A kind of hand-held bluetooth PCR instrument being integrated with photoelectric detection systemTechnical field
The invention belongs to molecular biology and medical detecting Instrument technical field, be integrated with Photoelectric Detection particularly to oneHand-held bluetooth PCR (Polymerase Chain Reaction, the polymerase chain reaction) instrument of device.
Background technology
Bio information be regulation and control vital movement signal, be constitute organism three big key elements (material, energy,One of information), organism existence, breeding and metabolism etc. are all played an important role.The wide range that bio information comprises, generallyRefer to DNA, the RNA etc. comprising hereditary information in the narrow sense.These vary with each individual with the sequence of hereditary information, it is also possible to by thingReason, the impact of the factor such as chemical, biological and undergo mutation, or make a mistake during self replication, and some duplications, transcribeMistake or sudden change can cause the various diseases of human body, such as cancer, thus are analyzed bio information, find out abrupt information in advanceSurvey and the important in inhibiting that diagnoses the illness.Analyzing and processing bio information has often required that a certain amount of sample just can carry out gene surveyThe work such as sequence, but the gene needed for some Disease-causing gene sequences especially early-stage cancer examination of human body extracting directly gainedThe amount of sequence is often the least, therefore needs it is carried out amplification in vitro to reach the magnitude can observed and analyze.Currently, loseThe amplification in vitro passing information sequence is mainly by carrying out polymerase chain reaction in gene-amplificative instrament (PCR instrument)(Polymerase Chain Reaction) obtains.
The semiconservative replication of DNA is biological evolution and the important channel passed on, and polymerase chain reaction be a kind of forAmplify the Protocols in Molecular Biology of amplification specific DNA fragments, be regarded as external special semiconservative DNA replication.Continue 1983United States Biochemical man K.B.Mullis advanced an idea and invented polymerase chain reaction in 1985 year, i.e. simple DNA expandsAfter increasing method, this technology sustainable development to the third generation, there is heat-resisting, stable archaeal dna polymerase Taq enzyme.PCR reacts firstUtilize the character of degeneration under the DNA high temperature of about 95 DEG C to be in vitro allowed to untwist into strand, then be cooled to about 60 DEG C, make twoUntwist with previous step respectively two strands obtaining of primer are combined by the principle of base pair complementarity, adjust the temperature to 72 the most againAbout DEG C (archaeal dna polymerase optimal reactive temperature), make archaeal dna polymerase can hold to the 3 ' directions held along 5 ' with primer for starting pointComplementary strand is synthesized with free base A, T, G, C.Often completing such circulation, the quantity of DNA sequence just doubles, manyAfter individual circulation, target sequence just can be expanded to considerable magnitude, using the sample as next step analysis of biological information.
Along with life sciences and the development of bioinformatics research, polymerase chain reaction technology and instrument also obtainMore concerns and development, be increasingly becoming part indispensable in life science and experiment.PCR instrument is generally divided into commonlyPCR instrument, grads PCR instrument, In situPCR instrument and real-time fluorescence quantitative PCR instrument four class.Amplified reaction of regular-PCR instrument can onlySet a specified temp, be generally used for specific target gene is expanded, grads PCR instrument then can set a series of notSame temperature (thermograde), finds optimum reaction temperature in once amplification, more effectively expands；In situPCRInstrument utilizes the aim sequence fragment that complete cell comes in amplifying cells as a reaction system, and before not destroying cellPut and detect intracellular amplified production by some particular detection means, it is adaptable to content less target sequence in detection pathological sectionRow；Real-time fluorescence quantitative PCR instrument is the regular-PCR instrument with fluorescence signal acquisition system and computer analysis process system, itsCarrying out primer isotope used, fluorescein etc. during PCR amplification labeled, amplification can pass through fluorescence signal acquisitionSystem Real-time Collection also is transferred in computer analysis process system process to obtain real-time quantized result output.
