3 copies/mL
  • amplification reagent 3.2 mM dNTP, 4 ⁇ L 10 ⁇ Fast Buffer I (Mg2+plus), 1U SpeedSTAR HS DNA polymerase, 0.4 ⁇ L 10 ⁇ M JxbUL54F1, 0.4 ⁇ L 10 ⁇ M JxbUL54R11, 5 ⁇ l detection template, total volume 40 ⁇ l, remaining The volume is made up with DEPC water.
  • nucleic acid amplification a.
  • the prepared amplification reagent is separately injected into a nucleic acid amplification reaction tube of the controllable liquid circulation path of the present invention, and a reaction tube without a circulation control function, and 10 ⁇ l of paraffin oil is added dropwise.
  • the amplification reagent is filled into the reaction vessel by centrifugation, vibration or other means;
  • the bottom temperature of the self-made nucleic acid amplification instrument is set to 95 ° C, the upper temperature is 60 ° C, and the amplification time is set to 30 minutes. Insert the above two reaction tubes containing the nucleic acid amplification reagent into the instrument, open the program, and take out the reaction tube after the end of the program.
  • Example 5 Comparison of amplification efficiency of reaction tubes with and without circulation control function
  • Instrument consumables self-made nucleic acid amplification instrument; a nucleic acid amplification reaction tube for controlling the liquid circulation path of Embodiment 1, a reaction tube without circulation control function (see application 201110360350.5), a gel electrophoresis apparatus, a gel imager ( Bio-Rad)
  • JxbUL54F1 GTGCCTTGACACTGTAC (SEQ ID NO. 1)
  • JxbUL54R11 CGACAAGTACTTTGAGCAGG (SEQ ID NO. 2)
  • Detection template 1 CMV viral DNA extraction template at a concentration of 10 3 copies/ml
  • amplification reagent 3.2 mM dNTP, 4 ⁇ L 10 ⁇ Fast Buffer I (Mg 2+ plus), 1 U SpeedSTAR HS DNA polymerase, 0.4 ⁇ L 10 ⁇ M JxbUL54F1, 0.4 ⁇ L 10 ⁇ M JxbUL54R11, 5 ⁇ l detection template, total volume 40 ⁇ l, The remaining volume is made up with DEPC water.
  • nucleic acid amplification a.
  • the prepared amplification reagent is separately injected into a nucleic acid amplification reaction tube of the controllable liquid circulation path of the present invention, and a reaction tube without a circulation control function, and 10 ⁇ l of paraffin oil is added dropwise.
  • the amplification reagent is filled into the reaction tube by centrifugation, vibration or other means;
  • setting the bottom temperature of the self-made nucleic acid amplification instrument to 95 ° C, the upper temperature is 60 ° C, and the amplification time is set to 15 minutes, 20 minutes, respectively. 25 minutes. Insert the above two reaction tubes containing the nucleic acid amplification reagent into the instrument, open the program, and take out the reaction tube after the end of the program.
  • Instrument consumables self-made nucleic acid amplification and real-time fluorescence detector (see application CN201110456811.9); a nucleic acid amplification reaction tube for controlling the liquid circulation path of Embodiment 1, and a reaction tube without circulation control function (see application 201110360350.5) ,
  • JxbUL54F1 GTGCCTTGACACTGTAC (SEQ ID NO. 1)
  • JxbUL54R11 CGACAAGTACTTTGAGCAGG (SEQ ID NO. 2)
  • JxbUL54P1 FAM-AGCCGGCTCCAAGTGCAAG-BHQ-1 (SEQ ID NO. 5)
  • Detection template 1 CMV virus DNA extraction template at a concentration of 10 6 copies/ml
  • Detection template 2 CMV virus DNA extraction template at a concentration of 10 5 copies/ml
  • amplification reagent 3.2 mM dNTP, 4 ⁇ L 10 ⁇ Fast Buffer I (Mg 2+ plus), 1 U SpeedSTAR HS DNA polymerase, 0.4 ⁇ L 10 ⁇ M JxbUL54F1, 0.4 ⁇ L 10 ⁇ M JxbUL54R11, 0.2 ⁇ L 10 ⁇ M JxbUL54P1, 5 ⁇ l detection template
  • the total volume is 40 ⁇ l, and the remaining volume is made up with DEPC water.
  • nucleic acid amplification a. Injecting the prepared amplification reagent into a controllable manner of the present invention a nucleic acid amplification reaction tube for the liquid circulation path and a reaction tube without circulation control function, 10 ⁇ l of paraffin oil is added dropwise, and the amplification reagent is filled into the reaction container by centrifugation, vibration or other means; b. setting the self-made nucleic acid amplification The bottom temperature of the instrument was 95 ° C, the upper temperature was 60 ° C, and the amplification time was set to 30 minutes.
  • the above two reaction tubes containing the nucleic acid amplification reagent are inserted into the self-made nucleic acid amplification and real-time fluorescence detector, and the program is started. After the program is finished, the reaction tube is taken out and the data is analyzed.
  • Fig. 8a The results of a reaction tube amplification using a novel controllable circulation path of the present invention are shown in Fig. 8a, and the result of the amplification of the reaction tube using the loopless control function is shown in Fig. 8b.
  • the results show that with the amplification of the present invention, the amplification curve of the same concentration sample is significantly more reproducible than the reaction tube without the circulation control function, thereby suggesting that the nucleic acid amplification reaction tube of the present invention can control the liquid circulation path. Quantitative detection of nucleic acid samples is possible.

