CN109957612A - A kind of real-time fluorescence quantitative PCR detection method and device - Google Patents
A kind of real-time fluorescence quantitative PCR detection method and device Download PDFInfo
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- CN109957612A CN109957612A CN201910257348.1A CN201910257348A CN109957612A CN 109957612 A CN109957612 A CN 109957612A CN 201910257348 A CN201910257348 A CN 201910257348A CN 109957612 A CN109957612 A CN 109957612A
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- 238000010257 thawing Methods 0.000 claims abstract description 8
- 238000004108 freeze drying Methods 0.000 claims description 22
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- 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
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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
- C12Q1/6851—Quantitative amplification
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- 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
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- 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
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Abstract
The present invention provides a kind of real-time fluorescence quantitative PCR detection method and device, comprising the following steps: built-in solid paraffin in detection reaction tube；Real-time fluorescence quantitative PCR detection reagent, template to be detected is added in detection reaction tube；Heating detection reaction tube, the solid paraffin heat melt and float on PCR reaction solution surface；The real-time fluorescence quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on PCR reaction solution surface and solidifies, and forms solid paraffin sealant.The real-time fluorescence quantitative PCR detection method method and apparatus can guarantee to detect the leakproofness in space, prevent fluorescence quantitative PCR detection reaction system from aerosol phenomenon occurs, real-time fluorescence quantitative PCR detection reagent caused by avoiding because of careless manipulation leaks, detection environmental pollution is avoided, guarantees the accuracy of template detection result to be detected.
Description
Technical field
The present invention relates to a kind of real-time fluorescence quantitative PCR detection method and device, in particular to one kind can effectively prevent gas
The real-time fluorescence quantitative PCR detection method and device that colloidal sol is formed.
Background technique
Based on quantitative fluorescent PCR (realtime fluorescence quantitative PCR, RTFQ PCR) side
The nucleic acid detection method of method has obtained great development and popularization in fields such as biology, medicine, food and medicine analysis, has had
High sensitivity, detection is accurate, the advantages such as detection efficiency height.But there is also certain problems in practical applications, one of them is prominent
Out the problem of is the generation of Aerosol Pollution.
Since experience multiple high temp recycles during the reaction for PCR reaction, the DNA segment formed in simultaneous reactions pipe is often
Concentration is very high, in the reaction or after reaction, once careless manipulation, is easy to cause fluid seepage and forms gas in detection environment
Colloidal sol pollution.The formation of Aerosol Pollution carries out the appearance of false positive when same detection detection after will lead to, seriously affect
The real effectiveness of testing result.
To avoid this problem of Aerosol Pollution, laboratory is often by sample treatment area, and reagent prepares area and detection zone is only
It stands as different physical isolation spaces, to avoid the diffusion of pollution.Although this method effectively, needs the detection of larger area
Space and the biggish laboratory finishing investment of the amount of money, limit the promotion and popularization based on fluorescent quantitative PCR detection method.Such as
In some combination areas of city and country, remote districts, or the test point temporarily set up, it all has no idea to set up standardized qualified detection
Environment, and Aerosol Pollution once occurs, the authenticity and validity of the detection carried out are all affected.
So how to effectively prevent the generation of aerosol, have for the promotion and popularization of fluorescent quantitative PCR detection method
Important meaning.
Therefore, the prior art is defective, to be further improved and developed.
Summary of the invention
(1) goal of the invention: to solve above-mentioned problems of the prior art, the object of the present invention is to provide glimmering in real time
Fluorescent Quantitative PCR detection method and device, the real-time fluorescence quantitative PCR detection method and device can guarantee to detect the sealing in space
Property, the leakage of PCR reaction solution is prevented, the generation of Aerosol Pollution is avoided, guarantees the accuracy of template detection result to be detected.
