CN106442454A - Quick gene amplification detection device and quick gene amplification detection method based on fluorescent quantitation - Google Patents
Quick gene amplification detection device and quick gene amplification detection method based on fluorescent quantitation Download PDFInfo
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- CN106442454A CN106442454A CN201611023872.5A CN201611023872A CN106442454A CN 106442454 A CN106442454 A CN 106442454A CN 201611023872 A CN201611023872 A CN 201611023872A CN 106442454 A CN106442454 A CN 106442454A
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- 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
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Abstract
The invention discloses a quick gene amplification detection device and a quick gene amplification detection method based on fluorescent quantitation. The detection device comprises a PCR (polymerase chain reaction) test tube and a temperature control module. The detection device is characterized by further comprising heat-conducting silica gel, a fluorescent detection module, a first transmission mechanism, a first driving mechanism, a connection mechanism, a second transmission mechanism and a second driving mechanism. The problems such as long amplification time, poor temperature uniformity of sample liquor and low fluorescent collection efficiency in the prior art are solved, and temperature control requirements of the gene amplification detection device are reduced. The quick gene amplification detection device and the quick gene amplification detection method based on fluorescent quantitation have the advantages of high sample liquor temperature conversion speed, high uniformity of temperatures among samples, high fluorescent signal collection efficiency, simplicity in temperature control, reliability, low energy consumption and the like.
Description
Technical field
The present invention relates to biological and medical field, particularly a kind of fluorescent quantitation gene rapid amplifying detection means and amplification
Detection method.
Background technology
(Polymerase Chain Reaction, hereinafter referred to as PCR) is a kind of important base for polymerase chain reaction
Because of external rapid amplifying detection technique.The reproduction process of the biological internal DNA of round pcr simulation, under suitable temperature conditionss, profit
With raw materials such as the template required for amplification, primer, polymerases, target dna or RNA fragment is made to prolong through degeneration, annealing and polymerization
Stretch the continuous circulation of three processes, obtain several times to millions of times of target dna or RNA fragment of amplification.Quantitative fluorescent PCR skill
Art is exactly to add specific fluorophor in the course of reaction of PCR, and the fluorescence signal by sample in collection amplification procedure is strong
Degree, obtains the content of target dna or RNA in sample.Fluorescent quantitative PCR technique achieve to micro target dna in sample or
The rapid, high volume amplification of RNA fragment and detection, have important using value in biology and medical domain.
In quick augmentation detection outside genosome, the control of sample temperature and the detection of sample fluorescence signal are two big cores
Technology.Sample temperature regulation and control are typically realized by metal derby, meet target gene sample body by the temperature changing metal derby
The requirement to temperature change for the outer amplification.
Formerly technology " fluorescence quantitative PCR detection system based on bottom scan detection " (Chinese invention patent, Granted publication
Number：CN101328503A) a kind of fluorescence quantitative PCR detection system based on bottom scan detection, temperature in this invention are proposed
Control device includes metal derby, semiconductor cooler and fan etc..In this invention, the temperature of sample solution is by semiconductor cooler
Control, to the detection of sample solution fluorescence signal in test tube by bear or lay optical fiber and realize.This
Bright technical scheme has the following disadvantages：
First, proliferation time is long.This invention only carries out temperature control with a metal derby, when need change sample solution temperature
When spending, it is necessary for changing the temperature of metal derby, and the temperature changing metal derby generally requires cost longer time, therefore sample
Amplification needs longer time.
Second, the poor temperature uniformity of the sample solution of diverse location.In this invention, the temperature of sample solution is by quasiconductor
Refrigerator controls, and semiconductor cooler is typically only placed in the lower center position of metal derby, therefore in heating or refrigeration
There is a problem of that middle strong and edge is weak, thus leading to meso sample solution inconsistent with the temperature of edge sample solution, entering
And the inconsistent of meso sample and the amplification efficiency of edge sample can be caused；
3rd, sample solution fluorescent collecting efficiency is low.If this invention is by the way of directly punching in bottom, can lead to examine
The sensor surveying fluorescence signal cannot be close to the sample in test tube, thus reducing exciting and collecting efficiency of fluorescence signal；As
By the way of optical fiber, fluorescence signal can be limited fruit by optical fiber clear aperature, also reduces exciting and gathering of fluorescence signal
Efficiency.