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Abstract

公开了一种可控制液体环流路径的核酸扩增反应管，包含该反应管的核酸扩增反应装置和使用该反应管扩增核酸的方法。还公开了包含该反应管的试剂盒以及该反应管用于制备试剂盒的用途。

Description

一种可控制液体环流路径的核酸扩增反应管 技术领域
本发明属于分子生物学领域，特别是核酸扩增领域。具体而言，本发明涉及一种核酸扩增反应管，特别是一种可控制液体环流路径的核酸扩增反应管。此外，本发明还涉及一种扩增核酸的方法，其包括使用本发明的可控制液体环流路径的核酸扩增反应管。此外，本发明还涉及一种包含所述反应管的核酸扩增反应装置。此外，本发明还涉及包含所述反应管的试剂盒，以及所述反应管用于制备试剂盒的用途。
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。PCR是体外大量获得目的DNA片段的方法，便于对核酸分子做进一步的分析和检验。
目前，主流的PCR扩增技术的反应装置一般以温控金属块加热塑料制成的PCR反应管，通过金属块的加热、冷却，达到平衡温度后将热量通过反应管传递至PCR反应液。这种装置的缺陷是：反应体积较大，即系统通常具有较大的体积和热容，常规PCR完成30个循环一般 需要2-3小时，其中大部分时间消耗于加热和冷却过程，即将金属块达到平衡温度并将热通过反应管传递至PCR反应液，因此，PCR难以实现快速高效。
2002年Madhavi Krishnan等人报导的雷诺本纳德(Rayleigh-Benard)PCR(简称RB-PCR)方法，基于热传导及热对流原理，利用上下两个恒温热源建立具自下而上温度梯度的封闭反应内腔，并因此使腔内的PCR试剂发生自发的对流运动反复流经不同温度区域从而完成扩增。该方法扩增速度快、仪器较传统PCR仪简单，但扩增试剂须充满整个封闭腔，造成上样难度大、易泄漏、易污染等问题。
台湾大学Chou等在RB-PCR技术的基础上做了改进，将封闭的反应内腔变成开放式的具特定规格的反应试管，并利用单一恒温热源加热试管底部驱动管内试剂自发循环，从而完成扩增。该方法解决了RB-PCR易泄漏、污染的问题。
但现有的对流PCR扩增方法均存在共同的缺陷，即管内液体流动路径复杂。管内流路为近同心椭圆型的多层流路(图1a)，这种多层次的复杂流路在扩增中存在以下问题：
1.扩增效率偏低：
(a)变性的效率：如图1a所示，从不低于模板或扩增子所需的变性温度的D1区经过的模板或扩增子，可发生有效的变性；而从位置高于D1区的D2区经过的模板或扩增子，则无法发生有效的变性反应，导致总体变性效率偏低；
(b)退火的效率：如图1a所示，从不高于模板或扩增子所需的退火温度的A1区经过的单链模板及引物，才有可能发生有效的退火反应；而从位置低于A1区的A2区经过的单链模板及引物，则无法发生有效的退火反应，导致总体退火效率偏低。
2.扩增的特异性不好：
在对流PCR中，因没有固定温度的退火区域及时段，因此通常会通过温度场及流场的控制进而使得反应管上端的温度低于引物的退火温度，用以保障引物能够尽可能充分的退火。但当单链模板和(/或)引 物在环流中经过过低的温度区域时，会导致退火的特异性降低，容易形成引物内部或引物间或引物与模板(/扩增子)之间的非特异性的配对，并进而发生延伸反应，形成非特异性扩增产物。
3.平行反应在扩增中可能存在管间差异：
(a)终点定性检测，在结果上主要表现为扩增效率及产物组成的差异：上述第2点中描述的非特异性扩增产物经变性后，又会变成下一轮非特异性扩增的模板，从而使得非特异性扩增不断放大，并与正确的扩增竞争引物、酶、dNTP等反应组分，使得正确扩增受到抑制，反应效率降低。而这种非特异性反应是否发生、发生的早晚、发生的比例都是不受控制的，即存在一定的随机性，这就会导致发生了此类非特异性扩增的反应管的扩增效率的不一致。非特异性扩增发生的较早、发生比例较高的反应管，其效率低于非特异性扩增发生的较晚、发生比例较低的反应管，并均低于未发生非特异性扩增的反应管。同样的，非特异性扩增发生的较早、发生比例较高的反应管，其管内产物组成中有较高比例的非特异性产物，非特异性扩增发生的较晚、发生比例较低的反应管，其管内产物组成中有较低比例的非特异性产物，未发生非特异性扩增的，其管内产物均为正确扩增产物。
(b)实时定量检测，在结果上主要表现为单位时间内有效扩增效率的差异：也就是说，无法进行实时定量检测。同上述1、2两点的描述，当对流PCR反应起始，双链模板能否经过有效地变性区域、单链模板和引物能否经过有效地退火区域、以及退火中是否发生了非特异性的反应，都是不受控制的，因此在反应起始就有可能造成不同管的产物组成存在差异，这些差异不仅会导致上述3(a)中描述的问题，还使得单位时间内，不同反应管的产物(模板)数量存在差异，进入指数扩增期的时间点就会不同，因此无法用传统的实时监测方法进行模板的定量。
发明内容
本发明的目的是提供一种新型的核酸扩增反应管以及核酸扩增方法，以解决现有的对流PCR技术中存在的扩增效率偏低、特异性不好、 管间差异大、定量不准确等问题。
本发明的第一方面提供一种核酸扩增反应管，包括一端封闭的管体，所述管体包括储液区以及位于储液区下方的核酸扩增区，其特征在于，所述核酸扩增区内设置有上下悬空的插片。当反应管内注入试剂时，通过插片的物理阻隔，使得反应管内试剂在外力或内力的作用下，形成围绕插片上方及下方的环流路径。
优选地，所述插片沿管体的中轴线设置，并且插片的两侧与核酸扩增区的内壁连接。插片沿管体的中轴线方向将核酸扩增区分隔成第一区域和第二区域，第一区域和第二区域在核酸扩增区的上部和下部连通。
优选地，所述插片下端与管体底部之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区高度的1/2；更优选地，所述插片下端与管体底部之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区高度的1/3；进一步优选地，所述插片下端与管体底部之间的距离大于0mm(例如大于等于1mm)并且小于等于4mm。
优选地，所述插片上端与核酸扩增区上端之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区高度的1/2；更优选地，所述插片上端与核酸扩增区上端之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区高度的1/3；进一步优选地，所述插片上端与核酸扩增区上端之间的距离大于0mm(例如大于等于1mm)并且小于等于3mm。
