CN107356576A - A kind of device and method for real-time fluorescence quantitative PCR - Google Patents
A kind of device and method for real-time fluorescence quantitative PCR Download PDFInfo
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- CN107356576A CN107356576A CN201710600147.8A CN201710600147A CN107356576A CN 107356576 A CN107356576 A CN 107356576A CN 201710600147 A CN201710600147 A CN 201710600147A CN 107356576 A CN107356576 A CN 107356576A
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- thermally conductive
- conductive sheet
- microchannel
- temperature
- shuttle
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention provides a kind of device and method for real-time fluorescence quantitative PCR, the device includes pressurizer (1), shuttle (3), microchannel (4), thermally conductive sheet (5) and thermostat (6), multiple thermally conductive sheets (5) are located on thermostat (6), thermostat (6) heating thermally conductive sheet (5) simultaneously makes the Temperature Distribution on the surface of thermally conductive sheet (5) include high-temperature portion and low temperature portion, the temperature of high-temperature portion includes the high-temperature denatured temperature of PCR amplifications, and the temperature in low temperature portion includes the low temperature renaturation temperature of PCR amplifications；Circularly pass through high-temperature portion and the low temperature portion of thermally conductive sheet (5) with preset times with the one-to-one microchannel (4) of thermally conductive sheet (5)；Pressurizer (1) is used for inflating pressure in the system of multiple shuttles (3) and multiple microchannels (4) composition.Device volume provided by the invention is small, cost is low, and easy to operate.
Description
Technical field
The present invention relates to PCR quantitative techniques field, more particularly to a kind of device for real-time fluorescence quantitative PCR and side
Method.
Background technology
PCR (PCR) is a kind of Protocols in Molecular Biology for being used to amplify the specific DNA fragmentation of amplification, real
When quantitative fluorescent PCR principle be：After the probe for being marked with fluorescein is mixed with template DNA, template DNA is completed high temperature and become
Property, low temperature renaturation, the thermal cycle of thermophilic extension, with the increase of cycle-index, the target gene fragment being amplified exponentially is advised
Rule increases, by detect in real time it is corresponding with amplification and the fluorescence signal intensity changed, try to achieve Ct value (fluorescence signal intensities
Reach the period undergone during given threshold), while compared using the standard items of several known template concentration, you can draw
The copy number of sample target gene to be measured.
At present, carry out real-time fluorescence quantitative PCR to usually require to use complicated temperature control system to realize thermal cycle, temperature
Spend that the universal volume of control system is larger, cost is higher, complex operation, therefore, how to provide that a kind of small volume, cost be low, easy behaviour
The device for real-time fluorescence quantitative PCR made, becomes those skilled in the art's technical problem urgently to be resolved hurrily.
The content of the invention
In view of this, the invention provides a kind of device and method for real-time fluorescence quantitative PCR, the device volume
It is small, cost is low, and easy to operate.
In order to achieve the above object, the present invention provides following technical scheme：
A kind of device for real-time fluorescence quantitative PCR, including：
Thermostat；
Multiple thermally conductive sheets on the thermostat, the thermostat are used to heat the thermally conductive sheet, the thermally conductive sheet
The Temperature Distribution on surface include high-temperature portion and low temperature portion, the temperature of the high-temperature portion includes the high-temperature denatured temperature of PCR amplifications,
The temperature in the low temperature portion includes the low temperature renaturation temperature of PCR amplifications；
Multiple microchannels corresponding with the quantity of the thermally conductive sheet, the microchannel contact with the surface of the thermally conductive sheet,
And the microchannel circularly passes through high-temperature portion and the low temperature portion of the thermally conductive sheet with preset times；
Multiple shuttles corresponding with the quantity of the thermally conductive sheet, the shuttle correspond with the microchannel
Connection；
The pressurizer connected with multiple shuttles, the pressurizer are used for multiple shuttles and multiple
Inflating pressure in the system of the microchannel composition.
Preferably, in said apparatus, the preset times are 20 times~80 times.
Preferably, in said apparatus, the microchannel winding is laid on the thermally conductive sheet, and the microchannel circulates
Reciprocally pass through the upper and lower surface of the thermally conductive sheet；
The upper surface of the thermally conductive sheet is the high-temperature portion, and lower surface is the low temperature portion, or the thermally conductive sheet is upper
Surface is the low temperature portion, and lower surface is the high-temperature portion.