Content of the invention
The purpose of the present invention is to propose to a kind of fluorescent quantitation gene rapid amplifying detection means and amplification detection method, this
The bright amplification required time length not only solving formerly technology presence, sample solution poor temperature uniformity, fluorescent collecting efficiency are low
The problems such as, also reduce the requirement to temperature control algorithm for the gene amplification detection means.This invention has sample solution temperature and turns
Throw-over degree is fast, sample room temperature homogeneity is good, fluorescence signal acquisition efficiency high, temperature control are simple, reliability, energy consumption are low excellent
Point.
The technical solution of the present invention is as follows：
A kind of fluorescent quantitation gene rapid amplifying detection means, including PCR test tube, temperature control modules, its feature is
Also have heat conductive silica gel, fluoroscopic examination module, the first connecting gear, the first drive mechanism, bindiny mechanism, the second connecting gear and the
Two drive mechanisms, described temperature control modules include more than one isothermal block, between each two isothermal block by heat insulation layer every
Open and be arranged in order composition, described heat conductive silica gel is arranged on the outside of described PCR test tube, described temperature control modules
Isothermal block is placed on around described heat conductive silica gel；The top of described PCR test tube is fixed on by described bindiny mechanism
On the second described connecting gear, under the driving of the second drive mechanism, the second described connecting gear drives described PCR examination
Pipe moves in the vertical direction；Described fluoroscopic examination module is placed in the described top surface of PCR test tube, bottom surface or side, described
Fluoroscopic examination module is constituted and is fixed on the first connecting gear by excitation source, detection light path, photoelectric detection unit, the first drive
Motivation structure drives the first connecting gear to drive described fluoroscopic examination block motion.
The first described connecting gear is screw mandrel or conveyer belt.
Described PCR test tube is single test tube, single platoon test tube or multiple rows of test tube.
Sample solution is positioned in described PCR test tube, places all in same one-time detection in the PCR test tube of different hole positions
The consistent identical sample solution of Thermal cycling conditions needed for sample amplification or different sample solutions.
The height of described heat conductive silica gel is more than sample solution in the described invisible spectro height of PCR it is ensured that sample can
It is obtained described heat conductive silica gel to coat completely.
One PCR test tube, two PCR test tubes, multiple PCR test tube or whole PCR test tube correspond to a temperature control mould
Block.
Described fluoroscopic examination module includes more than one fluorescence detection device, and each fluorescence detection device detects one
The fluorescence of above launch wavelength, described fluoroscopic examination module can detect and be placed on different samples in described PCR test tube
The identical or different fluorescence that solution sends.
The structure of described fluoroscopic examination module is：
Using an excitation source, detection light path, non-image light electric transducer constitute be placed in all PCR test tubes 1 top surface,
Bottom surface or lateral location, described non-image light electric transducer includes photodiode, light cell, photocell, photomultiplier tube,
Described point excitation source once irradiating simultaneously excites a PCR sample solution, and described non-image light electric transducer is realized to one
Sample solution fluorescence signal in individual PCR test tube is detected；
Or the top being placed in all PCR test tubes 1 is constituted using face excitation source, detection light path dough-making powder array image sensor
Face, bottom surface or lateral location, the described disposable full illumination of face excitation source simultaneously excites all of PCR sample solution simultaneously,
Described array image sensor detects to the sample solution fluorescence signal in all PCR test tubes simultaneously.
Using the fluoroscopic examination module more than one described, it is arranged respectively at top surface, bottom surface or the side of all PCR test tubes
Face, realizes the fluorescence signal of the sample solution in corresponding PCR test tube is detected.