优选地，所述管体的底部通过与管体相互配合的底塞实现一端封闭。优选地，所述管体与底塞之间通过旋转螺纹结构，或环状卡口结构，或凸点锁扣结构相互密闭连接，也可以采用现有技术中其它的密闭连接方式。
优选地，所述管体还包括与其配合的管盖。优选地，所述管体与管盖之间通过旋转螺纹结构，或环状卡口结构，或凸点锁扣结构相互密闭连接，当然也可以采用现有技术中其它的密闭连接方式。
优选地，所述核酸扩增区的高度/内径的比值为3～12，更优选地， 所述核酸扩增区的高度/内径的比值为6～9。
优选地，所述核酸扩增区的容积为30～200μl，更优选地，所述核酸扩增区的容积为40～150μl。
优选地，所述管体和插片由耐热材料制成，所述耐热材料例如选自玻璃、聚碳酸酯、聚丙烯、聚醚砜和聚砜。
此外，优选地，所述管体的内壁可通过牛血清白蛋白(BSA)或硅烷化试剂等做钝化处理，从而降低对核酸及反应试剂中某些成分的吸附。
在一个优选的实施方案中，所述核酸扩增区的内腔为上下内径相等的柱状中空结构，也可以为横截面上宽下窄的锥状中空结构或多层梯形中空结构，核酸的扩增、RNA的转录、实时检测信号的采集均位于此区域。
在另一个优选的实施方案中，所述核酸扩增区上可设有可见的体积刻度标识。
本发明的另一方面提供一种核酸扩增反应装置，其包括本发明第一方面任一项的反应管和一个或多个可提供或移走热量的温度控制装置，所述温度控制装置设置于所述反应管的外部或内部。
本发明的还一方面提供一种试剂盒，其包含本发明第一方面任一项的反应管。
本发明的还一方面提供一种扩增样品中目的核酸的方法，其包括使用本发明第一方面任一项的反应管或本发明任一项的核酸扩增反应装置，
优选地，所述核酸是DNA或RNA；
优选地，所述扩增是PCR反应或逆转录反应。
优选地，所述方法包括以下步骤：
1)将核酸扩增反应试剂注入本发明第一方面任一项的反应管中；
2)通过震动、离心或其他方式，使反应试剂完全填充核酸扩增区；任选地，还包括用不易挥发的物质(例如石蜡油或低熔点的蜡)覆盖于试剂表面或用管盖封闭反应管的步骤；
3)用温度控制装置对所述反应管的特定部位提供或移走热量，以 完成RNA逆转录和/或DNA扩增反应；
4)任选地，在进行核酸扩增的同时或扩增结束后，还包括对扩增产物进行检测的步骤。
本发明还提供本发明第一方面任一项的反应管或本发明任一项的核酸扩增反应装置用于核酸扩增的用途。
本发明还提供本发明第一方面任一项的反应管用于制备试剂盒的用途，所述试剂盒用于进行核酸扩增。
发明的有益效果
本发明通过插片的物理阻隔，使得管内试剂在外力或内力驱动的运动中，仅能从插片的下方及上方经过，并通过一个或多个设于所述反应管的外部或内部的温度控制装置，对反应管的特定区域进行加热，使得环流从插片下方一定区域经过时，均可发生变性反应，环流从插片上方区域经过时，均可发生退火反应。该种环流运动路径及温度控制方式对PCR反应带来的好处是：
1.提高扩增效率：(a)提高变性的效率：如图1b所示，受到插片2的物理阻隔，环流运动到反应管下端时，仅能从插片2的下方的D1区经过，而该区域在所述温度控制装置的作用下，可维持高于变性所需的温度，因此，环流从反应管插片2下方的D1区经过时，均可发生有效的变性反应；(b)提高退火的效率：如图1b所示，受到插片2的物理阻隔，环流运动到反应管上端时，仅能从插片2上方的A1区经过，而该区域在所述温度控制装置的作用下，可维持满足特定引物退火所需的温度，因此，环流从反应管插片2上方经过时，均可发生有效的退火反应。
2.保障扩增的特异性：如图1b所示，受到插片2的物理阻隔，环流运动到反应管上端后，仅能从插片2上方的A1区经过，而插片2上方区域在所述温度控制装置的作用下，可维持正好满足特定引物退火所需的温度，而不会过低于特定引物的退火温度，因此，环流从该区域经过时，引物内部或引物间或引物与模板(/扩增子)之间不会形成非特异 性的配对，并造成非特异性扩增。
3.提高管间的一致性：
(a)通过提高特异性，保障扩增的一致性：如上段第2点所述，因不会再随机的形成非特异性产物，并与正确扩增竞争引物、酶、dNTP等反应组分，因此，正确扩增的效率不会受到非特异性扩增的影响，因而可提高管间在单位时间内扩增的一致性；(b)通过提高扩增效率，保障扩增的一致性：对流PCR反应起始后，受到反应管内插片2的物理阻隔作用及所述温度控制装置控制，管内的双链模板均能经过有效地变性区域发生解链反应，且环流运动到反应管上端时，单链模板和引物均能经过有效地退火区域发生退火反应，不会再因随机的环流路径而造成无效循环(即环流运动至下方时，未从满足变性条件的温度区域经过，和(/或)环流运动至上方时，未从满足退火条件的温度区域经过)，从而保障管间在单位时间内扩增的一致性。(c)扩增的一致性得到了保障，因而可提高平行反应中，终点检测时扩增产物的一致性；及平行反应中，单位时间内扩增的一致性，并进而使得对流PCR扩增也可通过实时荧光检测的方法实现对初始模板的定量。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1a示意了自然对流状态下的环流轨迹；
图1b示意了通过本发明的反应管控制后的液体环流轨迹；
图2a所示为一种可控制液体自发环流路径的反应管的正视图；
图2b所示为一种可控制液体自发环流路径的反应管的侧视图；
图2c所示为一种可控制液体自发环流路径的反应管的正视分解图；
图2d所示为一种可控制液体自发环流路径的反应管的俯视图；
图3示意了一种对可控制液体自发环流路径的反应管进行加热及荧光检测的装置图；
图4所示为应用本发明的反应管对DNA模板进行扩增后产物进行琼脂糖凝胶电泳检测的结果；
图5所示为应用本发明的反应管对RNA模板进行扩增后产物进行琼脂糖凝胶电泳检测的结果；
图6a所示为使用一种可控制液体自发环流路径的反应管进行核酸扩增后，产物进行琼脂糖凝胶电泳检测的结果；
图6b所示为使用无环流控制功能反应管进行核酸扩增后，产物进行琼脂糖凝胶电泳检测的结果；
图7a所示为不同扩增时间下，使用一种可控制液体自发环流路径的反应管进行核酸扩增后，产物进行琼脂糖凝胶电泳检测的结果；
图7b所示为不同扩增时间下，使用无环流控制功能反应管进行核酸扩增后，产物进行琼脂糖凝胶电泳检测的结果；
图8a所示为使用一种可控制液体自发环流路径的反应管进行核酸扩增并同时进行实时荧光检测的结果；
图8b所示为使用无环流控制功能反应管进行核酸扩增，并同时进行实时荧光检测的结果。
具体实施方式
