CN104130933B - A kind of fluorescence constant temperature PCR amplification instrument - Google Patents
A kind of fluorescence constant temperature PCR amplification instrument Download PDFInfo
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- CN104130933B CN104130933B CN201410377035.7A CN201410377035A CN104130933B CN 104130933 B CN104130933 B CN 104130933B CN 201410377035 A CN201410377035 A CN 201410377035A CN 104130933 B CN104130933 B CN 104130933B
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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
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
- B01L7/5255—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones by moving sample containers
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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
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/02—Water baths; Sand baths; Air baths
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The present invention relates to a kind of fluorescence constant temperature PCR amplification instrument, it is made up of three constant temperature water baths and a measuring cylinder, guide the sample in sample hose load plate to realize pcr amplification reaction in three constant temperature water baths by stepper-motor, ball screw, line slideway, and in measuring cylinder, carry out fluoroscopic examination after the completion of reaction.It is for the specific DNA fragmentation that increases in vitro, utilizes the change of fluorescent signal to detect the change of the amount of pcr amplification reaction final product simultaneously.
Description
Technical field:
The present invention relates to a kind of fluorescence constant temperature PCR amplification instrument, it is made up of three constant temperature water baths and a measuring cylinder, guide the sample in sample hose load plate to realize pcr amplification reaction in three constant temperature water baths by stepper-motor, ball screw, line slideway, and in measuring cylinder, carry out fluoroscopic examination after the completion of reaction.It is for the specific DNA fragmentation that increases in vitro, utilizes the change of fluorescent signal to detect the change of the amount of pcr amplification reaction final product simultaneously.
Prior art:
People find in testing in vitro, and DNA sex change can occur when high temperature and unwinds, after temperature reduces, renaturation can become double-strand again.Therefore, coordinate biological means by the denature and renature of temperature variation controls DNA, and the circulation of repeatedly repetition " sex change unwind-anneal-synthesize extension " just to be increased specific gene in a large number with geometricprogression.The important channel that the semiconservative replication of DNA is organic evolution and goes down to posterity.In the evolution of round pcr, the discovery of heat-stable DNA polymerase and the automatization of temperature cycle enormously simplify pcr amplification program.Fluorescent quantitative PCR technology adds fluorophor in PCR reaction system, along with the accumulation of its fluorescence signal intensity of circulation of PCR reaction, for the whole PCR process of Real-Time Monitoring.
At present, PCR amplification instrument is generally divided into regular-PCR amplification instrument and fluorescent PCR amplification instrument, air-flowing type PCR amplification instrument, metal module formula PCR amplification instrument and water-bath PCR amplification instrument etc. are had by heat-conduction medium difference point, their principles are similar, but control to be determine that PCR reacts the key of success due to accurate temperature, therefore different in apparatus structure, heating cooling mode and fluoroscopic examination mode etc., to adapt to various requirement.The application of PCR amplification instrument has: the molecular diagnosis of infectivity, heredopathia and malignant tumour and research, paternity test, clone gene etc.
Summary of the invention:
Do not detect after existing thermostatic type PCR amplification instrument amplification terminates, the detection of the terminal product of its amplified reaction is realized by miscellaneous equipment, this just seems pretty troublesome to the pcr amplification of a small amount of sample, and thermostatic type PCR amplification instrument has inexpensive feature easy and simple to handle, it has more the superiority such as temperature-controlled precision is high, transformation temperature is quick than other form amplification instrument.Non-thermostatic type PCR amplification instrument is detected by the final product of this amplification instrument to amplified reaction after amplification terminates.Current fluorescent PCR amplification instrument adopts very low temperature CCD imaging, this system is once to multiple spot imaging but sensitivity low accuracy is poor, the employing optical system separately had scanning samples detecting one by one, but its scanning system complexity involves great expense, detection speed is slow, and the amplification therefore for most of a small amount of sample is very uneconomic.The present invention introduces a kind of fluorescence constant temperature PCR amplification instrument, it is characterized in that it is made up of the constant temperature water bath of three differing tempss and a measuring cylinder, the mechanical arm consisted of stepper-motor I, ball screw, line slideway guides sample hose load plate to move, sample hose is made sequentially to be dipped in three constant temperature water baths, sample realizes the pcr amplification reaction of different steps in three constant temperature water baths, thus complete sex change, renaturation and extension three processes, and carry out fluoroscopic examination in measuring cylinder after amplified reaction terminates.Sample hose is the residence time and the movement between groove in each groove, is all to be controlled by controller.This fluorescence constant temperature PCR amplification instrument temperature is adjustable, constant-temperature precision is high, homogeneous temperature, cheap, is applicable to laboratories and uses.