Side target dna or RNA being expanded and being detected using above-mentioned fluorescent quantitation gene rapid amplifying detection means
Method, comprises the steps：
1) the required sample solution of amplification is added described PCR test tube；
2) number of specified temp point according to needed for target dna or RNA amplification and numerical value, select and arrange corresponding temperature
Degree control module, determines amplification cycles number of times；
3) the second driving machine drives the second connecting gear to drive described PCR test tube to move to needed for DNA or RNA amplification
The corresponding isothermal block position of first temperature spot, makes the temperature of the sample solution in PCR test tube reach the first described temperature value,
Stop gene first proliferation time again, then the second drive mechanism drives the second connecting gear to drive described PCR test tube to move
To the corresponding isothermal block position of second temperature point needed for DNA or RNA amplification, the temperature of the sample solution in PCR test tube is made to reach
Described second temperature value, then stop gene second proliferation time, then the second drive mechanism drives the second connecting gear to drive
Described PCR test tube moves to next temperature spot corresponding isothermal block position needed for DNA or RNA amplification, makes in PCR test tube
The temperature of sample solution reaches next described temperature value, then stops next proliferation time of gene, until complete target dna or
All temperature values needed for the RNA amplification cycle and corresponding proliferation time；
4) the second driving machine described in drives the second connecting gear to drive described PCR test tube to move to described temperature control
Carry out fluorescence signal detection outside molding block：
If adopting the described fluoroscopic examination module of non-image light electric transducer, described fluoroscopic examination mould in detection means
Block is fixed on the first connecting gear, and the first drive mechanism drives the first connecting gear to drive described fluoroscopic examination module fortune
Dynamic, point excitation source excites a PCR sample solution, and described non-image light electric transducer is realized in a PCR test tube
Sample solution fluorescence signal is detected, is done step-by-step and the sample solution fluorescence signal in all PCR test tubes is detected；
If adopt imageing sensor in detection means, the face excitation source of described fluoroscopic examination module is disposably whole
Irradiate and excite all of PCR sample solution simultaneously, described array image sensor is simultaneously to the sample in all PCR test tubes
Solution fluorescence signal is detected；
5) repeat step 3), 4), complete another amplification cycles and fluorescence signal detection, until complete last amplification
Circulation and fluorescence signal detection；
6) utilize above-mentioned measurement result to draw the amplification curve of the fluorescence signal of sample solution, obtain by existing analysis method
Content to sample solution target dna or RNA.
Compared with formerly technology, the present invention has the following technical effect that：
First, amplification rate is fast.Formerly in technology when needing to change sample solution temperature, it is necessary for changing metal derby
Temperature, and the temperature changing metal derby needs to spend longer time.The temperature of such as metal derby rises to 95 degree again from 60 degree
Dropping to 60 degree needs one minute about, completes 40 cyclic amplifications and generally requires 40 minutes about.And in the present invention, described
Temperature control modules in comprise the isothermal block of multiple different temperatures, and the temperature of each isothermal block is needed for gene amplification
Specified temp.Sample solution to complete temperature transition by mobile between the isothermal block of different temperatures, need not repeatedly heat and
Refrigerated constant temperature block, has therefore saved the most of the time required for heating and cooling, so the present invention has the advantages that amplification rate is fast.
Second, the uniformity of the sample solution temperature of diverse location is good.It is in same amplification when needing control all samples
During temperature, formerly technology to control sample temperature using the lower center position mounting semiconductor refrigerator in metal derby, therefore
Can there is a problem of that in heating or refrigeration middle strong and edge is weak, thus leading to meso sample solution and edge sample solution
Temperature has the temperature contrast more than 0.3 degree.And the temperature to the sample solution in single PCR test tube can be realized in the present invention
It is controlled, the difference of the temperature between the sample solution of diverse location can be eliminated, the temperature contrast between all samples is less than 0.2
Degree, improves the uniformity of sample solution temperature.
3rd, fluorescence detection efficiency is high.Formerly technology fluoroscopic examination adopts bear or using the side installing optical fiber
Formula.If by the way of directly punching in bottom, can lead to detect fluorescence signal sensor cannot close to the sample in test tube,
Thus reducing exciting and collecting efficiency of fluorescence signal；If by the way of optical fiber, fluorescence signal can be subject to optical fiber light hole
The restriction in footpath, also reduces exciting and collecting efficiency of fluorescence signal.The present invention to control sample using isothermal block and heat conductive silica gel
The temperature of product solution, when needing detection sample solution fluorescence, drives PCR test tube to move by the second drive mechanism, makes in PCR
Sample solution moves to outside temperature control modules, facilitates the exciting and detecting to sample solution fluorescence of fluoroscopic examination module, because
This improves the fluorescence detection efficiency of sample solution.
4th, temperature control is simple.Formerly in technology when needing to change sample solution temperature, it is necessary for changing metal derby
Temperature, in order to meet the fast two high aspect demands of temperature-controlled precision simultaneously of warming and cooling rate, temperature control algorithm needs to examine simultaneously
Consider dynamic performance index and steady-state behaviour index.And in temperature control modules described in the present invention, comprise multiple different temperatures
The isothermal block of point, and the temperature of each isothermal block is the specified temp needed for gene amplification, and sample solution passes through in difference
Move between the isothermal block of temperature to complete temperature transition, temperature control only needs to consider Index For Steady-state.Therefore the present invention has temperature
Degree controls simple advantage.