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“扩增”应从广义上来理解，其包括从RNA或DNA获得DNA的过程，并且包括但不限于PCR反应，逆转录反应，以及其各种变型(例如实时PCR反应)。
如本文中所使用的，术语“核酸”包括核糖核酸(RNA)和脱氧核糖核酸(RNA)。
图1a所示意的即为在自然对流状态下，反应管内环流的运动轨迹。反应管内只需在特定区间内形成一定的温度差异，即可形成对流。因此，在反应管的空间中，环流轨迹并非是单一的，而是呈现多层次、多方向的特点。那么，在这种多层环流中，(1)从不低于模板或扩增子所需的变性温度的D1区经过的模板或扩增子，可发生有效的变性；而从位置高于D1区的D2区经过的模板或扩增子，则无法发生有效的变性反应，导致总体变性效率偏低；(2)从不高于模板或扩增子所需的退火温度的A1区经过的单链模板及引物，才有可能发生有效的退火反应；而从位置低于A1区的A2区经过的单链模板及引物，则无法发生有效的退火反应，导致总体退火效率偏低；(3)当单链模板和(/或)引物在环流中经过过低的温度区域时，会导致退火的特异性降低，容易形成引物内部或引物间或引物与模板(/扩增子)之间的非特异性的配对，并进而发生延伸反应，形成非特异性扩增产物；(4)上述非特异性扩增产物经变性后，又会变成下一轮非特异性扩增的模板，从而使得非特异性扩增不断放大，并与正确的扩增竞争引物、酶、dNTP等反应组分，使得正确扩增受到抑制，反应效率降低。而这种非特异性反应是否发生、发生的早晚、发生的比例都是不受控制的，即存在一定的随机性，这就会导致发生了此类非特异性扩增的反应管的扩增效率的不一致；(5)上述扩增的不一致，在实时定量检测中将表现为单位时间内有效扩增效率的差异，从而导致无法应用传统荧光定量PCR方法制作标准曲线对核酸模板进行相对定量。
图1b所示的环流轨迹，为在自然对流的基础之上，通过物理阻隔使环流路径得到控制，而形成的单方向、相对集中、规则的环流轨迹。在该种可控制液体环流路径的反应管中，因反应管内的插片2的物理性阻隔，环流运动到反应管下端时，仅能从插片2的下方经过，而该区域在温度控制装置的作用下，可维持高于变性所需的温度，因此，环流从 反应管插片2下方经过时，均可发生有效的变性反应；同样因反应管内的插片2的物理性阻隔，环流运动到反应管上端时，仅能从插片2的上方经过，而该区域在温度控制装置的作用下，可维持满足特定引物退火所需的温度，因此，环流从反应管内的插片2上方经过时，均可发生有效的退火反应。
参见图2a、图2b、图2c、图2d，为实践上述方法，本发明首先提供一种可实践的可控制液体环流路径的核酸扩增反应管的优选实施例，包括一端封闭的管体1，所述管体1包括储液区4以及位于储液区下方的核酸扩增区3；所述核酸扩增区3内设置有上下悬空的插片2，当反应管内注入试剂时，通过插片2的物理阻隔，使得反应管内试剂在外力或内力的作用下，形成围绕插片上方及下方的环流路径。
优选地，所述插片2沿管体1的中轴线设置，并且插片的两侧a和b与核酸扩增区3的内壁连接；进一步优选地，插片的两侧a和b与核酸扩增区3的内壁密封连接。插片2沿管体1的中轴线方向将核酸扩增区3分隔成第一区域3-1和第二区域3-2，第一区域3-1和第二区域3-2在核酸扩增区3的上部3-A和下部3-B连通。
优选地，所述插片2下端d与管体1底部之间的距离(即，核酸扩增区3的下部3-B的高度)大于0mm(例如大于等于1mm)并且小于核酸扩增区3高度的1/2；更优选地，所述插片2下端d与管体1底部之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区3高度的1/3；进一步优选地，所述插片2下端d与管体1底部之间的距离大于0mm(例如大于等于1mm)并且小于等于4mm。
优选地，所述插片2上端c与核酸扩增区3上端之间的距离(即，核酸扩增区3的上部3-A的高度)大于0mm(例如大于等于1mm)并且小于核酸扩增区3高度的1/2；更优选地，所述插片2上端c与核酸扩增区3上端之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区3高度的1/3；进一步优选地，所述插片2上端c与核酸扩增区3上端之间的距离大于0mm(例如大于等于1mm)并且小于等于3mm。
优选地，所述管体1的底部通过与管体1相互配合的底塞1-1实现一端封闭。例如，所述管体与底塞之间通过旋转螺纹结构，或环状卡口结构，或凸点锁扣结构相互密闭连接，也可以采用现有技术中其它的密闭连接方式。
优选地，所述管体1还包括与其配合的管盖。所述管体1与管盖之间通过旋转螺纹结构，或环状卡口结构，或凸点锁扣结构相互密闭连接，当然也可以采用现有技术中其它的密闭连接方式。
优选地，所述核酸扩增区3的高度/内径的比值为3～12，更优选地，所述核酸扩增区3的高度/内径的比值为6～9，例如为7～8。进一步优选地，所述核酸扩增区3的内径小于等于10mm，例如小于等于5mm，且所述核酸扩增区3的内径小于所述储液区4的内径。本发明的此种尺寸及比例结构，能够有效地保障和促进反应管的管内液体自发地形成连续而稳定的对流。在本发明中，管体1上方内径较大的区域作为储液区4，因为核酸扩增区3的内径相对较小，不方便将移液尖插入底部，也无法让液体自流至底部，所以反应试剂可先暂时贮存于储液区4中，然后再通过离心、震荡或其它方式使上方储液区4的反应试剂进入下方的核酸扩增区3，在核酸扩增区3完成扩增反应或荧光信号采集等。且储液区4相对于核酸扩增区3具有较大的直径，更容易握、拿，因此为操作者在配液中提供了极大的便利性。
优选地，所述核酸扩增区3的容积为30～200μl，更优选地，所述核酸扩增区3的容积为40～150μl。
进一步地，所述核酸扩增区3的内腔为横截面上宽下窄的锥状中空结构或多层梯形中空结构，核酸的扩增、RNA的转录、实时检测信号的采集均位于此区域。本发明核酸扩增区3内腔上宽下窄的内径设计优点为：当试剂因建立的上下温度梯度而发生对流时，反应管上方较宽内径的区域会增加试剂在该温度区域的路径，即相当于增加PCR反应中“延伸”步骤的时间，有利于长片段的扩增。当然，为了制造方便，所述核酸扩增区3的内腔也可以为上下内径相等的柱状中空结构。
优选地，所述管体1和插片2由耐热材料制成，所述耐热材料例 如选自玻璃(glass)、聚碳酸酯(PC)、聚丙烯(PE)、聚醚砜(PES)和聚砜(PSF)。
此外，优选地，所述管体1的内壁可通过牛血清白蛋白(BSA)或硅烷化试剂等做钝化处理，从而降低对核酸及反应试剂中某些成分的吸附。