The consequence all the more shown in life science and medical treatment detection due to PCR instrument and good marketProspect, many Medical Equipment Plants commercial city has been enter into PCR instrument market, and actively seizes the market share, is wherein no lack of renowned company such asThe ABI U.S. applies biotech firm, ROCHE Roche Diagnistics, MJ, Bole etc., and day, the bright base in Hangzhou, Shanghai are won in domestic HangzhouMaple ridge, Xiamen peace Puli etc., driven developing rapidly of global vitro detection market.
Mostly existing PCR instrument, especially domestic equipment, be that build is big towards laboratory and big-and-middle-sized medical institutions, valencyLattice are high, family and small-sized medical institutions, as community hospital etc. still has no ability to be equipped with.But, due to China's large population base,Medical resource is the deficientest, and big-and-middle-sized medical institutions can only solve the problem of seeing a doctor of fraction people, and most people cannot enjoyCarried out disorder in screening or diagnosis by this part medical resource, delay the state of an illness and best occasion for the treatment.Meanwhile, receive along with its peopleEntering and growth in the living standard, people pay close attention to all the more healthy, the most growing to the demand of precisely medical treatment.
In sum, PCR instrument etc. is convenient, effective medical diagnostic equipment transfers to basic medical treatment service organization and familyIn, PCR instrument effectiveness and market thereof could be maximized, the most effectively solve different medical unit and family's disease detection and diagnosisThe problem of difficulty.
On Vehicles Collected from Market, the PCR instrument volume of main flow is relatively big, heaviness not Portable belt, and operation complexity needs Specialty Experiment personnel to graspMake, and the testing time is averagely more than 2 hours.The dyestuff tool of the temporary planless data management system of this kind of PCR instrument and EB dyeingHigh carcinogenic, price is had to be also up to several ten thousand RMB.Thus, a kind of volume portable little, light, rapid low one-tenth simple to operateThis, the trend having the PCR instrument device of data management function can be market demand development, replace traditional PCR instrument the most at last and play moreAct on widely.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, it is an object of the invention to provide one and be integrated with photoelectric detection systemHand-held bluetooth PCR instrument, control heating and cooling device based on PID regulation reaction temperature regulated in real time, coordinate bluetoothDevice realizes the real-time data transmission between computer or cell-phone customer terminal, and utilizes photoelectric detection system Rapid identification experiment knotReally, its feature includes operation efficient, easy, accurately controls, and be avoided that to react failed product carry out the special of next step againProperty analyze, cause time and cost loss.
To achieve these goals, the technical solution used in the present invention is:
A kind of hand-held bluetooth PCR instrument being integrated with photoelectric detection system, including:
One metal reaction platform 1 placing PCR reaction tube, including lower base and top Re Gai；
One control circuit 2 based on PID/feedback regulation；
One or more heater 3；
One or more heat sink 4；
One or more general temperature sensor 5；
One or more photoelectric detection system 6；
One blue-tooth device 7, for sending the relevant informations such as temperature in real time to receiving device；
It is characterized in that: described control circuit 2 utilizes PID regulation to control heater 3 and heat sink 4 to being placed withThe metal reaction platform 1 of PCR reaction tube carries out heating or lowering the temperature, and utilizes the feedback of temperature sensor 5 to carry out real time temperature to repairJust, real time temperature information is sent to receiving device so that monitoring in real time by blue-tooth device 7, and utilizes the most afterwardsPhotoelectric detection system 6 detects content of predetermined substance in reactant liquor to be changed with Preliminary Identification reaction result.
Described metal reaction platform 1 is made up of the metal of good heat conductivity, includes but not limited to aluminum, aluminium alloy or silver-plated aluminium blockDeng, its as medium heat conductivity than air evenly, than liquid more faster, modes upper and lower, four thorough encirclements can guarantee that again insteadShould pipe variations in temperature evenly.