Preferably, in said apparatus, a part for the thermally conductive sheet is overlapped on the thermostat, and remainder is suspended in
Outside the thermostat, the microchannel is laid on the another side of relatively described thermostat of the thermally conductive sheet, and described micro-
Pipeline is circularly by the thermally conductive sheet and the thermostat part overlapped and the part being suspended in outside the thermostat.
Preferably, in said apparatus, the exit end of the microchannel is communicated with collector, is installed on the collector
There is air bleeding valve.
Preferably, in said apparatus, the shuttle is dropper.
Preferably, in said apparatus, the pressurizer is syringe.
Preferably, in said apparatus, breather valve is provided between the pressurizer and the shuttle.
A kind of method for real-time fluorescence quantitative PCR, methods described use the device as disclosed in above-mentioned any one,
Methods described is used to demarcate described device, and tool flow includes：
The different nucleic acid solution of the concentration known but concentration value of preset dose is respectively put into multiple shuttles；
Adjust the position of the shuttle, make the nucleic acid solution in the shuttle do not block the shuttle with
The connector of the microchannel, by the pressurizer into the inner chamber of the shuttle and the system of microchannel composition
Gas is filled with, the internal pressure of the system is higher than external pressure；
The position of the shuttle is adjusted again, nucleic acid solution is blocked the company of the shuttle and the microchannel
Interface, by the turned on outside of the exit end of the microchannel and the system, described in the gas push in the shuttle
Nucleic acid solution flows through the microchannel, detects fluorescence signal intensity and records core when fluorescence signal intensity reaches given threshold
The cycle-index of the microchannel corresponding to acid solution position, complete demarcation.
It can be seen from above-mentioned technical proposal, provided by the present invention in the device of real-time fluorescence quantitative PCR, thermally conductive sheet position
In on thermostat, oven heated thermally conductive sheet simultaneously makes the Temperature Distribution on the surface of thermally conductive sheet include high-temperature portion and low temperature portion, wherein,
The temperature of high-temperature portion includes the high-temperature denatured temperature of PCR amplifications, and the temperature in low temperature portion includes the low temperature renaturation temperature of PCR amplifications,
And circularly pass through high-temperature portion and the low temperature portion of thermally conductive sheet with preset times with the microchannel that the surface of thermally conductive sheet contacts, institute
To form thermal cycle spatially in microchannel, with the temperature control for being used to realize temporal thermal cycle in the prior art
System is compared, and device volume provided by the invention is smaller, cost is lower, and heating source is thermostat, only need to set single temperature
Angle value, it is more convenient to operate.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 is a kind of schematic diagram of device for real-time fluorescence quantitative PCR provided in an embodiment of the present invention；
Fig. 2 is the schematic diagram for testing the experimental provision for selecting suitable part 5；
Fig. 3 is the schematic diagram of part 4 and another arrangement form of part 5 in Fig. 1.
In figure mark for：
1st, pressurizer；2nd, breather valve；3rd, shuttle；4th, microchannel；5th, thermally conductive sheet；6th, thermostat；7th, air bleeding valve；8th, receive
Storage；9th, syringe；10th, nucleic acid solution.
Embodiment
In order to make it easy to understand, the invention will be further described below in conjunction with the accompanying drawings.
, should for a kind of schematic diagram of device for real-time fluorescence quantitative PCR provided in an embodiment of the present invention referring to Fig. 1
Device includes pressurizer 1, shuttle 3, microchannel 4, thermally conductive sheet 5 and thermostat 6.
Wherein, the quantity of thermally conductive sheet 5 is multiple that thermostat 6 is used to heat thermally conductive sheet 5, the temperature point on the surface of thermally conductive sheet 5
Cloth includes high-temperature portion and low temperature portion, and the temperature of high-temperature portion includes the high-temperature denatured temperature (such as 95 DEG C) of PCR amplifications, low temperature portion
Temperature includes the low temperature renaturation temperature (such as 65 DEG C) of PCR amplifications；
The quantity of microchannel 4 is corresponding with the quantity of thermally conductive sheet 5, and microchannel 4 contacts with the surface of thermally conductive sheet 5, and micro-pipe
Road 4 circularly passes through high-temperature portion and the low temperature portion of thermally conductive sheet 5 with preset times；
Multiple shuttles 3 are corresponded with microchannel 4 and connected；
Pressurizer 1 connects with multiple shuttles 3, and pressurizer 1 is used for 4 groups of multiple shuttles 3 and multiple microchannels
Into system in inflating pressure.