在上述的反应管中可以含有：待检样本核酸、DNA聚合酶、三磷酸腺嘌呤脱氧核苷酸、三磷酸胞嘧啶脱氧核苷酸、三磷酸鸟嘧啶脱氧核苷酸、三磷酸胸腺嘧啶脱氧核苷酸、反应缓冲液、二价镁离子、非主要成分的PCR添加剂(如：甜菜碱、牛血清白蛋白、DMSO等)和至少两条与待检核酸序列特异互补的寡核苷酸引物，以及任选地与双链DNA结合的荧光染料或特异性荧光探针等。其后，用低密度的不易挥发物质(如石蜡油或者各种低熔点的蜡)覆盖于试剂表面或采用管盖封闭反应管以防止蒸发。
同时本发明还提供一种核酸扩增反应装置，其包括本发明任一项的反应管和一个或多个可提供或移走热量的温度控制装置，所述温度控制装置设置于所述反应管的外部或内部。该温度控制装置的作用是，基于雷诺本纳德(Rayleigh-Benard)原理，建立反应管内试剂的温度梯度及密度梯度，从而驱动反应试剂在反应管内的自发循环流动；控制反应管及管内试剂特定部位的温度；并进而通过试剂的自发循环及温度控制，实现聚合酶链式反应等核酸扩增反应。能够建立反应管内试剂的温度梯度及密度梯度的温度控制装置为本领域所公知，例如可参考发明专利CN103173434A，CN1571849A，CN101983236A。
本发明温度控制装置的优选实施例如图3所示，优选装置含有上加热模块4和下加热模块5，分别用于对反应管的底部与上部提供或移走热量，并通过这种温度控制，对上述可通过控制液体环流路径实现PCR高效扩增的反应管的底部提供合适的变性温度，以保障从插片下方的流经的试剂可高效的发生变性反应；对反应管的上部提供合适的退火温度，以保障从插片上方流经的试剂可高效的发生退火反应。此外，在上、下两个金属加热模块中间，构造一块热传导系数较低的模块6，用于包裹 反应管的非直接加热区域，可避免该区域因暴露在外界空气中而受到干扰，还可避免因多通量模块中部与外部散热能力差异较大而导致不同孔中反应管内温度场分布的差异。
优选地，本装置还含有实时荧光信号检测模组。该模组是由激发光源7、滤光片8及感光检测装置9组成的有机整体，能在毫秒级时间内完成多个标本快速均衡扫描检测。
本发明不局限于图2与图3所描述的反应管与检测装置，加热方式的改变与容器形状的改变皆属本发明的范畴。
图4出示了应用本发明的一种可控制液体自发环流路径的核酸扩增反应管对DNA模板进行扩增后的琼脂糖凝胶电泳检测结果。应用时，在反应管中含有：待检DNA模板、DNA聚合酶、三磷酸腺嘌呤脱氧核苷酸、三磷酸胞嘧啶脱氧核苷酸、三磷酸鸟嘧啶脱氧核苷酸、三磷酸胸腺嘧啶脱氧核苷酸、反应缓冲液、二价镁离子、非主要成分的PCR添加剂(如：甜菜碱、牛血清白蛋白、DMSO等)和至少两条与待检核酸序列特异互补的寡核苷酸引物。并用低密度的不易挥发物质(如石蜡油或者各种低熔点的蜡)覆盖于试剂表面或用管盖封闭反应管，以防止蒸发。扩增时，反应管置于加热装置中，位于反应管底部的加热模块设置为95℃，位于反应管上部的加热模块设置为60℃，反应时间设置为30分钟。反应管内试剂将在该温差的驱动下连续流动，并在反应管中插片的物理阻隔下，仅从插片的底部与上方经过，并在流经插片底部时发生变性反应，在流经插片上方时，发生退火反应，并在聚合酶活性温度区间内进行延伸反应。扩增结束后，从管内取5μl产物进行琼脂糖凝胶电泳分析。泳道1与泳道2为阳性样本扩增结果，泳道3与泳道4为阴性对照(DEPC水)扩增结果。从结果中可以看出，本发明可实现对DNA模板的扩增。
图5出示了应用本发明的一种可控制液体自发环流路径的核酸扩增反应管对RNA模板进行扩增后的琼脂糖凝胶电泳检测结果。与DNA扩增不同的是：反应管内除含有上述DNA扩增所需试剂外，还含有反转录酶，用于实现以RNA为模板的cDNA合成。此外，反应模块的温度 设置也有所不同：位于反应管底部的加热模块首先被设置为60℃，维持20分钟后，再升温至95℃，维持30分钟；位于反应管上部的加热模块仍设置为恒温60℃，50分钟。同样的，扩增结束后，从管内取5μl产物进行琼脂糖凝胶电泳分析。泳道1与泳道2为阳性样本扩增结果，泳道3与泳道4为阴性对照(DEPC水)扩增结果。从电泳图中可以看出，本发明还可实现对RNA模板的扩增。
图6说明应用本发明的一种可控制液体自发环流路径的核酸扩增反应管相对于先前对流PCR方法可提高不同管间扩增的一致性与特异性。用相同加热装置分别在本发明的一种控制液体自发环流路径的反应管与无环流控制功能反应管中，对4管重复的巨细胞病毒(CMV)阳性样本的提取模板(CMV DNA浓度为103copies/tube)及4管CMV阴性的HBV阳性样本的提取模板(HBV DNA浓度为106copies/tube)进行扩增。应用本发明的一种控制液体自发环流路径的反应管扩增的结果如图6a所示，应用无环流控制功能反应管扩增的结果如图6b所示。泳道1-4为4管相同CMV核酸阳性样本的重复扩增结果，泳道5-8为CMV核酸阴性HBV核酸阳性样本的对照。结果显示，应用本发明进行4组阳性样本的平行检测，其终点检测结果的一致性(图6a，泳道1-4)明显优于应用无环流控制功能反应管进行扩增的结果(图6b，泳道1-4)。说明本发明的一种控制液体自发环流路径的反应管可提高不同管间扩增的一致性。此外，应用本发明进行4组阴性样本的平行扩增，其终点检测结果(图6a，泳道5-8)中，非特异性扩增(如引物二聚体)较无环流控制功能反应管结果(图6b，泳道5-8)有极大的改善，说明本发明的一种控制液体自发环流路径的反应管可提高扩增的特异性。
图7说明应用本发明的一种可控制液体自发环流路径的核酸扩增反应管相对于先前对流PCR方法可提高扩增的速率。用相同加热装置分别在本发明的一种控制液体自发环流路径的反应管与无环流控制功能反应管中，对浓度为103copies/ml的CMV DNA进行3组扩增(每组4个重复样本及1个模板为DEPC水的阴性对照)，3组扩增时间分别设定为15分钟，20分钟，25分钟。应用本发明的一种控制液体自发环流 路径的反应管扩增的结果如图7a所示，应用无环流控制功能反应管扩增的结果如图7b所示。结果显示，应用本发明的扩增，在扩增20分钟后，阳性管即可观察到弱条带，在扩增25分钟后，阳性管即可观察到强条带；而应用无环流控制功能的反应管，则需扩增25分钟，阳性反应管才可观察到弱条带。该结果说明应用本发明的一种可控制液体自发环流路径的核酸扩增反应管相对于先前对流PCR方法可提高扩增的效率。
图8说明应用本发明的一种可控制液体自发环流路径的核酸扩增反应管相对于先前对流PCR方法可提高定量检测的准确性。用相同加热装置分别在本发明的一种控制液体自发环流路径的反应管与无环流控制功能反应管中，对浓度为106copies/tube、105copies/tube的人巨细胞病毒(CMV)DNA及阴性样本(DEPC水)进行扩增，并用taqman水解探针对扩增进行实时检测。结果显示，应用本发明的检测，其相同浓度样本的重复性明显高于用无环流控制功能的反应管。