5th, power consumption is few.Formerly in technology when needing to change sample solution temperature, it is necessary for changing the temperature of metal derby
Degree, and the quick gentle temperature-fall period of liter repeatedly needs to consume big energy.And in heretofore described temperature control modules
Comprise the isothermal block of multiple different temperature points, and the temperature of each isothermal block is the specified temp needed for gene amplification, sample
Product solution by the isothermal block in different temperatures between move to complete temperature transition, need not repeatedly heat or freeze, if consumption
Little energy is maintaining the temperature constant of described isothermal block.The temperature control system power consumption of the therefore present invention is few.
Brief description
Fig. 1 is the single PCR test tube schematic diagram of fluorescent quantitation gene rapid amplifying detection means of the present invention.
Fig. 2 is the multiple PCR test tube schematic diagram of fluorescent quantitation gene rapid amplifying detection means of the present invention.
Fig. 3 is in schematic diagram during the first isothermal block for inventive samples solution.
Fig. 4 is in schematic diagram during the second isothermal block for inventive samples solution.
Fig. 5 is the embodiment of the present invention one schematic diagram.
Fig. 6 is the embodiment of the present invention two schematic diagram.
Fig. 7 is the embodiment of the present invention three schematic diagram.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and examples, but should not limit the protection model of the present invention with this
Enclose.One skilled in the art would recognize that present invention encompasses potentially included in Claims scope is all alternative
Scheme, improvement project and equivalents.
Refer to Fig. 1-2, Fig. 1 is the single PCR test tube schematic diagram of fluorescent quantitation gene rapid amplifying detection means of the present invention,
Fig. 2 is the multiple PCR test tube schematic diagram of fluorescent quantitation gene rapid amplifying detection means of the present invention.Refering to Fig. 1, fluorescence of the present invention is fixed
Amount gene rapid amplifying detection means, including PCR test tube 1, heat conductive silica gel 2, temperature control modules 3, fluoroscopic examination module 4, the
One connecting gear 5, the first drive mechanism 6, bindiny mechanism 7, the second connecting gear 8, the second drive mechanism 9.Temperature control modules 3
It is made up of the first isothermal block 301, heat insulation layer 302 and the second isothermal block 303.The position relationship of above-mentioned part is as follows：
Place sample solution in described PCR test tube 1, can place in same one-time detection in the PCR test tube 1 of different hole positions
The consistent identical sample solution of the required Thermal cycling conditions of all samples amplification or different sample solutions, described
PCR test tube 1 is single test tube, single platoon test tube or multiple rows of test tube, and wherein Fig. 1 is single PCR test tube；Fig. 2 is single platoon PCR examination
Pipe.
Described PCR test tube 1 is fixed on the second described connecting gear 8 by described bindiny mechanism 7, by described
Second drive mechanism 9 drives the second described connecting gear 8 to drive described PCR test tube 1 mobile.
Described PCR test tube 1 can move to the outside of described temperature control modules 3.
Described heat conductive silica gel 2 is placed on the outside of described PCR test tube 1, the constant temperature in described temperature control modules 3
Block is placed on described heat conductive silica gel 2 around.
Described heat conductive silica gel 2 againsts described PCR test tube 1, now described heat conductive silica gel 2 and described PCR test tube 1
Sufficient heat transfer can be carried out it is ensured that in test tube the temperature of sample solution and the temperature of heat conductive silica gel identical.Described thermal conductive silicon
The height of glue 2 is more than sample solution in the described invisible spectro height of PCR it is ensured that sample can completely be coated.
Described heat conductive silica gel 2 is located between the isothermal block in described PCR test tube 1 and described temperature control modules 3,
Described heat conductive silica gel 2 is attached to the outer wall of described PCR test tube 1, passes through between described isothermal block and described PCR test tube 1
Described heat conductive silica gel 2 realizes heat transfer.
Each described PCR test tube 1 correspond to one described in temperature control modules 3 or two, multiple or
All PCR test tube corresponds to temperature control modules 3.
Described temperature control modules 3 comprise more than one isothermal block, are adiabatic layer between the isothermal block of different temperatures
Separate, prevent from occurring heat exchange between the isothermal block of different temperatures.