(2) technical solution:
A kind of real-time fluorescence quantitative PCR detection method, comprising the following steps:
First, built-in solid paraffin in reaction tube is detected；
Secondly, real-time fluorescence quantitative PCR detection reagent, template to be detected is added in detection reaction tube；
Finally, heating detection reaction tube, the solid paraffin heat melt and float on PCR reaction solution surface；The real-time fluorescence
Quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on the PCR reaction solution surface and solidifies, and forms solid-state
Paraffin sealant.
The real-time fluorescence quantitative PCR detection method, in which: the built-in solid paraffin in the detection reaction tube, tool
Body the following steps are included:
The detection reaction tube includes tube body and Guan Gai,
In the accommodating space that the inboard wall of tube body that liquid paraffin after solid paraffin is melted is added to detection reaction tube is formed, and wait liquid
State paraffin；
Or be added to after melting solid paraffin in the cap wall of detection reaction tube, wait liquid paraffins to solidify；
Or solid paraffin is direct plungeed into the accommodating space of detection reaction tube tube body.
The real-time fluorescence quantitative PCR detection method, in which: the solid paraffin added in the detection reaction tube, institute
Stating the volume after solid paraffin melts is 10uL to 100uL.
The real-time fluorescence quantitative PCR detection method, in which: the accommodating for direct plungeing into detection reaction tube tube body is empty
Between in solid paraffin shape be ball, cube, stereo-flower.
The real-time fluorescence quantitative PCR detection method, in which: the template to be detected is DNA profiling.
The real-time fluorescence quantitative PCR detection method, wherein the solid paraffin heat is melted and to swim in PCR anti-
Liquid surface, the process of the real-time fluorescence quantitative PCR detection reagent and template reaction to be detected are answered, the detection reaction tube does not cover
Pipe lid.
The real-time fluorescence quantitative PCR detection method, wherein the PCR of the real-time fluorescence quantitative PCR after reaction
A temperature descending section is added in condition setting, the PCR reaction solution is allowed to cool to room temperature, guarantees the solidification of liquid paraffin layer.
A kind of real-time fluorescence quantitative PCR detection method, comprising the following steps:
Firstly, will test solid paraffin built in reaction tube and freeze-drying detection reagent；
Secondly, template to be detected is added in detection reaction tube；
Finally, heating detection reaction tube, the solid paraffin heat is melted, and floats on PCR reaction solution surface；The real-time fluorescence
Quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on the PCR reaction solution surface and solidifies, and forms solid-state
Paraffin sealant.
The real-time fluorescence quantitative PCR detection method, in which:
In solid paraffin built in the detection reaction tube, specifically includes the following steps:
The detection reaction tube includes tube body and Guan Gai,
In the accommodating space that the inboard wall of tube body that liquid paraffin after solid paraffin is melted is added to detection reaction tube is formed, and wait liquid
State paraffin；
Or be added to after melting solid paraffin in the cap wall of detection reaction tube, wait liquid paraffins to solidify；
Or solid paraffin is direct plungeed into the accommodating space of detection reaction tube tube body.
The real-time fluorescence quantitative PCR detection method, in which: the solid paraffin added in the detection reaction tube, institute
Stating the volume after solid paraffin melts is 10uL to 100uL.
The real-time fluorescence quantitative PCR detection method, in which: the accommodating for direct plungeing into detection reaction tube tube body is empty
Between in solid paraffin shape be ball, cube, stereo-flower.
The real-time fluorescence quantitative PCR detection method, in which: the real-time fluorescence quantitative PCR detection reagent is freeze-drying
Solid reagent, form are sphere, cube, stereo-flower.
The real-time fluorescence quantitative PCR detection method, wherein the PCR of the real-time fluorescence quantitative PCR after reaction
A temperature descending section is added in condition setting, reaction solution is allowed to cool to room temperature, guarantees the solidification of liquid paraffin layer.
A kind of real-time fluorescence quantitative PCR detection device, including detection reaction tube, wherein further include being placed in the detection
Solid paraffin and freeze-drying detection reagent in reaction tube.