Each temperature sensor installing the platinum resistance thermometer sensor, of 1 band protective tube in the constant temperature water bath of three differing tempss; the compensating resistance of platinum resistance thermometer sensor, adopts accurate type wire wound resistor; 3 to 4 accurate type wire wound resistor are housed in the controller simultaneously; its resistance value is looked into platinum resistance thermometer sensor, phasing meter according to the temperature near the design temperature point of DNA sex change, renaturation and extension three links and is obtained, due to its temperature of reaction difference of different design of primers therefore many accurate type wire wound resistor.Temperature value record corresponding for accurate type wire wound resistor is kept in the non-volatile memory of controller, for instrument calibration temperature.In the laggard trip temperature error check of each start once, it realizes verification by single multichannel analog electronic switch timesharing gating accurate type wire wound resistor access input signal amplifying circuit II to controller.Got the measuring error of each platinum resistance thermometer sensor, temperature during verification by timing, and temperature shown by accurate type wire wound resistor and described be kept in non-volatile memory should the difference of displays temperature, by controller, platinum resistance thermometer sensor, measured temperature is compensated.Each constant temperature water bath its outer wall all rounded is provided with electric heating tube, installs an electric fan I for cooling at three constant temperature water bath immediate vicinity.And having conditioner at the indoor location installing fluorescence constant temperature PCR amplification instrument, in watch-keeping cubicle, temperature-stable is in three constant temperature water baths below lowest set temperature.Another is measuring cylinder, an electric fan II and low-power heating device are housed bottom measuring cylinder, and fill the temperature sensor of 1 small platinum thermal resistance, measuring cylinder temperature is controlled 30 DEG C to 35 DEG C scopes, wherein low-power heating device is for accelerating moisture evaporation in measuring cylinder, electric fan II is for discharging moisture in air, to keep measuring cylinder inner drying.This electric fan II is closed when measuring, and other proliferation time is opened.The position aiming at sample hose in measuring cylinder is provided with the proofing unit be made up of excitation light source and optical pickup apparatus.Its excitation light source is made up of LED photodiode, optical filtering I and reflection cup, and monochromatic LED photodiode price is low, less energy consumption, life-span are long, but needing different LED photodiodes to join optical filtering again could realize different excitation wavelength better.Optical pickup apparatus is made up of optical filtering II and photomultiplier, and the optical filtering in excitation light source and optical pickup apparatus is all the impact for getting rid of surround lighting and useless optical signal, in order to improve light accuracy of detection.Sample hose load plate drives rotation to make sample sequentially enter measuring position by stepper-motor II.Fix a shading strip shut out the light in sample hose load plate outer, be provided with optoelectronic switch simultaneously in measuring cylinder, when shading strip is by optoelectronic switch gap, optoelectronic switch action is No. 1 sample hose on positioning sample QC load plate.Namely when No. 1 sample hose enters measuring position, its shading strip present position should make optoelectronic switch just action, thus starts and detect, and then stepper-motor II sequentially turns over a corner, realizes the sample in sequentially detection and localization various kinds QC.Stepper-motor II transmits the corner of stepper-motor II by transmission shaft, and transmission shaft two ends are fixed with the axle of stepper-motor II and sample hose load plate respectively.Ball screw there is a feed screw nut be fixed on straight-line guide rail slide block makes it linearly guide rail and move, except the hole coordinated with screw mandrel, an axis hole coordinated with vertical transmission shaft is also had in this feed screw nut, transmission shaft is that a kind of axle of hierarchic structure realizes dynamic cooperation with feed screw nut by copper sheathing, on transmission shaft, also fill a tooth bar simultaneously, the hole of tooth bar realizes being slidably matched by copper sheathing and transmission shaft, little axle by vertical direction between tooth bar with feed screw nut is connected location, tooth bar is only moved as vertical direction, one end of tooth bar by transmission shaft the spacing the other end of shoulder by axle sleeve and Xiao spacing, like this when stepper-motor III is moved as vertical direction by gear driven tooth bar, just drive the sample hose load plate be fixed on transmission shaft to do vertical direction to move, thus realize sample hose load plate and move between three constant temperature water baths and a measuring cylinder.
Ball screw line slideway can outsourcing.A stepper-motor I is filled for accurately being transmitted the sample hose load plate revolution of translocation distance and corner in the horizontal direction by ball screw and feed screw nut in ball screw one end.Respectively be fixed on support at line slideway two ends.In order to prevent liquid in reaction system from producing steam, sample hose load plate installing additional a lid, has covered strip electrical-heater and made lid temperature slightly higher than constant temperature water bath.