Described PCR test tube 1 can move between the isothermal block of different temperatures.Temperature setting by described isothermal block is
Required a kind of, the two kinds or more of specific temperature values of target gene amplification, described PCR test tube 1 is in the perseverance of different temperatures
Move back and forth between warm block, realize the temperature cycles needed for target gene rapid amplifying.
The number of described isothermal block is consistent with the quantity of the specified temp completing needed for gene amplification.
Refering to Fig. 2, described fluoroscopic examination module 4 is placed on the bottom of described PCR test tube 1, described fluoroscopic examination
The photodetector unit of module 4 adopts imageing sensor or non-image light electric transducer, and described non-image light electric transducer includes
Photodiode, light cell, photocell, photomultiplier tube.
Using the fluoroscopic examination module 4 of non-image light electric transducer, including excitation source, detection light path, non-image photoelectricity
Sensor constitutes top surface, bottom surface or the lateral location being placed in all PCR test tubes 1, and described point excitation source once irradiating is simultaneously
Excite a PCR sample solution, described non-image light electric transducer is realized to the sample solution fluorescence letter in a PCR test tube
Number detected.Described fluoroscopic examination module 4 is fixed on the first connecting gear 5, and the first drive mechanism 6 drives the first transmission
Mechanism 5 drives described fluoroscopic examination module 4 to move, and realizes the sample solution fluorescence signal in all PCR test tubes is examined
Survey.
Face excitation source, detection light path dough-making powder system of battle formations picture is adopted to pass using the fluoroscopic examination module 4 of image photoelectric sensor
Sensor constitutes top surface, bottom surface or the lateral location being placed in all PCR test tubes 1, and described face excitation source disposably all shines
Penetrate and excite simultaneously all of PCR sample solution, described array image sensor is simultaneously molten to the sample in all PCR test tubes
Liquid fluorescence signal is detected.
In the present invention, it would however also be possible to employ more than one described fluoroscopic examination module 4, it is arranged respectively at all PCR examinations
The top surface of pipe 1, bottom surface or side, realize the fluorescence signal of the sample solution in corresponding PCR test tube is detected.
Using fluorescent quantitation gene rapid amplifying detection means of the present invention, target dna or RNA are carried out with the side of augmentation detection
Method, comprises the steps：
1) the required sample solution of amplification is added described PCR test tube；
2) number of specified temp point according to needed for target dna or RNA amplification and numerical value, select and arrange corresponding temperature
Degree control module 3；
3) the second driving machine drives the second connecting gear to drive described PCR test tube to move to needed for DNA or RNA amplification
The corresponding isothermal block position of first temperature spot, makes the temperature of the sample solution in PCR test tube reach the first described temperature value,
Stop gene first proliferation time again, then the second drive mechanism drives the second connecting gear to drive described PCR test tube to move
To the corresponding isothermal block position of second temperature point needed for DNA or RNA amplification, the temperature of the sample solution in PCR test tube is made to reach
Described second temperature value, then stop gene second proliferation time, then the second drive mechanism drives the second connecting gear to drive
Described PCR test tube moves to next temperature spot corresponding isothermal block position needed for DNA or RNA amplification, makes in PCR test tube
The temperature of sample solution reaches next described temperature value, then stops next proliferation time of gene, until complete target dna or
All temperature values needed for the RNA amplification cycle and corresponding proliferation time.
4) the second driving machine described in drives the second connecting gear to drive described PCR test tube to move to described temperature control
Outside molding block 3.
If adopting the described fluoroscopic examination module of non-image light electric transducer, described fluoroscopic examination mould in detection means
Block 4 is fixed on the first connecting gear 5, and the first drive mechanism 6 drives the first connecting gear 5 to drive described fluoroscopic examination module
4 motions, realize the sample solution fluorescence signal in all PCR test tubes is detected.
If adopting the described fluoroscopic examination module of image sensor, the fluorescence of described employing imageing sensor in detection means
The disposable full illumination of face excitation source in detection module 4 simultaneously excites all of PCR sample solution simultaneously, the described face system of battle formations
As sensor detects to the sample solution fluorescence signal in all PCR test tubes simultaneously.
5) repeat step 3,4), until completing last amplification cycles and fluorescence signal detection.
6) utilize above-mentioned measurement result to draw the amplification curve of the fluorescence signal of sample solution, obtain by existing analysis method
Content to sample solution target dna or RNA.