实施例
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：一种控制液体自发环流路径的反应管
如图2a、2b、2c、2d所示，本发明的一种可控制液体环流路径的核酸扩增反应管包括一端封闭的管体1，所述管体1包括储液区4以 及位于储液区下方的核酸扩增区3，所述核酸扩增区3内设置有上下悬空的插片2，当反应管内注入试剂时，通过插片2的物理阻隔，使得反应管内试剂在外力或内力的作用下，形成围绕插片上方及下方的环流路径。
优选地，所述插片2沿管体1的中轴线设置，并且插片的两侧a和b与核酸扩增区3的内壁连接。插片2沿管体1的中轴线方向将核酸扩增区3分隔成第一区域3-1和第二区域3-2，第一区域3-1和第二区域3-2在核酸扩增区3的上部3-A和下部3-B连通。
优选地，所述插片2下端d与管体1底部之间的距离(即，核酸扩增区3的下部3-B的高度)大于0mm(例如大于等于1mm)并且小于核酸扩增区高度的1/2，例如小于1/3，例如小于等于4mm。
优选地，所述插片2上端c与核酸扩增区3上端之间的距离(即，核酸扩增区3的上部3-A的高度)大于0mm(例如大于等于1mm)并且小于核酸扩增区3高度的1/2，例如小于1/3，例如小于等于3mm。
优选地，所述管体1的底部通过与管体1相互配合的底塞1-1实现一端封闭。所述管体与底塞之间可通过旋转螺纹结构，或环状卡口结构，或凸点锁扣结构相互密闭连接，也可以采用现有技术中其它的密闭连接方式。
优选地，所述核酸扩增区3的高度/内径的比值为3～12，进一步优选为6～9。
优选地，所述核酸扩增区3的容积为30～200μl，进一步优选为40～150μl。
优选地，所述核酸扩增区3的内腔可以为横截面上宽下窄的锥状中空结构或多层梯形中空结构，也可以为上下内径基本相等的柱状中空结构。核酸的扩增、RNA的转录、实时检测信号的采集均位于此区域。
所述管体1和插片2可以由耐热材料制成，所述耐热材料例如选自玻璃(glass)、聚碳酸酯(PC)、聚丙烯(PE)、聚醚砜(PES)和聚砜(PSF)。
此外，优选地，所述管体1的内壁可通过牛血清白蛋白(BSA)或硅烷化试剂等做钝化处理，从而降低对核酸及反应试剂中某些成分的吸附。
在上述反应管中含有：待检样本核酸、DNA聚合酶、三磷酸腺嘌呤脱氧核苷酸、三磷酸胞嘧啶脱氧核苷酸、三磷酸鸟嘧啶脱氧核苷酸、三磷酸胸腺嘧啶脱氧核苷酸、反应缓冲液、二价镁离子、非主要成分的PCR添加剂(如：甜菜碱、牛血清白蛋白、DMSO等)和至少两条与待检核酸序列特异互补的寡核苷酸引物，以及任选地与双链DNA结合的荧光染料或特异性荧光探针等。其后，用低密度的不易挥发物质(如石蜡油或者各种低熔点的蜡)覆盖于试剂表面或用管盖封闭反应管，以防止蒸发。
实施例2：应用实施例1的一种可控制液体环流路径的核酸扩增反应管进行的、以DNA为模板的扩增与检测
1、实验材料
化学试剂：SpeedSTAR HS DNA polymerase(TaKaRa)，10×Fast Buffer I(Mg2+plus)(TaKaRa)，dNTP(TaKaRa)，DEPC水，石蜡油，6×DNA上样缓冲液(含Sybr Green)
仪器耗材：自制核酸扩增仪(参见申请CN201110456811.9)；实施例1的一种可控制液体环流路径的核酸扩增反应管、凝胶电泳仪、凝胶成像仪(Bio-Rad)
引物：JxbUL54F1：GTGCGCCTTGACACTGTAC(SEQ ID NO.1)
JxbUL54R11：CGACAAGTACTTTGAGCAGG(SEQ ID NO.2)
检测模板1：CMV病毒DNA提取物，浓度为103copies/mL
检测模板2：DEPC水
2、实验方法：
(1)扩增试剂的配制：3.2mM dNTP，4μL 10×Fast Buffer I(Mg2+plus)，1U SpeedSTAR HS DNA polymerase，0.4μL 10μM JxbUL54F1， 0.4μL 10μM JxbUL54R11，5μl检测模板，总体积40μl，剩余体积用DEPC水补足。
(2)核酸扩增：a.将配制好的扩增试剂注入本发明的一种可控制液体环流路径的核酸扩增反应管，滴加10μl石蜡油，并通过离心、振动或其他方式，使扩增试剂充满反应容器；b.设置自制核酸扩增仪底部温度为95℃，上部温度为60℃，扩增时间设置为30分钟。将装有核酸扩增试剂的反应管插入核酸扩增仪中，开启程序，程序运行结束后取出反应管。
(3)扩增结果电泳检测：从反应管中取出5μl扩增产物，与1μl上样缓冲液混合后，使用3％的琼脂糖凝胶电泳检测扩增产物。
3、实验结果：如图4所示，泳道1与泳道2为阳性样本扩增结果，泳道3与泳道4为阴性对照(DEPC水)扩增结果。从结果中可以看出，本发明可实现以DNA为模板的扩增，阴性对照正常说明无非特异性扩增。
实施例3：应用实施例1的一种可控制液体环流路径的核酸扩增反应管进行的、以RNA为模板的扩增与检测
1、实验材料
化学试剂：SpeedSTAR HS DNA polymerase(TaKaRa)，反转录酶MMLV(Transgen)，10×Fast Buffer I(Mg2+plus)(TaKaRa)，dNTP(TaKaRa)，DEPC水，石蜡油，6×DNA上样缓冲液(含Sybr Green)
仪器耗材：自制核酸扩增仪(参见申请CN201110456811.9)；实施例1的一种可控制液体环流路径的核酸扩增反应管、凝胶电泳仪、凝胶成像仪(Bio-Rad)
引物：
CA16-WJ-F6-1：CAAGTAYTACCYACRGCTGCCAA(SEQ ID NO.3)
CA16-WJ-R6-1：CAACACACAYCTMGTCTCAATGAG(SEQ ID  NO.4)
检测模板1：柯萨奇病毒A16型(CA16病毒)RNA提取物，浓度为103copies/mL
检测模板2：DEPC水
2、实验方法：
(1)扩增试剂的配制：
3.2mM dNTP，4μL 10×Fast Buffer I(Mg2+plus)，1U SpeedSTAR HS DNA polymerase，0.4U MMLV，0.4μL 10μM JxbUL54F1，0.4μL 10μM JxbUL54R11，5μl检测模板，总体积40μl，剩余体积用DEPC水补足。