In specific practical application, the preset times of high-temperature portion and low temperature portion of the microchannel 4 Jing Guo thermally conductive sheet 5 can be 20 times
~80 times.
Fig. 1 and Fig. 3 respectively show the arrangement form of two kinds of microchannels 4 and thermally conductive sheet 5, and in Fig. 1, microchannel 4 is wound
It is laid on thermally conductive sheet 5, and microchannel 4 circularly passes through the upper and lower surface of thermally conductive sheet 5, then thermally conductive sheet 5
Upper surface is high-temperature portion, and lower surface is low temperature portion, or the upper surface of thermally conductive sheet 5 is low temperature portion, and lower surface is high-temperature portion.
And in figure 3, a part for thermally conductive sheet 5 is overlapped on thermostat 6, and remainder is suspended in outside thermostat 6, micro-pipe
Road 4 is laid on the another side of relative thermostat 6 of thermally conductive sheet 5, and microchannel 4 circularly passes through thermally conductive sheet 5 and constant temperature
The part that device 6 overlaps and the part being suspended in outside thermostat 6, then the part that thermally conductive sheet 5 overlaps with thermostat 6 is high-temperature portion,
It is low temperature portion to be suspended in the part outside thermostat 6.
As shown in figure 1, in the present embodiment, the exit end of microchannel 4 is communicated with collector 8, the row of being provided with collector 8
Air valve 7.Shuttle 3 selects dropper, and pressurizer 1 selects syringe, breather valve is provided between pressurizer 1 and shuttle 3
2。
Provided by the present invention in the device of real-time fluorescence quantitative PCR, thermally conductive sheet 5 is located on thermostat 6, thermostat 6
Heating thermally conductive sheet 5 simultaneously makes the Temperature Distribution on the surface of thermally conductive sheet 5 include high-temperature portion and low temperature portion, wherein, the temperature bag of high-temperature portion
Include the high-temperature denatured temperature of PCR amplifications, the temperature in low temperature portion includes the low temperature renaturation temperature of PCR amplifications, and with the table of thermally conductive sheet 5
The microchannel 4 of face contact circularly passes through high-temperature portion and the low temperature portion of thermally conductive sheet 5 with preset times, so in microchannel 4
Thermal cycle spatially is inside formd, compared with being used to realize the temperature control system of temporal thermal cycle in the prior art,
Device volume provided by the invention is smaller, cost is lower, and heating source is thermostat, only need to set single temperature value,
It is more convenient to operate.
Based on device provided by the present invention, the invention provides a kind of method for real-time fluorescence quantitative PCR, the party
Method is used to demarcate device, and tool flow comprises the following steps：
S1, the different nucleic acid solution of the concentration known but concentration value of preset dose is respectively put into multiple shuttles 3.
So that there are four droppers as in case of shuttle 3 shown in Fig. 1,50 are put into four droppers respectively
Microlitre original content, 10 times of diluted concentrations, the nucleic acid solution of 100 times of diluted concentrations and 1000 times of diluted concentrations.
S2, the position of shuttle 3 is adjusted, the nucleic acid solution in shuttle 3 is not blocked shuttle 3 and microchannel
4 connector, gas is filled with into the inner chamber of shuttle 3 and the system of the composition of microchannel 4 by pressurizer 1, makes system
Internal pressure is higher than external pressure.
In case of pressurizer 1 is from volume 50ml syringe, dropper is laid flat, then syringe is by 50ml positions
Put and shift 20ml positions and fixation onto, complete pressurization inflation.
S3, the position of shuttle 3 is adjusted again, make the connector of nucleic acid solution closure shuttle 3 and microchannel 4,
By the exit end of microchannel 4 and the turned on outside of system, the gas push nucleic acid solution in shuttle 3 flows through microchannel 4,
Detection fluorescence signal intensity simultaneously records microchannel 4 corresponding to nucleic acid solution position when fluorescence signal intensity reaches given threshold
Cycle-index, complete demarcation.