Embodiment one
Refer to Fig. 3-5, Fig. 5 is fluorescent quantitation gene rapid amplifying detection means of the present invention and detection method embodiment one
Schematic diagram.
The present embodiment includes multiple PCR test tubes 1, heat conductive silica gel 2, temperature control modules 3, fluoroscopic examination module 4, first biography
Send mechanism 5, the first drive mechanism 6, bindiny mechanism 7, the second connecting gear 8, the second drive mechanism 9.Described temperature control mould
Block 3 includes the first isothermal block 301, heat insulation layer 302 and the second isothermal block 303.In the present embodiment, described in sample solution is placed on
Multiple PCR test tubes 1 in, described fluoroscopic examination module 4 is placed on the bottom of described PCR test tube 1.Described PCR test tube 1
Surrounded by temperature control modules 3, a fluoroscopic examination module 4.Described temperature control modules 3 include the first isothermal block
301 and second isothermal block 303, the first isothermal block 301 is high temperature constant temperature block, and the second isothermal block 303 is cryogenic thermostat block.Described
Separated by described heat insulation layer 302 between first isothermal block 301 and the second isothermal block 303, prevent the first described isothermal block 301
There is heat exchange and the second described isothermal block 303 between.Described the first isothermal block 301 keeping temperature is constant, and its temperature
Angle value is the high-temperature needed for gene amplification；Described the second isothermal block 303 keeping temperature is constant, and its temperature value is gene
The required low temperature of amplification.Refering to Fig. 3-5, described PCR test tube 1 is permanent in the first described isothermal block 301 and described second
Move between warm block 303 and to change the temperature of sample solution, the perseverance in described PCR test tube 1 and described temperature control modules 3
The heat exchange of warm block is to be completed by described heat conductive silica gel 2.The fluorescence of the employing non-image light electric transducer described in 1
Detection module 4 is placed in the bottom surface of PCR test tube 1.Described fluoroscopic examination module 4 is fixed on the first connecting gear 5, the first driving
Mechanism 6 drives the first connecting gear 5 to drive described fluoroscopic examination module 4 to move, and realizes molten to the sample in all PCR test tubes
Liquid fluorescence signal is detected, by analyzing the change curve of the fluorescence signal of each circulation of sample solution, obtains mesh in sample
The content of mark DNA or RNA.
Embodiment two
Refer to Fig. 6, Fig. 6 is the schematic diagram of fluorescent quantitation gene rapid amplifying detection means embodiment two of the present invention.This
Embodiment includes 8 PCR test tubes 1, heat conductive silica gel 2, temperature control modules 3, fluoroscopic examination module 4, the first connecting gear 5, institute
The first drive mechanism 6 of stating, bindiny mechanism 7, the second connecting gear 8, the second drive mechanism 9.Described temperature control modules 3 wrap
Include the first isothermal block 301, heat insulation layer 302 and the second isothermal block 303.Described fluoroscopic examination module 4 is placed on described PCR examination
The side of pipe 1.All PCR test tubes 1 are surrounded by same described temperature control modules 3, are furnished with 8 fluoroscopic examination modules 4.Institute
It is provided with the first isothermal block 301 and the second isothermal block 303, the first described isothermal block 301 is height in the temperature control modules 3 stated
Warm isothermal block, the second described isothermal block 303 is cryogenic thermostat block.By institute between first isothermal block 301 and the second isothermal block 303
The heat insulation layer 302 stated separates, and prevents from occurring heat exchange between the first isothermal block 301 and the second isothermal block 303.First isothermal block
301 keeping temperatures are constant, and its temperature value is the high-temperature needed for gene amplification；Second isothermal block 303 keeping temperature is constant,
And its temperature value is the low temperature needed for gene amplification.Refering to Fig. 3-4, described PCR test tube 1 is in the first isothermal block 301 He
Move between second isothermal block 303 and to change the temperature of sample solution, described PCR test tube 1 and described temperature control modules 3
In the heat exchange of isothermal block be to be completed by described heat conductive silica gel 2.The fluoroscopic examination mould of 8 non-image light electric transducers
Block 4 is arranged respectively at the side of 8 PCR test tubes 1.Described fluoroscopic examination module 4 is fixed on the first connecting gear 5, and first
Drive mechanism 6 drives the first connecting gear 5 to drive described fluoroscopic examination module 4 to move, and realizes to the sample in all PCR test tubes
Product solution fluorescence signal is detected, by analyzing the change curve of the fluorescence signal of each circulation of sample solution, obtains sample
Middle target dna or the content of RNA.