It is arranged in lower base and the delivery position of top heat lid for placing the reactive tank 101 of PCR reaction tube, it may be assumed that whenWhen PCR reaction tube is arranged on reactive tank 101, one part is positioned in lower base, and another part is positioned in the heat lid of top.ReactionGroove 101 is cannelure, and reaction platform top half, i.e. top heat cover and be provided with a PCR reaction tube in each reactive tank 101The fixing spring 102 of lid, with ensure PCR reaction tube can be fixed on reactive tank 101 closely in and be heated evenly.Metal reaction is put downPlatform 1 main body is cylinder or cuboid or other turntable structure, and outside arranges rotating disk and fixes bayonet socket to 103.Reactive tank 101 is with circleRadially it is uniformly distributed centered by the axis of a cylinder heart or is evenly distributed in cuboid in a row, to ensure differential responses groove 101It is heated evenly.
Described control circuit 2 is with MCU as control centre, by writing control program to MCU, to heater 3, cooling dressPutting 4, temperature sensor 5, photoelectric detection system 6 and blue-tooth device 7 to be scheduling controlling, described MCU passes through USB interface with outerIf or host computer directly communicates；MCU carries out internal storage reading by ISP (In-System Programming) interfaceWrite and replacement to self, and the data of ISP interface are total by SPI (Serial Peripheral Interface) on sheetLine is transmitted；On sheet, clock frequency is defined by external crystal oscillator；This MCU can pass through USB with the data transmission of peripheral hardware or host computerCommunication module realizes；Control circuit employs power management module input voltage is changed, for MCU, USB interface andOther module for power supply.
When the regulation of described control circuit 2 controls reaction temperature, the mode that temperature is arranged includes but not limited to specific amplificationObject sets fixing degeneration, annealing and renaturation temperature, or sets a series of thermograde and find in once amplificationSuitable reaction temperature, in order to follow-up more effectively expand.
Described blue-tooth device 7 is connected with described MCU, receives the data that MCU processed, by communication by dataReal-time Transmission to mobile phone etc. equipped with the equipment of Bluetooth receptions device, it is simple to monitor response situation in real time in device clients.
Described control circuit 2 uses PID control principle to be precisely controlled reaction temperature, when temperature sensor 5 gathersThe set-point desired by dut temperature deviation time, the deviation measuring signal and set-point that PID controls according to feeding back to is carried outRatio P, integration I, differential D computing, thus export certain suitable control signal to heater 3 or heat sink 4, promote to surveyValue returns to set-point, reaches the effect automatically controlled.
Described heater 3 include but not limited to heating electric stopband or film heating resistance, described heat sink 4 include butBeing not limited to fan, thermoelectric refrigerator or liquid cooling system, described temperature sensor 5 includes but not limited to temperature-sensitive device or digital temperatureSensor.
When heater 3 is multiple, should ensure that its heating power is consistent, and be evenly distributed on metal reaction platform 1Bottom and top, to ensure that reaction platform entirety heats up uniformly.
When heat sink 4 is multiple, should be in parallel to ensure multiple work and power is identical simultaneously.
Described PCR reaction tube is the center arrangement of cincture metal reaction platform 1, heater 3 cloth on metal reaction platform 1Putting two side position up and down at metal reaction platform 1, position is positioned at upside and the downside of lower base of top heat lid respectively；DescribedHeat sink 4 is arranged in metal reaction platform 1 and the outside of heater 3, lay respectively at corresponding heater 3 top orBelow person, described temperature sensor 5 is arranged in the edge of metal reaction platform 1 and the surface location at center.Due to metal reactionPlatform 1 is different with PCR reactant liquor heating rate, therefore still needs to measure in real time and instead after reaction platform temperature reaches period setpointFeedback, controls reaction platform temperature and continues for some time to ensure that reactant liquor reaches the temperature consistent with reaction platform.
The judgement of reaction result is to utilize the spectral characteristic of PCR product to enter its concentration by photoelectric detection system 6Row detection obtains, and described photoelectric detection system 6 carries out Preliminary Identification to product content and (should the most first detectOnce reflection product assay is as benchmark group, this value is generally the least or be zero), to judge whether the reaction result terminated meetsExpection requirement, if product is quantitatively positioned at zone of reasonableness, thinks that reaction is successfully completed, under carrying out productOne step specificity analyses, if product quantity seldom even without; think that reaction is unsuccessful, then need again to carry out to react withThe result.