As shown in figure 1, four breather valves 2 of dropper front end are closed, and four droppers are placed vertically floods nucleic acid solution
No burette exit port, slowly open tail end venting valve 7, the nucleic acid solution of four various concentrations is respectively enterd microchannel 4 and carry out instead
Should, record various concentrations nucleic acid solution flows through the number of turns of microchannel 4 when producing the fluorescence signal intensity of given threshold, complete mark
It is fixed.
, can be according to the step similar with above step S1~S3 come to unknown concentration nucleic acid solution after device completes demarcation
Real-time fluorescence quantitative PCR is carried out, for example, the nucleic acid solution of 50 microlitres of unknown concentrations is put into dropper, to the core of unknown concentration
Acid solution carries out fluoroscopic examination, and the number of turns of microchannel 4 is flowed through when recording the fluorescence signal intensity that it produces given threshold, by with
Mark the number of turns corresponding to the concentration known nucleic acid solution of time recording to compare, you can determine the concentration of unknown concentration nucleic acid solution
Scope, complete to quantify unknown concentration nucleic acid solution.
It is the schematic diagram for testing the experimental provision for selecting suitable thermally conductive sheet 5, it is necessary to explanation referring to Fig. 2,
" suitable thermally conductive sheet 5 " is to refer to produce good Temperature Distribution, so as to more expeditiously carry out real-time fluorescence quantitative PCR
Thermally conductive sheet 5.Below by taking the thermally conductive sheet 5 made of copper powder and silica gel solution as an example, illustrate and tested using the experimental provision
Select the process of suitable thermally conductive sheet 5：
Copper powder is mixed from silica gel solution according to different mass ratios, is placed in vacuum chamber and coagulates after stirring
Gu the thermally conductive sheet 5 that length degree is 70mm, 30mm, 10mm is cut into after solidification, wherein, the mass ratio of copper powder can divide
Wei 20%, 40%, 60% and 80%；200 microlitres of nucleic acid solutions 10 are put into volume 50ml syringe 9, by syringe 9
Piston is placed on 50ml positions, by the access system of syringe 9 and is laid flat, and closes tail end venting valve 7.Then by syringe 9 by
50ml shifts position 20ml positions onto, and syringe 9 is placed to the outlet for making nucleic acid solution 10 enter syringe 9 vertically, slowly opened
Air bleeding valve 7, nucleic acid solution 10 is entered inside microchannel 4 and reacted.The PCR in four microchannels 4 is carried out respectively real-time
Fluoroscopic examination, by observe corresponding to which thermally conductive sheet 5 produced at first in microchannel 4 fluorescence signal for setting intensity or which
Most strong fluorescence signal is produced in microchannel 4 corresponding to thermally conductive sheet 5, the thermally conductive sheet 5 is suitable thermally conductive sheet to be selected.
The foregoing description of the disclosed embodiments, professional and technical personnel in the field are enable to realize or using the present invention.
A variety of modifications to embodiment will be apparent for those skilled in the art, as defined herein general
Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not
Embodiment illustrated herein can be restricted to, and is to fit to consistent with principles disclosed herein and features of novelty most wide
Scope.
Claims (9)
- A kind of 1. device for real-time fluorescence quantitative PCR, it is characterised in that including：Thermostat (6)；Multiple thermally conductive sheets (5) on the thermostat (6), the thermostat (6) are used to heat the thermally conductive sheet (5), institute Stating the Temperature Distribution on the surface of thermally conductive sheet (5) includes high-temperature portion and low temperature portion, and the temperature of the high-temperature portion includes the height of PCR amplifications Warm denaturation temperature, the temperature in the low temperature portion include the low temperature renaturation temperature of PCR amplifications；Multiple microchannels (4) corresponding with the quantity of the thermally conductive sheet (5), the microchannel (4) and the table of the thermally conductive sheet (5) Face contacts, and the microchannel (4) circularly passes through high-temperature portion and the low temperature portion of the thermally conductive sheet (5) with preset times；Multiple shuttles (3) corresponding with the quantity of the thermally conductive sheet (5), the shuttle (3) and the microchannel (4) Correspond connection；The pressurizer (1) connected with multiple shuttles (3), the pressurizer (1) are used for multiple shuttles (3) and multiple microchannels (4) composition system in inflating pressure.