The outer circle of sample hose load plate circumferentially equal distribution for placing the hole of sample hose, outer in the hole of sample hose, a shading strip is fixed by spiral shell fourth, to identify No. 1 hole of sample hose, controller is made up of single multichannel analog electronic switch, input signal amplifying circuit I, input signal amplifying circuit II, input modulate circuit I, input modulate circuit II, input multiselect one switch, A/D change-over circuit, micro-chip, RS232 circuit, heating power supply, stepper motor driving circuit.Wherein comprise by precision operational-amplifiers such as high-precision LT1014 or OPA121 in input signal amplifying circuit I, it and input modulate circuit I form dedicated optical signal amplification circuit.The temperature-voltage signal amplifying circuit that input signal amplifying circuit II is made up of OP07 and input modulate circuit II thereof are formed.RS232 circuit is used for communicating with upper computer, and it, by sending Computer Analysis to study after fluorescence signal acquisition and process, draws the real-time results of quantification.
Under the controller control wherein of described a kind of fluorescence constant temperature PCR amplification instrument, three constant temperature water bath heating temperatures constant temperature water baths set by link are reacted according to PCR, it is with low power heating or heat radiation that its constant temperature keeps, and temperature controlled return difference is remained in the scope of pcr amplification permission.
Accompanying drawing illustrates:
Fig. 1 is a kind of structural representation of fluorescence constant temperature PCR amplification instrument.
Fig. 2 is a kind of formation block diagram of fluorescence constant temperature PCR amplification instrument.
Embodiment:
A kind of formation schematic diagram of fluorescence constant temperature PCR amplification instrument as shown in Figure 1.It is by constant temperature water bath 13, measuring cylinder 18, mechanical arm, and the sample hose load plate assembly be made up of sample hose load plate 10, lid 25, strip electrical-heater 19, and electric fan I 16, conditioner 14 and power source circuit composition.Wherein constant temperature water bath 13 has three each constant temperature water bath outer walls to be provided with electric heating tube 12, the platinum resistance thermometer sensor, 15 of in-built 1 the band protective tube of constant temperature water bath 13.An electric fan II 20 and low-power heating device 19 are housed bottom measuring cylinder 18, and fill 1 small platinum thermal resistance for dry environment.The proofing unit being also provided with optoelectronic switch 23 in measuring cylinder 18 and being made up of excitation light source 22 and optical pickup apparatus 21.Its excitation light source 22 is made up of LED photodiode and reflection cup, optical filtering II, and optical pickup apparatus 21 is made up of optical filtering I and photomultiplier, and reflection cup can adopt the reflection cup of LED LED torch.Mechanical arm is made up of ball screw 2, line slideway 3, guide rail slide block 4, feed screw nut 5, stepper-motor I 1, stepper-motor II 8, stepper-motor III 9, transmission shaft 7, tooth bar 6, support 24.The hole of feed screw nut 5 is coordinated with transmission shaft 7 by copper sheathing, transmission shaft 7 is equipped be connected location by little axle between tooth bar 6 with feed screw nut 5 and make it to be limited to vertical direction and move, and stepper-motor III 9 is moved as vertical direction by gear driven tooth bar 6.Tooth bar 6 the other end is located on transmission shaft 7 by axle sleeve and Xiao.The outer circle of sample hose load plate 10 circumferentially equal distribution 24 holes for placing sample hose 11.By single multichannel analog electronic switch, input signal amplifying circuit II, the input amplifying circuit inputting the temperature-voltage signal that conditioning II forms and the light input signal amplifying circuit be made up of input signal amplifying circuit I, input conditioning I in controller 17, and input multiselect one switch, A/D change-over circuit, micro-chip, RS232 circuit and stepper motor driving circuit composition.Wherein input signal amplifying circuit I input amplifying circuit II of temperature-voltage signal that is made up of high precision LT1014 amplifying circuit and is made up of OP07.RS232 circuit is used for communicating with upper computer.