Described photoelectric detection system 6 includes being arranged in metal reaction platform 1 lower base and the light of Re Gai junction, topElectricity emitter 601 and photelectric receiver 602, wherein photoemitter 601 is positioned at edge, is specifically arranged in metal reaction platform 1With between PCR instrument shell, photelectric receiver 602 is positioned at metal reaction platform 1 center, and photoemitter 601 is towards opto-electronic receiverDevice 602, along center position from radial direction to metal reaction platform 1 launch specific wavelength light wave, photelectric receiver 602 towardsPhotoemitter 601 direction receives light wave；Light wave, through PCR reaction tube and wherein reactant liquor, judges anti-with the light intensity receivedAnswer the liquid absorbing state to this light, and then judge the content of product.
Photoemitter 601 and photelectric receiver 602 are fixed in lower base, are positioned at the bottom of metal reaction platform 1Base and top hot lid intersection, lower edge is concordant with interface, before starting the reaction and terminate after, open top Re Gai, lightThe light wave that electricity emitter 601 is launched just can be unobstructed, through PCR reaction tube directive photelectric receiver 602 direction；Photoelectric DetectionCentre-height also can be arranged on lower base and top hot lid intersection by device 6, i.e. photoelectric detection system 6 half is in lower bottomIn Zuo, half is in the heat lid of top, but so arranges, need to be corresponding each between photoemitter 601 and photelectric receiver 602Reactive tank 101 reserves an optical channel, not only increases manufacturing cost, is also unfavorable for the gentle fall of 1 liter of whole metal reaction platformThe uniformity of temperature.
Described photoemitter 601 includes but not limited to laser diode or light emitting diode, described photelectric receiver 602Include but not limited to light sensitive diode, photoconductive resistance or photomultiplier tube, the light wave of photoemitter 601 fixed transmission 490nm,The amount of byproduct of reaction pyrophosphoric acid (PPi) in detection reactant liquor, to judge reaction result.
Hand-held PCR instrument small in volume of the present invention, can gripping with one hand, have portability, relative like product more portableBand, and simple to operate, test is rapidly, is suitable for basic medical unit and family uses.Control circuit 2 based on PID/feedback regulationCan realize the accurate regulation of reaction temperature, the integrated of blue-tooth device 7 makes course of reaction to be monitored in real-time, photoelectric detection systemThe result of 6 energy anticipation reactions, it is to avoid carry out follow-up detection and specificity analyses in the case of not obtaining expection product,Reduce detection time and cost to a certain extent.
Compared with prior art, the present invention is integrated with the hand-held bluetooth PCR instrument of photoelectric detection system, by reactionIn real time, it is precisely controlled the application with low cost device and detection means, while realizing high speed, efficient amplification reaction, reachesDevice miniaturization and the human-oriented target of price are arrived, for PCR equipment basic unit with enter family's important in inhibiting.
Accompanying drawing explanation
Fig. 1 is that operation during PCR instrument of the present invention application performs flow chart.
Fig. 2 is the integrally-built generalized section of one of the present invention.
Fig. 3 is the cross section elevational schematic view of a kind of heat lid of the present invention.
Fig. 4 is the sectional overhead schematic view of a kind of PCR instrument main body of the present invention.
Fig. 5 is the circuit diagram of control system of the present invention.
Fig. 6 is the principle schematic of the PID temperature control regulation that the present invention uses.
Fig. 7 is the PID temperature control regulation execution unit schematic diagram that the present invention uses.
Fig. 8 is a kind of heater of using of the present invention and circuit diagram thereof.
Fig. 9 is the structural representation of electro-heat equipment main body in Fig. 8.
Detailed description of the invention
Embodiments of the present invention are described in detail below in conjunction with the accompanying drawings with embodiment.