(2)核酸扩增：a.将配制好的扩增试剂注入本发明的一种可控制液体环流路径的核酸扩增反应管，滴加10μl石蜡油，并通过离心、振动或其他方式，使扩增试剂充满反应容器；b.仪器底部加热模块程序为60℃ 20分钟，95℃ 30分钟；仪器上部加热模块设置为恒温60℃，50分钟。将装有核酸扩增试剂的反应管插入仪器中，开启程序，程序运行结束后取出反应管。
(3)扩增结果电泳检测：从反应管中取出5μl扩增产物，与1μl上样缓冲液混合后，使用3％的琼脂糖凝胶电泳检测扩增产物。
3、实验结果：如图5所示，泳道1与泳道2为阳性样本扩增结果，泳道3与泳道4为阴性对照(DEPC水)扩增结果。从结果中可以看出，本发明可实现以RNA为模板的扩增，阴性对照正常说明无非特异性扩增。
实施例4：有、无环流控制功能反应管的扩增的均一性与特异性比较
1、实验材料
化学试剂：SpeedSTAR HS DNA polymerase(TaKaRa)，10×Fast Buffer I(Mg2+plus)(TaKaRa)，dNTP(TaKaRa)，DEPC水，石蜡油，6×DNA上样缓冲液(含Sybr Green)
仪器耗材：自制核酸扩增仪；实施例1的一种可控制液体环流路径的核酸扩增反应管、无环流控制功能的反应管(参见申请201110360350.5)、凝胶电泳仪、凝胶成像仪(Bio-Rad)
引物：JxbUL54F1：GTGCGCCTTGACACTGTAC(SEQ ID NO.1)
JxbUL54R11：CGACAAGTACTTTGAGCAGG(SEQ ID NO.2)
检测模板1：CMV病毒DNA提取物，浓度为103copies/mL
检测模板2：DEPC水
2、实验方法：
(1)扩增试剂的配制：3.2mM dNTP，4μL 10×Fast Buffer I(Mg2+plus)，1U SpeedSTAR HS DNA polymerase，0.4μL 10μM JxbUL54F1，0.4μL 10μM JxbUL54R11，5μl检测模板，总体积40μl，剩余体积用DEPC水补足。
(2)核酸扩增：a.将配制好的扩增试剂分别注入本发明的一种可控制液体环流路径的核酸扩增反应管、及无环流控制功能的反应管，滴加10μl石蜡油，并通过离心、振动或其他方式，使扩增试剂充满反应容器；b.设置自制核酸扩增仪底部温度为95℃，上部温度为60℃，扩增时间设置为30分钟。将上述两种装有核酸扩增试剂的反应管插入仪器中，开启程序，程序运行结束后取出反应管。
(3)扩增结果电泳检测：从反应管中取出5μl扩增产物，与1μl上样缓冲液混合后，使用3％的琼脂糖凝胶电泳检测扩增产物。
3、实验结果：阳性扩增如图6泳道1-4所示，用本发明的一种可控制液体环流路径的核酸扩增反应管扩增的结果(图6a)，4管条带的强度明显优于用无环流控制功能的反应容器扩增的结果(图6b)。阴性扩增如图6泳道5-8所示，用本发明的一种可通过控制液体环流路径实现PCR高效扩增的反应管扩增的结果(图6a)，背景干净，没有引物二聚体等非特异性扩增，而用无环流控制功能的反应管扩增的结果(图6b)，有明显的引物二聚体。上述结果说明本发明的一种控制液体自发环流路径的反应管具有提高不同管间扩增的一致性与特异性的作用。
实施例5：有、无环流控制功能反应管的扩增效率比较
1、实验材料
化学试剂：SpeedSTAR HS DNA polymerase(TaKaRa)，10×Fast Buffer I(Mg2+plus)(TaKaRa)，dNTP(TaKaRa)，DEPC水，石蜡油，6×DNA上样缓冲液(含Sybr Green)
仪器耗材：自制核酸扩增仪；实施例1的一种可控制液体环流路径的核酸扩增反应管、无环流控制功能的反应管(参见申请201110360350.5)、凝胶电泳仪、凝胶成像仪(Bio-Rad)
引物：JxbUL54F1：GTGCGCCTTGACACTGTAC(SEQ ID NO.1)
JxbUL54R11：CGACAAGTACTTTGAGCAGG(SEQ ID NO.2)
检测模板1：CMV病毒DNA提取模板，浓度为103copies/ml
检测模板2：DEPC水
2、实验方法：
(1)扩增试剂的配制：3.2mM dNTP，4μL 10×Fast Buffer I(Mg2+plus)，1U SpeedSTAR HS DNA polymerase，0.4μL 10μM JxbUL54F1，0.4μL 10μM JxbUL54R11，5μl检测模板，总体积40μl，剩余体积用DEPC水补足。
(2)核酸扩增：a.将配制好的扩增试剂分别注入本发明的一种可控制液体环流路径的核酸扩增反应管、及无环流控制功能的反应管，滴加10μl石蜡油，并通过离心、振动或其他方式，使扩增试剂充满反应管；b.设置自制核酸扩增仪底部温度为95℃，上部温度为60℃，扩增时间分别设定为15分钟，20分钟，25分钟。将上述两种装有核酸扩增试剂的反应管插入仪器中，开启程序，程序运行结束后取出反应管。
(3)扩增结果电泳检测：从反应管中取出5μl扩增产物，与1μl上样缓冲液混合后，使用3％的琼脂糖凝胶电泳检测扩增产物。
3、实验结果：
应用本发明的一种可控制液体自发环流路径的核酸扩增反应管扩 增的结果如图7a所示，应用无环流控制功能反应管扩增的结果如图7b所示。结果显示，应用本发明的扩增，在扩增20分钟后，阳性管即可观察到弱条带，在扩增25分钟后，阳性管即可观察到强条带；而应用无环流控制功能的反应管，则需扩增25分钟，阳性反应管才可观察到弱条带。该结果说明应用本发明相对于先前对流PCR方法可提高扩增的效率。
实施例6：有、无环流控制功能反应管的实时荧光检测比较
1、实验材料
化学试剂：SpeedSTAR HS DNA polymerase(TaKaRa)，10×Fast Buffer I(Mg2+plus)(TaKaRa)，dNTP(TaKaRa)，DEPC水，石蜡油
仪器耗材：自制核酸扩增与实时荧光检测仪(参见申请CN201110456811.9)；实施例1的一种可控制液体环流路径的核酸扩增反应管、无环流控制功能的反应管(参见申请201110360350.5)、
引物：JxbUL54F1：GTGCGCCTTGACACTGTAC(SEQ ID NO.1)
JxbUL54R11：CGACAAGTACTTTGAGCAGG(SEQ ID NO.2)
探针：JxbUL54P1：FAM-AGCCGGCTCCAAGTGCAAG-BHQ-1(SEQ ID NO.5)
检测模板1：CMV病毒DNA提取模板，浓度为106copies/ml
检测模板2：CMV病毒DNA提取模板，浓度为105copies/ml
检测模板3：DEPC水
2、实验方法：