The real-time fluorescence quantitative PCR detection device, in which: the detection reaction tube includes tube body and pipe cap, tube body
The accommodating space of inner wall and pipe cap inner wall cooperation closed tube；Adhere to solid paraffin on the inboard wall of tube body；Or pipe lid is interior
Wall adheres to solid paraffin；Or solid paraffin direct plunges into the accommodating space of detection reaction tube inboard wall of tube body.
The real-time fluorescence quantitative PCR detection device, in which: the solid paraffin is ball, cube, stereo-flower.
The real-time fluorescence quantitative PCR detection device, in which: the freeze-drying detection reagent is ball, cube, solid
Flower.
The real-time fluorescence quantitative PCR detection device, in which:
The freeze-drying detection reagent is that real-time fluorescence quantitative PCR detection reagent adds protective agent to be lyophilized, and the real-time fluorescence is fixed
Measuring PCR detection reagent includes taq enzyme, buffer, primer, probe, dNTP.
(3) the utility model has the advantages that a kind of real-time fluorescence quantitative PCR detection method provided by the invention and device, by solid paraffin
It is fabricated to microballoon and is placed in detection reaction tube, PCR reaction solution surface is melted and floated on to the solid paraffin heat, reacts PCR
Liquid surface is sealed, and prevents from generating the aerosol with template component when fluorescence quantitative PCR detection, to avoid since gas is molten
The detection mistake that glue generates.Real-time fluorescence quantitative PCR detection method and device provided by the invention, reduce to fluorescent quantitation
PCR detects the demand of environment, can detecte whenever and wherever possible, improves the ease for use of real-time fluorescence quantitative PCR detection device, simplifies
Operation, convenient for promoting.
Detailed description of the invention
Fig. 1 is the flow diagram of real-time fluorescence quantitative PCR detection method first preferred embodiment of the present invention；
Fig. 2 is the flow diagram of real-time fluorescence quantitative PCR detection method third preferred embodiment of the present invention；
Fig. 3 is the structural schematic diagram of real-time fluorescence quantitative PCR detection device of the present invention.
Specific embodiment
The present invention is described in further details below with reference to preferred embodiment, is elaborated in the following description more
Details to facilitate a thorough understanding of the present invention, still, the present invention obviously can be come with a variety of other modes different from this description
Implement, those skilled in the art can without violating the connotation of the present invention according to practical situations make it is similar promote,
It deduces, therefore should not be limited the scope of the invention with the content of this specific embodiment.
Fig. 1-3 is the schematic diagram of the embodiment of the present invention, it should be noted that this attached drawing only as an example, not be by
It draws, and should not be construed as limiting in this, as to actual requirement protection scope of the invention according to the condition of equal proportion.
A kind of real-time fluorescence quantitative PCR detection method and device provided by the invention, are applied to fluorescence for solid paraffin and determine
Prevent aerosol from being formed to realize in amount PCR reaction.
The real-time fluorescence quantitative PCR detection method needs to use fluorescence quantitative PCR detection reaction tube (PCR pipe), fluorescence
Quantitative PCR detecting reagent (PCR reagent), template to be detected；Fluorescence quantitative PCR detection reagent and the mixture of template to be detected claim
For PCR reaction solution.
Real-time fluorescence quantitative PCR reaction is generally divided into two-step method and three-step approach.It is limited with doctor of precious day biotechnology (Beijing)
The article No. of company's sale is for RR006Q reagent, two-step method is arranged, the first step, and 98 degrees Celsius, 10 seconds；Second step, 68
Degree Celsius, 1 minute；The setting of its three-step approach, the first step, 98 degrees Celsius, 10 seconds；Second step, 55 degrees Celsius, 30 seconds；Third
Step, 72 degrees Celsius, 1 minute.No matter two steps or three-step approach, 30 thermal cycles are all carried out.The present invention is in real-time fluorescence quantitative PCR
Solid paraffin is added in detection, haves no need to change existing thermal cycle setting, reason is temperature used in each circulation step of PCR
Degree is all higher than the fusing point of solid paraffin used.In the first step heating process in first circulation of reaction, with temperature
Raising solid paraffin is melted.Liquid paraffin after thawing, since its density is light compared to water, so it is anti-to float on PCR
Liquid surface is answered, and forms the sealant of a liquid in real-time fluorescence quantitative PCR reaction process.The real time fluorescent quantitative
PCR reaction terminates, and reaction temperature can reduce, and typically drop to 55 degrees Celsius or less.The present invention can be in the real-time fluorescence
A temperature descending section is added in the PCR condition setting of quantitative PCR after reaction and cools to room temperature by reaction solution, to ensure liquid stone
Wax layer solidification.