Claims (2)
1. a fluorescence constant temperature PCR amplification instrument, its feature it be made up of the constant temperature water bath of three differing tempss and a measuring cylinder, by stepper-motor I, ball screw, the mechanical arm that line slideway is formed guides sample hose load plate to move, sample hose is made sequentially to be dipped in three constant temperature water baths, sample realizes the pcr amplification reaction of different steps in three constant temperature water baths, thus complete sex change, renaturation and extension three processes, and fluoroscopic examination is carried out in measuring cylinder after amplified reaction terminates, sample hose is the residence time and the movement between groove in each groove, all controlled by controller, each platinum resistance thermometer sensor, installing 1 band protective tube in the constant temperature water bath of three differing tempss, its compensating resistance adopts accurate type wire wound resistor, 3 to 4 accurate type wire wound resistor are housed in the controller simultaneously, its resistance value is according to DNA sex change, temperature near the design temperature point of renaturation and extension three links is looked into platinum resistance thermometer sensor, phasing meter and is obtained, temperature value record corresponding for accurate type wire wound resistor is kept in the non-volatile memory of controller, for instrument calibration temperature, controller in the laggard trip temperature error check of each start once, it realizes verification by single multichannel analog electronic switch timesharing gating accurate type wire wound resistor access input signal amplifying circuit II, got the measuring error of each platinum resistance thermometer sensor, temperature by timing during verification, and temperature shown by accurate type wire wound resistor and described be kept in non-volatile memory should the difference of displays temperature, by controller, platinum resistance thermometer sensor, measured temperature is compensated, each constant temperature water bath its outer wall all rounded is provided with electric heating tube, an electric fan I is installed for cooling at three constant temperature water bath immediate vicinity, and have conditioner at the indoor location installing fluorescence constant temperature PCR amplification instrument, in watch-keeping cubicle, temperature-stable is in three constant temperature water baths below lowest set temperature, another is measuring cylinder, an electric fan II and low-power heating device are housed bottom measuring cylinder, and fill the temperature sensor of 1 small platinum thermal resistance, wherein low-power heating device is for accelerating moisture evaporation in measuring cylinder, electric fan II is for discharging moisture in air, to keep measuring cylinder inner drying, the position aiming at sample hose in measuring cylinder is provided with the proofing unit be made up of excitation light source and optical pickup apparatus, its excitation light source is by LED photodiode, optical filtering I and reflection cup are formed, different LED photodiodes joins optical filtering again could realize different excitation wavelength better, optical pickup apparatus is made up of optical filtering II and photomultiplier, sample hose load plate drives rotation to make sample sequentially enter measuring position by stepper-motor II, a shading strip shut out the light is fixed in sample hose load plate outer, in measuring cylinder, optoelectronic switch is installed simultaneously, when shading strip is by optoelectronic switch gap, optoelectronic switch action is No. 1 sample hose on positioning sample QC load plate, namely when No. 1 sample hose enters measuring position, its shading strip present position should make optoelectronic switch just action, thus start detection, then stepper-motor II sequentially turns over a corner, realize the sample in sequentially detection and localization various kinds QC, transmission shaft two ends are fixed with the axle of stepper-motor II and sample hose load plate respectively, ball screw there is a feed screw nut be fixed on straight-line guide rail slide block makes it linearly guide rail and move, except the hole coordinated with screw mandrel, an axis hole coordinated with vertical transmission shaft is also had in this feed screw nut, transmission shaft is that a kind of axle of hierarchic structure realizes dynamic cooperation with feed screw nut by copper sheathing, on transmission shaft, also fill a tooth bar simultaneously, the hole of tooth bar realizes being slidably matched by copper sheathing and transmission shaft, little axle by vertical direction between tooth bar with feed screw nut is connected location, tooth bar is only moved as vertical direction, one end of tooth bar by the shoulder of transmission shaft come the spacing the other end by axle sleeve and Xiao Lai spacing, like this when stepper-motor III is moved as vertical direction by gear driven tooth bar, just drive the sample hose load plate be fixed on transmission shaft to do vertical direction to move, thus realize sample hose load plate and move between three constant temperature water baths and a measuring cylinder, a stepper-motor I is filled for accurately being transmitted the sample hose load plate revolution of translocation distance and corner in the horizontal direction by ball screw and feed screw nut in ball screw one end, steam is produced in order to prevent liquid in reaction system, sample hose load plate installs additional a lid, having covered strip electrical-heater makes lid temperature slightly higher than constant temperature water bath, the outer circle of sample hose load plate circumferentially equal distribution for placing the hole of sample hose, outer in the hole of sample hose, a shading strip is fixed by spiral shell fourth, to identify No. 1 hole of sample hose, temperature controls three constant temperature water bath heating temperatures constant temperature water baths set by PCR reaction link, it is with low power heating or heat radiation that its constant temperature keeps, temperature controlled return difference is remained in the scope of pcr amplification permission.
2. a kind of fluorescence constant temperature PCR amplification instrument according to claim 1, characterized by further comprising:
Described controller is by single multichannel analog electronic switch, input signal amplifying circuit I, input signal amplifying circuit II, input modulate circuit I, input modulate circuit II, input multiselect one switch, A/D change-over circuit, micro-chip, RS232 circuit, heating power supply, stepper motor driving circuit forms, wherein comprise by precision operational-amplifiers such as high-precision LT1014 or OPA121 in input signal amplifying circuit I, it and input modulate circuit I form dedicated optical signal amplification circuit, the temperature-voltage signal amplifying circuit that input signal amplifying circuit II is made up of OP07 and input modulate circuit II thereof are formed, RS232 circuit is used for communicating with upper computer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017213590A1 (en) * | 2016-06-10 | 2017-12-14 | Star Array Pte Ltd | Rapid thermal cycling for sample analyses and processing |
| CN109609608A (en) * | 2019-01-17 | 2019-04-12 | 浙江大学 | The linear quick dual temperature PCR amplification automatic control device of one kind and control method |
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| CN104130933A (en) | 2014-11-05 |
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