(1)扩增试剂的配制：3.2mM dNTP，4μL 10×Fast Buffer I(Mg2+plus)，1U SpeedSTAR HS DNA polymerase，0.4μL 10μM JxbUL54F1，0.4μL 10μM JxbUL54R11，0.2μL 10μM JxbUL54P1，5μl检测模板，总体积40μl，剩余体积用DEPC水补足。
(2)核酸扩增：a.将配制好的扩增试剂分别注入本发明的一种可控 制液体环流路径的核酸扩增反应管、及无环流控制功能的反应管，滴加10μl石蜡油，并通过离心、振动或其他方式，使扩增试剂充满反应容器；b.设置自制核酸扩增仪底部温度为95℃，上部温度为60℃，扩增时间设定为30分钟。将上述两种装有核酸扩增试剂的反应管插入自制核酸扩增与实时荧光检测仪中，开启程序，程序运行结束后取出反应管，并分析数据。
3、实验结果：
应用本发明的一种可控制液体自发环流路径的反应管扩增的结果如图8a所示，应用无环流控制功能反应管扩增的结果如图8b所示。结果显示，应用本发明的扩增，其相同浓度样本扩增曲线的重复性明显优于无环流控制功能的反应管，从而提示应用本发明的一种可控制液体环流路径的核酸扩增反应管可进行核酸样本的定量检测。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (16)

  1. 一种核酸扩增反应管，包括一端封闭的管体(1)，所述管体(1)包括储液区(4)以及位于储液区下方的核酸扩增区(3)，其特征在于，所述核酸扩增区(3)内设置有上下悬空的插片(2)。
  2. 权利要求1的反应管，其特征在于，当反应管内注入试剂时，通过插片(2)的物理阻隔，使得反应管内试剂在外力或内力的作用下，形成围绕插片上方及下方的环流路径。
  3. 权利要求1的反应管，其特征在于，所述插片(2)沿管体(1)的中轴线设置，并且插片的两侧(a，b)与核酸扩增区(3)的内壁连接，
    优选地，插片插片(2)沿管体(1)的中轴线方向将核酸扩增区分隔成第一区域(3-1)和第二区域(3-2)，第一区域(3-1)和第二区域(3-2)在核酸扩增区的上部(3-A)和下部(3-B)连通。
  4. 权利要求1至3任一项的反应管，其特征在于，所述插片(2)下端(d)与管体底部之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区高度的1/2；
    优选地，所述插片(2)下端(d)与管体底部之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区(3)高度的1/3；
    进一步优选地，所述插片(2)下端(d)与管体底部之间的距离大于0mm(例如大于等于1mm)并且小于等于4mm。
  5. 权利要求1至3任一项的反应管，其特征在于，所述插片(2)上端(c)与核酸扩增区(3)上端之间的距离大于0mm(例如大于等于1mm)并且小于核酸扩增区(3)高度的1/2；
    优选地，所述插片(2)上端(c)与核酸扩增区(3)上端之间的 距离大于0mm(例如大于等于1mm)并且小于核酸扩增区(3)高度的1/3；
    进一步优选地，所述插片(2)上端(c)与核酸扩增区(3)上端之间的距离大于0mm(例如大于等于1mm)并且小于等于3mm。
  6. 权利要求1至3任一项的反应管，其特征在于，所述管体(1)的底部通过与管体(1)相互配合的底塞(1-1)实现一端封闭。
  7. 权利要求1至3任一项的反应管，其特征在于，所述管体(1)还包括与其配合的管盖。
  8. 权利要求1至7任一项的反应管，其特征在于，所述核酸扩增区(3)的高度/内径的比值为3～12，优选地，所述核酸扩增区(3)的高度/内径的比值为6～9。
  9. 权利要求1至7的反应管，其特征在于，所述核酸扩增区(3)的容积为30～200μl，优选地，所述核酸扩增区(3)的容积为40～150μl。
  10. 权利要求1-9任一项的反应管，其特征在于，所述管体(1)和插片(2)由耐热材料制成，所述耐热材料例如选自玻璃、聚碳酸酯、聚丙烯、聚醚砜和聚砜。
  11. 一种核酸扩增反应装置，其包括权利要求1-10任一项的反应管和一个或多个可提供或移走热量的温度控制装置，所述温度控制装置设置于所述反应管的外部或内部。
  12. 一种试剂盒，其包含权利要求1-10任一项的反应管。
  13. 一种扩增样品中目的核酸的方法，其包括使用权利要求1-10 任一项的反应管或权利要求11的反应装置，
    优选地，所述核酸是DNA或RNA；
    优选地，所述扩增是PCR反应或逆转录反应。
  14. 权利要求13的方法，其包括以下步骤：
    1)将核酸扩增反应试剂注入权利要求1-10任一项的反应管中；
    2)通过震动、离心或其他方式，使反应试剂完全填充核酸扩增区(3)；任选地，还包括用不易挥发的物质(例如石蜡油或低熔点的蜡)覆盖于试剂表面或用管盖封闭反应管的步骤；
    3)用温度控制装置对所述反应管的特定部位提供或移走热量，以完成RNA逆转录和/或DNA扩增反应；
    4)任选地，在进行核酸扩增的同时或扩增结束后，还包括对扩增产物进行检测的步骤。
  15. 权利要求1-10任一项的反应管或权利要求9的反应装置用于核酸扩增的用途。
  16. 权利要求1-10任一项的反应管用于制备试剂盒的用途，所述试剂盒用于进行核酸扩增。
PCT/CN2016/081649 2015-05-12 2016-05-11 一种可控制液体环流路径的核酸扩增反应管 WO2016180333A1 (zh)

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US15/573,381 US10487301B2 (en) 2015-05-12 2016-05-11 Reaction tube for nucleic acid amplification capable of controlling liquid circulation path
ES16792183T ES2798287T3 (es) 2015-05-12 2016-05-11 Tubo de reacción de amplificación de ácido nucleico capaz de controlar la trayectoria de circulación de líquido
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