The present invention may be that heating stepses are separately provided in solid paraffin, not limit here.
First preferred embodiment of real-time fluorescence quantitative PCR detection method of the present invention, includes the following steps
First, solid paraffin built in reaction tube is detected；
Secondly, real-time fluorescence quantitative PCR detection reagent, template to be detected is added in detection reaction tube；
Finally, heating detection reaction tube, the solid paraffin heat melt and float on PCR reaction solution surface；The real-time fluorescence
Quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on the PCR reaction solution surface and solidifies, and forms solid-state
Paraffin sealant.
The step of built-in solid paraffin, can specifically include following in the detection reaction tube:
The detection reaction tube includes tube body and pipe cap, and tube body and pipe cap cooperate the accommodating space of closed tube, the tube body
Accommodating space is detected for solid paraffin, real-time fluorescence quantitative PCR detection reagent and template to be detected react
Space
After solid paraffin is melted, certain volume such as 10uL to 100uL liquid paraffin is directly added to the pipe of detection reaction tube
The accommodating space that internal wall is formed, uses after waiting liquid paraffins to solidify.Or after melting solid paraffin, directly by certain volume
Such as 10uL to 100uL liquid paraffin is added to the cap wall of detection reaction tube, uses after waiting liquid paraffins to solidify.The detection
The accommodating space that the cap wall face inboard wall of tube body of reaction tube is formed, for blocking the accommodating space of inboard wall of tube body formation.Also
Solid paraffin can be direct plungeed into the accommodating space of detection reaction tube, concrete form is with no restrictions.
The fluorescence quantitative PCR detection box includes fluorescence quantitative PCR detection agent, and the fluorescence quantitative PCR detection agent includes
Taq enzyme, buffer (buffer), primer, probe, ingredient required for the fluorescent quantitations such as dNTP react.In the detection box
Fluorescence quantitative PCR detection agent needs subzero 20 degrees Celsius of temperature transports below and storage.Preferably, fluorescence of the present invention
Quantitative PCR detection module can be DNA profiling.
Second preferred embodiment of the invention, further, the solid paraffin can be 10uL to 100uL's after melting
Volume, the solid paraffin can be made the shapes such as ball, cube, stereo-flower, here with no restrictions.The solid paraffin is done
At ball, paraffin microballoon further can also be.Specifically includes the following steps:
Firstly, the solid paraffin microballoon with certain volume, the volume of the solid paraffin microballoon is added in detection reaction tube
For 10uL to 100uL, preferably 20uL-25uL, can need to select according to specific detection.
Secondly, real-time fluorescence quantitative PCR detection reagent is added in the detection reaction tube with solid paraffin microballoon, then
It adds and needs the template to be detected that detects, i.e. DNA profiling, the present invention, which can make to detect reaction tube not cover, to be detected.
Finally, reacting the detection with solid paraffin microballoon, real-time fluorescence quantitative PCR detection reagent and template to be detected
Pipe is heated, and during heating, real-time fluorescence quantitative PCR detection reagent and template to be detected start Thermal Cycling, and
During heating, it is liquid paraffin that paraffin microballoon heat, which is melted,.It is also possible to real-time fluorescence quantitative PCR detection reagent and to be checked
Surveying the heat that template autoreactivity generates melts paraffin microballoon, here with no restrictions.Liquid paraffin compared to PCR due to reacting
The density of liquid (aqueous solution) is low, will float on above PCR reaction solution, can prevent PCR reaction solution during the reaction due to high temperature
The problem of volatilization, and since liquid paraffin is to the buffer action of air, the formation of DNA aerosol under high temperature can be prevented.PCR
After reaction, reaction temperature reduces, and paraffin is condensed into solid-state to the cold, forms one layer of solid stone on the surface of PCR reaction solution
Wax sealant can prevent the leakage of PCR reaction solution, play the function of detection reaction vial closure.