Embodiment three
Refer to Fig. 7, Fig. 7 is the schematic diagram of fluorescent quantitation gene rapid amplifying detection means embodiment three of the present invention.This
Embodiment include multiple PCR test tubes 1, heat conductive silica gel 2,3, fluoroscopic examination module 4 of temperature control modules, bindiny mechanism 7,
Two connecting gears 8, the second drive mechanism 9.Described temperature control modules 3 include the first isothermal block 301, heat insulation layer 302 and
Two isothermal blocks 303.In the present embodiment, described fluoroscopic examination module 4 is placed on the top of described PCR test tube 1.All
Isothermal block in temperature control modules 3 described in for the PCR test tube 1 surrounds, the fluoroscopic examination module 4 described in.Described
Temperature control modules 3 in be provided with the first described isothermal block 301 be high temperature constant temperature block, the second described isothermal block 303 is
Cryogenic thermostat block.Separated by described heat insulation layer 302 between first isothermal block 301 and the second isothermal block 303.Prevent the first constant temperature
There is heat exchange between block 301 and the second isothermal block 303.First isothermal block 301 keeping temperature is constant, and its temperature value is base
High-temperature needed for gene-amplification；Second isothermal block 303 keeping temperature is constant, and its temperature value is the low temperature needed for gene amplification
Degree.Described PCR test tube 1 moves to change the temperature of sample solution between the first isothermal block 301 and the second isothermal block 303,
The heat exchange of the isothermal block in described PCR test tube 1 and described temperature control modules 3 be by described heat conductive silica gel 2
Complete.
Described fluoroscopic examination module 4 is constituted and is placed in institute by face excitation source, detection light path dough-making powder array image sensor
There is the top surface of PCR test tube 1, the described disposable full illumination of face excitation source simultaneously excites all of PCR sample solution, institute simultaneously
The array image sensor stated detects to the sample solution fluorescence signal in all PCR test tubes simultaneously, by analyzing sample
The change curve of the fluorescence signal of each circulation of solution, obtains the content of target dna or RNA in sample.
Experiment shows, it is equal that the present invention not only solves formerly the amplification required time length that technology exists, sample solution temperature
Even property is poor, the low problem of fluorescent collecting efficiency, also reduces the requirement to temperature control algorithm for the gene amplification detection means.This
Bright have that sample solution temperature transition speed is fast, sample room temperature homogeneity is good, the letter of fluorescence signal acquisition efficiency high, temperature control
List, reliability, low power consumption and other advantages.
Claims (10)
1. a kind of fluorescent quantitation gene rapid amplifying detection means, including PCR test tube (1), temperature control modules (3), its feature
It is also heat conductive silica gel (2), fluoroscopic examination module (4), the first connecting gear (5), the first drive mechanism (6), bindiny mechanism
(7), the second connecting gear (8) and the second drive mechanism (9), described temperature control modules (3) include more than one constant temperature
Block, is separated and be arranged in order by heat insulation layer between each two isothermal block and constitute, and described heat conductive silica gel (2) is arranged on described
The outside of PCR test tube (1), the isothermal block of described temperature control modules (3) is placed on described heat conductive silica gel (2) around；
The top of described PCR test tube (1) is fixed on described the second connecting gear (8) by described bindiny mechanism (7), the
Lower described second connecting gear (8) that drives of two drive mechanisms (9) drives described PCR test tube (1) to move in the vertical direction；
Described fluoroscopic examination module (4) is placed in the described top surface of PCR test tube (1), bottom surface or side, described fluoroscopic examination module
(4) constituted and be fixed on the first connecting gear (5), the first drive mechanism by excitation source, detection light path, photoelectric detection unit
(6) the first connecting gear (5) is driven to drive described fluoroscopic examination module (4) motion.
2. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described first biography
Mechanism (5) is sent to be screw mandrel or conveyer belt.
3. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described PCR test tube
(1) it is single test tube, single platoon test tube or multiple rows of test tube.
4. fluorescent quantitation gene rapid amplifying detection means according to claim 1, its feature sample solution is positioned over institute
In the PCR test tube (1) stated, in the PCR test tube (1) of different hole positions, place the temperature required with all samples amplification in one-time detection
The consistent identical sample solution of cycling condition or different sample solutions.
5. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described thermal conductive silicon
The height of glue (2) is more than sample solution in the described invisible spectro height of PCR it is ensured that sample can be obtained described thermal conductive silicon
Glue (2) coats completely.
6. fluorescent quantitation gene rapid amplifying detection means according to claim 1 it is characterised in that PCR test tube,
Two PCR test tubes, multiple PCR test tube or whole PCR test tube correspond to a temperature control modules (3).
7. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described fluorescence is examined
Survey module (4) and include more than one fluorescence detection device, each fluorescence detection device detects the glimmering of more than one launch wavelength
Light, described fluoroscopic examination module can detect be placed on that different sample solution in described PCR test tube (1) sends identical
Or different fluorescence.
8. fluorescent quantitation gene rapid amplifying detection means according to claim 1 is it is characterised in that described fluorescence is examined
Survey module (4) structure be：
Top surface, the bottom being placed in all PCR test tubes (1) is constituted using an excitation source, detection light path, non-image light electric transducer
Face or lateral location, described non-image light electric transducer includes photodiode, light cell, photocell, photomultiplier tube, institute
The point excitation source once irradiating stated simultaneously excites a PCR sample solution, and described non-image light electric transducer is realized to one
Sample solution fluorescence signal in PCR test tube is detected；
Or using face excitation source, detection light path dough-making powder array image sensor constitute be placed in all PCR test tubes (1) top surface,
Bottom surface or lateral location, the described disposable full illumination of face excitation source simultaneously excites all of PCR sample solution simultaneously, described
Array image sensor the sample solution fluorescence signal in all PCR test tubes is detected simultaneously.
9. fluorescent quantitation gene rapid amplifying detection means according to claim 7 is it is characterised in that adopt more than one
Described fluoroscopic examination module (4), is arranged respectively at top surface, bottom surface or the side of all PCR test tubes (1), realizes to corresponding
The fluorescence signal of the sample solution in PCR test tube is detected.
10. using the fluorescent quantitation gene rapid amplifying detection means described in claim 1, target dna or RNA are expanded
With the method detecting it is characterised in that comprising the steps：
1) the required sample solution of amplification is added described PCR test tube；
2) number of specified temp point according to needed for target dna or RNA amplification and numerical value, select and arrange corresponding temperature control
Molding block (3), determines amplification cycles number of times；
3) the second driving machine drives the second connecting gear to drive described PCR test tube to move to needed for DNA or RNA amplification first
The corresponding isothermal block position of individual temperature spot, makes the temperature of the sample solution in PCR test tube reach the first described temperature value, then stops
Stay gene first proliferation time, then the second drive mechanism drives the second connecting gear to drive described PCR test tube to move to DNA
Or the corresponding isothermal block position of second temperature point needed for RNA amplification, make described in the temperature arrival of sample solution in PCR test tube
Second temperature value, then stop gene second proliferation time, then the second drive mechanism drives described in the second connecting gear drive
PCR test tube moves to next temperature spot corresponding isothermal block position needed for DNA or RNA amplification, makes the sample in PCR test tube molten
The temperature of liquid reaches next described temperature value, then stops next proliferation time of gene, until completing target dna or RNA amplification
All temperature values needed for cycle and corresponding proliferation time；
4) the second driving machine described in drives the second connecting gear to drive described PCR test tube to move to described temperature control mould
Carry out fluorescence signal detection outside block (3)：
If adopting the described fluoroscopic examination module of non-image light electric transducer, described fluoroscopic examination module in detection means
(4) it is fixed on the first connecting gear (5), the first drive mechanism (6) drives the first connecting gear (5) to drive described fluorescence inspection
Survey module (4) motion, point excitation source excites a PCR sample solution, and described non-image light electric transducer is realized to one
Sample solution fluorescence signal in PCR test tube is detected, is done step-by-step to the sample solution fluorescence signal in all PCR test tubes
Detected；
If adopt imageing sensor in detection means, the face excitation source of described fluoroscopic examination module (4) is disposably whole
Irradiate and excite all of PCR sample solution simultaneously, described array image sensor is simultaneously to the sample in all PCR test tubes
Solution fluorescence signal is detected；
5) repeat step 3,4), complete another amplification cycles and fluorescence signal detection, until completing last amplification cycles
With fluorescence signal detection；
6) utilize above-mentioned measurement result to draw the amplification curve of the fluorescence signal of sample solution, obtain sample by existing analysis method
Product solution target dna or the content of RNA.
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Application publication date: 20170222 |