- 2. device according to claim 1, it is characterised in that the preset times are 20 times~80 times.
- 3. device according to claim 1, it is characterised in that microchannel (4) winding is laid in the thermally conductive sheet (5) On, and the microchannel (4) circularly passes through the upper and lower surface of the thermally conductive sheet (5)；The upper surface of the thermally conductive sheet (5) is the high-temperature portion, and lower surface is the low temperature portion, or the thermally conductive sheet (5) Upper surface is the low temperature portion, and lower surface is the high-temperature portion.
- 4. device according to claim 1 a, it is characterised in that part for the thermally conductive sheet (5) is overlapped on the constant temperature On device (6), remainder is suspended in outside the thermostat (6), and the microchannel (4) is laid in the relative of the thermally conductive sheet (5) On the another side of the thermostat (6), and the microchannel (4) is circularly by the thermally conductive sheet (5) and the constant temperature The part of device (6) overlap joint and the part being suspended in outside the thermostat (6).
- 5. device according to claim 1, it is characterised in that the exit end of the microchannel (4) is communicated with collector (8) air bleeding valve (7), is installed on the collector (8).
- 6. the device according to any one in Claims 1 to 5, it is characterised in that the shuttle (3) is dropper.
- 7. device according to claim 6, it is characterised in that the pressurizer (1) is syringe.
- 8. device according to claim 6, it is characterised in that set between the pressurizer (1) and the shuttle (3) It is equipped with breather valve (2).
- A kind of 9. method for real-time fluorescence quantitative PCR, it is characterised in that methods described is used as appointed in claim 1~8 Device described in meaning one, methods described are used to demarcate described device, and tool flow includes：The different nucleic acid solution of the concentration known but concentration value of preset dose is respectively put into multiple shuttles (3)；The position of the shuttle (3) is adjusted, the nucleic acid solution in the shuttle (3) is not blocked the shuttle (3) with the connector of the microchannel (4), by the pressurizer (1) to the shuttle (3) and the microchannel (4) Gas is filled with the inner chamber of the system of composition, the internal pressure of the system is higher than external pressure；The position of the shuttle (3) is adjusted again, nucleic acid solution is blocked the shuttle (3) and the microchannel (4) connector, by the turned on outside of the exit end of the microchannel (4) and the system, in the shuttle (3) Nucleic acid solution described in gas push flows through the microchannel (4), detects fluorescence signal intensity and records fluorescence signal intensity arrival The cycle-index of the microchannel (4) corresponding to nucleic acid solution position during given threshold, complete demarcation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710600147.8A CN107356576B (en) | 2017-07-21 | 2017-07-21 | Device and method for real-time fluorescence quantitative PCR |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710600147.8A CN107356576B (en) | 2017-07-21 | 2017-07-21 | Device and method for real-time fluorescence quantitative PCR |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108875314A (en) * | 2018-05-30 | 2018-11-23 | 朱运峰 | A kind of detection method of the target gene based on epigenetics modification difference |
| CN109097455A (en) * | 2018-09-03 | 2018-12-28 | 中国科学院长春光学精密机械与物理研究所 | A kind of polymerase chain reaction system |
| CN110305785A (en) * | 2018-09-29 | 2019-10-08 | 中国科学院长春光学精密机械与物理研究所 | A kind of PCR fluorescence detection device and its detection method |
-
2017
- 2017-07-21 CN CN201710600147.8A patent/CN107356576B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108875314A (en) * | 2018-05-30 | 2018-11-23 | 朱运峰 | A kind of detection method of the target gene based on epigenetics modification difference |
| CN108875314B (en) * | 2018-05-30 | 2021-11-09 | 朱运峰 | Target gene detection method based on epigenetics modification difference |
| CN109097455A (en) * | 2018-09-03 | 2018-12-28 | 中国科学院长春光学精密机械与物理研究所 | A kind of polymerase chain reaction system |
| CN109097455B (en) * | 2018-09-03 | 2022-11-11 | 中国科学院长春光学精密机械与物理研究所 | Polymerase chain reaction system |
| CN110305785A (en) * | 2018-09-29 | 2019-10-08 | 中国科学院长春光学精密机械与物理研究所 | A kind of PCR fluorescence detection device and its detection method |
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