Preferably, to prevent solid paraffin layer caused by external force damaged, can react in fluorescence quantitative PCR detection terminates
Afterwards, add pipe lid for detection reaction tube, pipe lid further prevents the generation of leakage.
The real-time fluorescence quantitative PCR detection reagent is made into freeze-drying detection examination by third preferred embodiment of the invention
Agent, the freeze-drying detection reagent can be made the shapes such as sphere, cube, stereo-flower, here with no restrictions.It specifically includes following
Step:
Firstly, solid paraffin and freeze-drying detection reagent are put into detection reaction tube, preferential be can be paraffin microballoon and freeze-drying
Microballoon detection reagent puts detection reaction tube into.
Secondly, template to be detected is added in detection reaction tube；
Finally, heating detection reaction tube, the solid paraffin heat is melted, and floats on PCR reaction solution surface；The real-time fluorescence
Quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on the PCR reaction solution surface and solidifies, and forms solid-state
Paraffin sealant.
The real-time fluorescence quantitative PCR detection reagent is made into freeze-drying detection reagent by the present invention, be may be implemented 2 degrees Celsius and is arrived
8 degrees Celsius of transport storages, the transport even under room temperature are kept.It, can be in addition, solid freeze-drying detection reagent and solid paraffin
It is previously positioned in the tube body of detection reaction tube as needed, or in the pipe lid of detection reaction tube, eliminates point when test
The step of dress operates, and simplifies testing process.The present invention can be not added pipe lid and operate, after reaction, liquid paraffin layer
It is frozen into solid, PCR reaction solution is fully sealed in detection reaction tube, air has been isolated, has effectively prevent the formation of aerosol.
To prevent solid paraffin layer caused by external force damaged, can add capped pipe lid again prevents the generation of PCR reaction solution leakage.
Solid paraffin in the present invention refers to and extracts from petroleum, coal or whale oil, or consolidated by what is be chemically synthesized
The mixture of state higher alkane, the molecular formula of main component are CnH2n+2, wherein n=20~40.Solid paraffin of the invention includes
But it is not limited to: n-eicosane icosane C20H42, n-docosane Docosane C22H46, lignocerane Tetracosane
C24H50, hexacosane hexacosane C26H54And positive octacosane octacosane C28H58、Nonacosane
nonacosane C29H60, melissane triacontane C30H62, positive hentriacontane hentriacosane C31H64, positive three
Dodecane dotriacontane C32H66、Tetratriacontane tetratriacontane C34H70, pentatriacontane
pentatriacontane C35H72, hexatriacontane Hexatriacontane C36H74.Solid paraffin in the present invention, also may be used
Finger adds other compositions on the basis of above-mentioned solid alkanes mixture, such as low-molecular weight polymer and manufactured mixture.
The present invention provides a kind of real-time fluorescence quantitative PCR detection device, including detection reaction tube, and is placed in the inspection
The solid paraffin and freeze-drying detection reagent in reaction tube are surveyed, as shown in figure 3, the freeze-drying detection reagent is real time fluorescent quantitative
PCR detection reagent adds protective agent to freeze.The real-time fluorescence quantitative PCR detection reagent includes taq enzyme, buffer
(buffer), primer, probe, ingredient required for the fluorescent quantitations reaction detection such as dNTP.