Operation when a kind of hand-held bluetooth PCR instrument being integrated with photoelectric detection system that the present invention proposes is applied performs streamJourney as it is shown in figure 1, when reactant liquor prepare complete, put into PCR reaction tube and be placed in reaction platform reactive tank in after, first callThe content of predetermined substance to be detected in reactant liquor is measured by photoelectric detection system, (under normal circumstances should as benchmark groupValue is usually zero or minimum)；Then, covering heat lid, energising starts PCR instrument；In control circuit, MCU is according to each rank of program settingSection reaction temperature value is transferred heater or heat sink and reaction platform is heated or lowered the temperature, temperature sensor the real time measureReaction temperature, this temperature value can be sent to receiving device by blue-tooth device and monitor in real time, need to feed back to MCU simultaneously,MCU amasss according to the difference of measurement temperature value with set temperature value, application percentage (P) regulation or proportional integral (PI) regulation or ratioDivide differential (PID) regulation, control heating or temperature-fall period to reach design temperature；When temperature sensor measurement temperature value with setWhen constant temperature angle value is consistent, MCU, according to the duration value of program setting, controls heating or heat sink makes the maintenance of reaction platform be somebody's turn to doThe temperature corresponding time；Judge that the current stage of reaction is degeneration, annealing or renaturation, if last stage of reaction completes, thenThink that this is taken turns reaction and terminates, otherwise continue next stage reaction, until epicycle reaction terminates；Each wheel reacts end to reaction wheelSecondary count, and compare with setting round, if not up to setting round, maintain or update each elementary reaction time andTemperature (time initial, each phase sets temperature is that fixed value then maintains, and then updates for thermograde), proceeds next round reaction,If reaching, react stopping；It is again started up photoelectric detection system, launches specific wavelength light wave and specific product content is detectedTentatively to judge reaction result, if product assay as expected, is thought and is reacted successfully, otherwise, then may need again to test to testCard result.
Accompanying drawing 2 provides a kind of overall structure figure of the present invention, the i.e. front view of this PCR instrument centre section, whole PCR instrumentStructurally being broadly divided into casing, top heat lid and three parts of sub-body, its middle and upper part heat lid and sub-body containAll functional parts.Specifically include: a metal reaction platform 1 placing PCR reaction tube；One based on PID/feedback regulationControl circuit 2；One or more heater 3；One or more heat sink 4；One or more general temperatureSensor 5；One or more photoelectric detection system 6；And a blue-tooth device 7.
The bosom parts of whole reaction instrument are metal reaction platform 1, are divided into upper and lower two by top heat lid and sub-bodyIndividual can the part of tight split, wherein reactive tank 101 be divided into similarly upper and lower two can the part of tight split, in reactionGroove 101 top have PCR reaction lid to fix PCR reaction tube that spring 102 fixes in reactive tank 101 is to ensure that it is anti-with metalPlatform 1 is answered to contact tightly；Reaction platform side is provided with photoemitter 601 (laser diode), solid to reaction Platform centerSurely launch specific wavelength light wave, then have a photelectric receiver 602 (photoconductive resistance) at reaction Platform center, be used for detecting wearingThe intensity of this light wave of PCR reaction tube and reactant liquor injection is tentatively to judge the result that PCR reacts thoroughly.Temperature sensor 5 pointsNot being placed in the diverse location of metal reaction platform 1, the temperature data measured applies related algorithm to calculate reaction after passing MCU backTemperature in pipe, and feeding back to realize being precisely controlled of reaction temperature as the regulation of control system PID.Heater 3, at thisFor the folding heating electric stopband uniformly coiled in metal reaction platform 1 upper and lower surface in example, directly connect with metal reaction platform 1Touching, by MCU control, at degeneration and the renaturation stepwise heating metal reaction platform 1 of reaction, upper and lower surface is symmetrically placed be can ensure thatHeat up evenly.Heat sink 4, is the fan outside metal reaction platform 1 and heater 3, equally by MCU in this exampleControlling, mainly give reaction platform cooling in the annealing steps of reaction, symmetrical above and below being positioned to makes cooling evenly, bottomFan can also give simultaneously and control control circuit 2 and lower the temperature.
PCR instrument of the present invention controls heater 3 by control circuit 2 and metal reaction platform 1 is added by heat sink 4Heat or cooling, measured reaction platform temperature simultaneously in real time, data transmitted in real time by blue-tooth device 7 by temperature sensor 5To receiving terminal so that user monitors reaction temperature in real time, and feed back to control circuit 2 so that it controls heater 3 and cooling dressPut 4 pairs of reaction temperatures precisely to regulate.After having reacted, before carrying out next step specificity analyses, right with photoelectric detection system 6Reaction result carries out preliminary judgement to avoid subsequent step unnecessary time and cost consumption.
Accompanying drawing 3 is the sectional view of a kind of heat lid of the present invention, and hot lid main body is metal reaction platform 1 top, is close to reactionThe heater of platform 3 layers and the fan of more top, in metal reaction platform 1, there is PCR reaction tube the top of reactive tank 101The fixing spring 102 of lid is in order to be tightly fixed to PCR reaction tube in reactive tank 101.
Accompanying drawing 4 is the sectional view of a kind of PCR instrument main body of the present invention, and metal reaction platform 1 lower base next-door neighbour be placed inOn heater 3 (directly contacting with reaction platform) layer, there is multiple spacing identical in the middle of reaction platform and in reaction platformThe heart is apart from identical reactive tank 101, and the diverse location of reaction platform is also equipped with temperature sensor 5 and reacts platform with real-time measurementTemperature, heater 3 then has non-close fan and control circuit 2 downwards.During owing to carrying out Photoelectric Detection after having reactedNeed successively the reactant liquor in multiple PCR reaction tubes to be detected, and photoelectric detection system is integrally fixed at cylinder reaction and puts downThe two ends of one radius of platform, therefore need metal reaction platform can rotate around axle center so that each reactive tank position turns to light successivelyIn the light path of electric detection means (even if each reactive tank place radius overlaps with photoelectric detection system place radius successively), and anti-Answer platform side equipped with buckle to 103 just can each corresponding inspection positions fix metal platform prevent Photoelectric Detection timePlatform rotates and produces skew, can change to down by hand rotation reaction platform after often having detected a PCR reaction tube and having received dataOne PCR reaction tube position and with buckle to 103 fixing reaction platforms.
Accompanying drawing 5 is the circuit diagram of control system of the present invention, and the center of this control system is MCU, and control program is writeEntering MCU and perform, electro-heat equipment, fan, temperature sensor, photoemitter, photelectric receiver, bluetooth module be not respectively byBeing connected and execution corresponding function under it is dispatched with MCU with interface, MCU can be communicated by USB module with peripheral hardware, host computer etc.,And the data such as temperature also can be sent by bluetooth module in real time, can check equipped with receiving on the equipment of Bluetooth Receiver,And control circuit employs power management module input voltage is changed, supply for MCU, USB interface and other modulesElectricity.
Accompanying drawing 6 provides the schematic diagram of the PID temperature control regulation that the present invention uses, and accompanying drawing 7 provides the signal of its execution unitFigure, PID control system is a kind of closed-loop feedback control system, and system initial input is that one or several relatively suitably temperature setsHolding time of definite value (thermograde) and corresponding temperature, Kp、Ki、KdRepresent that ratio (P), integration (I), differential (D) are made respectivelyCoefficient, three jointly acts on and obtains control system transfer function H (s), input quantity transmission function effect under obtain and defeatedGoing out controlled quentity controlled variable u, act on object, i.e. react platform, result y is real time temperature measured value, after being fed back to control system,PID controller recalculates and transfers actuator according to the difference of desired temperature with measured value and adjusts object temperatureJoint, until input value r and output valve y difference are zero.In the present invention, MCU assume responsibility for the work of PID controller, heater and fallTemperature device to object (reaction platform) to carry out heating or lowering the temperature as the actuator of control system under PID controller control,Temperature sensor assume responsibility for sending back object (reaction platform) real time temperature the work of MCU as feedback mechanism, and channel radioThe data such as temperature then can be sent to receiving device to realize monitoring in real time by letter module (blue-tooth device) in real time.