The solid paraffin being arranged in the detection reaction tube, the detection reaction tube includes tube body and pipe cap, inboard wall of tube body
With the accommodating space of pipe cap inner wall cooperation closed tube, the accommodating space of the tube body is for by solid paraffin, real-time fluorescence
Quantitative PCR detecting reagent and template to be detected react, the space detected.It can be certain volume such as 10uL to arrive
Liquid paraffin after 100uL melts is added drop-wise in the accommodating space for detecting the inboard wall of tube body of reaction tube, and liquid paraffins is waited to be set in
It is used after on inboard wall of tube body, that is, adheres to solid paraffin on inboard wall of tube body.It, directly will be certain or after melting solid paraffin
Volume such as 100uL to 100uL liquid paraffin is added drop-wise in the inner wall of the pipe lid of detection reaction tube, is made after waiting liquid paraffins to solidify
With, that is, in the inner wall attachment solid paraffin of pipe lid.Solid paraffin can also be direct plungeed into detection reaction tube inboard wall of tube body
Accommodating space in.
The solid paraffin placed in the accommodating space of the detection reaction tube tube body can be made various shape, can be made
The shapes such as ball, cube, stereo-flower, elliptical shape ball, annulus, here with no restrictions.The paraffin, which is made into ball, can also be stone
Wax microballoon.
The freeze-drying detection reagent also can be made the shapes such as ball, cube, stereo-flower, elliptical shape ball, annulus, here not
It is limited.The freeze-drying detection reagent is made into ball, can also be freeze-drying detection microballoon.
The real-time fluorescence quantitative PCR detection device, can be in the tube body with paraffin microballoon and freeze-drying detection microballoon
Template to be detected is added, the template to be detected includes DNA profiling.Later to paraffin microballoon, freeze-drying detection microballoon and to
The tube body of detection template is heated, and the freeze-drying detection microballoon and template to be detected are reacted, and solid paraffin microballoon receives
Heat heat is melted, and the upper layer of reaction solution is floated on, and liquid paraffin layer is hyaline layer at this time, does not influence reaction and the inspection of fluorescence signal
It surveys.
The problem of liquid paraffin can prevent PCR reaction solution from volatilizing during the reaction because of high temperature, and due to liquid
Paraffin can prevent the formation of DNA aerosol under high temperature to the buffer action of air.Fluorescence quantitative PCR detection after reaction,
Reaction temperature reduces, and paraffin is condensed into solid-state to the cold, forms one layer of solid paraffin sealant on the surface of PCR reaction solution, can
To prevent the leakage of PCR reaction solution, the function of detection reaction vial closure is played.
A kind of real-time fluorescence quantitative PCR detection method and device provided by the invention, are fabricated to microballoon for solid paraffin and put
It is placed in detection reaction tube, paraffin microballoon is melted as liquid paraffin, liquid paraffin can float on the top of PCR reaction solution, right
PCR reaction solution is sealed, and the aerosol with template component is generated when preventing fluorescence quantitative PCR detection, thus avoid due to
The detection mistake that aerosol generates.Real-time fluorescence quantitative PCR detection method and device provided by the invention, it is fixed to fluorescence to reduce
Demand of the PCR detection to environment is measured, can detecte whenever and wherever possible, improve the ease for use of real-time fluorescence quantitative PCR detection device,
Operation is simplified, convenient for promoting.
The above content is the explanations of the preferred embodiment to the invention, those skilled in the art can be helped more to fill
The technical solution for dividing ground to understand the invention.But these embodiments are merely illustrative, and it cannot be said that the invention
Specific embodiment be only limitted to the explanations of these embodiments.The invention person of an ordinary skill in the technical field is come
It says, without departing from the concept of the premise of the invention, several simple deductions and transformation can also be made, all shall be regarded as belonging to
The protection scope of the invention.
Claims (10)
1. a kind of real-time fluorescence quantitative PCR detection method, comprising the following steps:
First, built-in solid paraffin in reaction tube is detected；
Secondly, real-time fluorescence quantitative PCR detection reagent, template to be detected is added in detection reaction tube；
Finally, heating detection reaction tube, the solid paraffin heat melt and float on PCR reaction solution surface；The real-time fluorescence
Quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on the PCR reaction solution surface and solidifies, and forms solid-state
Paraffin sealant.
2. real-time fluorescence quantitative PCR detection method according to claim 1, it is characterised in that: in the detection reaction tube
In built-in solid paraffin, specifically includes the following steps:
The detection reaction tube includes tube body and Guan Gai,
In the accommodating space that the inboard wall of tube body that liquid paraffin after solid paraffin is melted is added to detection reaction tube is formed, and wait liquid
State paraffin；
Or be added to after melting solid paraffin in the cap wall of detection reaction tube, wait liquid paraffins to solidify；
Or solid paraffin is direct plungeed into the accommodating space of detection reaction tube tube body.
3. real-time fluorescence quantitative PCR detection method according to claim 1, it is characterised in that: the template to be detected is
DNA profiling.
4. real-time fluorescence quantitative PCR detection method according to claim 1, which is characterized in that the real time fluorescent quantitative
A temperature descending section is added in the PCR condition setting of PCR after reaction, the PCR reaction solution is allowed to cool to room temperature, guarantees liquid
Paraffin layer solidification.
5. a kind of real-time fluorescence quantitative PCR detection method, comprising the following steps:
Firstly, will test solid paraffin built in reaction tube and freeze-drying detection reagent；
Secondly, template to be detected is added in detection reaction tube；
Finally, heating detection reaction tube, the solid paraffin heat is melted, and floats on PCR reaction solution surface；The real-time fluorescence
Quantitative PCR reaction terminates, and temperature reduces, and the paraffin after the thawing floats on the PCR reaction solution surface and solidifies, and forms solid-state
Paraffin sealant.
6. real-time fluorescence quantitative PCR detection method according to claim 5, it is characterised in that:
In solid paraffin built in the detection reaction tube, specifically includes the following steps:
The detection reaction tube includes tube body and Guan Gai,
In the accommodating space that the inboard wall of tube body that liquid paraffin after solid paraffin is melted is added to detection reaction tube is formed, and wait liquid
State paraffin；
Or be added to after melting solid paraffin in the cap wall of detection reaction tube, wait liquid paraffins to solidify；
Or solid paraffin is direct plungeed into the accommodating space of detection reaction tube tube body.
7. real-time fluorescence quantitative PCR detection method according to claim 5, which is characterized in that the real time fluorescent quantitative
A temperature descending section is added in the PCR condition setting of PCR after reaction, the PCR reaction solution is allowed to cool to room temperature, guarantees liquid
Paraffin layer solidification.
8. a kind of real-time fluorescence quantitative PCR detection device, including detection reaction tube, which is characterized in that further include be placed in it is described
Detect the solid paraffin and freeze-drying detection reagent in reaction tube.
9. real-time fluorescence quantitative PCR detection device according to claim 8, it is characterised in that: the detection reaction tube packet
Include tube body and pipe cap, the accommodating space of inboard wall of tube body and pipe cap inner wall cooperation closed tube；Adhere to solid on the inboard wall of tube body
Paraffin；Or the inner wall of pipe lid adheres to solid paraffin；Or solid paraffin direct plunges into the accommodating of detection reaction tube inboard wall of tube body
In space.
10. real-time fluorescence quantitative PCR detection device according to claim 8, it is characterised in that:
The freeze-drying detection reagent is that real-time fluorescence quantitative PCR detection reagent adds protective agent to be lyophilized, and the real-time fluorescence is fixed
Measuring PCR detection reagent includes taq enzyme, buffer, primer, probe, dNTP.
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| JP2021029150A (en) * | 2019-08-21 | 2021-03-01 | ヤマトエスロン株式会社 | PCR container |
| CN113801922A (en) * | 2021-09-13 | 2021-12-17 | 青岛农业大学 | On-site rapid high-sensitivity differential diagnosis kit for porcine diarrhea virus pathogens and use method thereof |
| WO2022135616A1 (en) * | 2020-12-23 | 2022-06-30 | 广州达安基因股份有限公司 | Method for separating nucleic acid amplification system, and separator |
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