Accompanying drawing 8 provides a kind of heater and the circuit diagram thereof that the present invention uses, and electro-heat equipment main body is uniformThe folding heating electric stopband of coiling, as shown in Figure 9, it is evenly distributed on metal reaction platform upper and lower surface (directly contact),Every resistor is connected in parallel with a capacitor, a termination power, and one end is by complementary Darlington transistor ground connection, and Darlington transistor is opened as powerClosing, its base stage connects MCU pin, controls resistor work under MCU dispatches.
A kind of detailed description of the invention of the present invention is described as follows:
First reactant liquor is quantitatively adding multiple PCR reaction tube, reactant liquor comprise target masterplate DNA, primer, Taq DNA gatherSynthase, 4 kinds of dNTP mixture, amplification buffer, positive divalent magnesium ion and distilled water.PCR reaction tube is put into metal reaction put downIn reactive tank 101 in platform 1, switching on power, first start photoelectric detection system, photoemitter (laser diode) 601 is launchedThe light wave of 490nm wavelength, rotational response platform makes light wave sequentially pass through each PCR reaction tube, photelectric receiver (photoconductive resistance)602 perception projection light intensity substantially to determine the content of pyrophosphoric acid (PPi) in PCR reaction tube, this value will as with reaction resultThe benchmark (under normal circumstances, this value is usually zero or minimum) compared；Then, closing heat lid makes PCR reaction lid fixPCR reaction tube can be tightly fixed in reactive tank 101 by spring 102；According to the control program of write MCU, first start and add hot chargingPut 3 to preheat reaction platform with activating reaction thing, then continue to be heated to about 95 DEG C and maintain certain time, make reactionMasterplate DNA double chain degeneration in liquid is untwisted becomes two DNA strands separated；In annealing (renaturation) stage, MCU controls to add hot chargingPut 3 quit work, heat sink 4 (i.e. fan) work, reactant liquor is cooled to suitable temperature and maintains a period of time, shouldTemperature is generally about 55-60 DEG C, but different according to differential responses thing, at this moment primer and the complementary sequence of masterplate DNA strandRow combine pairing；In the renaturation stage, heat sink 4 quits work, heater 3 restarts in MCU control, carries out reactant liquorBeing again heated to about 72 DEG C and maintain a period of time, at this moment archaeal dna polymerase reaches most preferably to replicate temperature, thus at archaeal dna polymeraseWith 4 kinds of dNTP as reaction raw materials under effect, by base pair complementarity and semiconservative replication principle, close with product on last stageThe DNA double chain of Cheng Xin.During whole, reaction temperature detected by temperature sensor 5 and Real-time Feedback to MCU to carry out PID tuneJoint is also sent out by blue-tooth device, and the actual temp in each stage may be different, all because reactant is different with the persistent periodGiven by control program.Three processes of repetitive cycling " degeneration-annealing-extension " repeatedly, can be by required DNA cloning to being easy to detectionConcentration, this period is also given by program, and determine PCR amplification yield, general period is 25-30 time, works as circulationAfter number is more than 30, DNA polymerase activity can progressively reach saturated, and the amount of product the most no longer increases with period and increases,I.e. " plateau " occurs, therefore general period is less than 30.After reaction terminates, opening heat lid, MCU controls photoemitter 601Launching the light wave of 490nm wavelength, rotational response platform makes light wave sequentially pass through each PCR reaction tube, anti-in detection PCR reaction tubeAnswering the concentration of by-product pyrophosphoric acid (PPi), if reacting successfully and fully, enough PPi should be produced, owing to wavelength is by PPiThe light of 490nm has absorbability, and the light intensity through PCR reaction tube directive photelectric receiver 602 can die down, if reacting unsuccessful,Will not produce or only produce a small amount of PPi, 490nm light not had the strongest trap, then connects through PCR reaction tube directive photoelectricityThe light intensity receiving device 602 is the strongest.According to the characteristic of photoconductive resistance, it can show different resistance under varying strength illumination.ByThis can be with preliminary judgement reaction result, if reacting successfully, can carry out the subsequent steps such as electrophoresis, to carry out special to reaction resultProperty analyze, if reacting unsuccessful, need not to expend again the time and cost carry out electrophoresis, and may need to re-start PCR reactionWith compound nucleus reaction result.