CN107574120A - Fluorescence quantitative PCR detection system and method based on magnetomotive switching flat-temperature zone - Google Patents
Fluorescence quantitative PCR detection system and method based on magnetomotive switching flat-temperature zone Download PDFInfo
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- CN107574120A CN107574120A CN201711022774.4A CN201711022774A CN107574120A CN 107574120 A CN107574120 A CN 107574120A CN 201711022774 A CN201711022774 A CN 201711022774A CN 107574120 A CN107574120 A CN 107574120A
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Abstract
The present invention provides fluorescence quantitative PCR detection system and method based on magnetomotive switching flat-temperature zone, and it includes constant temperature heating device, sample motion guide rail device and fluorescence detection device.Wherein described constant temperature heating device includes the flat-temperature zone of at least two different temperatures；The sample motion guide rail device includes motor magnet assembly, sample application zone and guide rail, the sample application zone is polarity identical bipolar magnet block with being respectively arranged with opposite face on the motor magnet assembly, sample application zone is driven to be moved along guide rail by magnetic field repulsive force caused by bipolar magnet block, toggled between the flat-temperature zone of different temperatures, carry out sample amplification.The present invention makes the sample application zone for being placed with PCR reaction tubes toggle motion between different flat-temperature zones, greatly reduces the DNA cloning time, effectively increase detection efficiency by using magnet homopolar-repulsion principle.
Description
Technical field
The present invention relates to molecule to diagnose detection field, more particularly to the quantitative fluorescent PCR based on magnetomotive switching flat-temperature zone
Detecting system and method.
Background technology
PCR (PCR) instrument is the most commonly used application of molecule diagnosis, and it can realize full-automatic right
Nucleic acid fragment amplification is detected.Its principle is based on the complementary duplication principle of DNA double chain, and DNA is provided in thermal cycle module
The temperature of denaturation (95 DEG C)-annealing (55 DEG C)-extension (72 DEG C) three courses of reaction required for amplification.In PCR courses of reaction
In by fluorescent dye or the probe of fluorescence labeling, real-time mark is carried out to PCR primer, then detected by Systems for optical inspection glimmering
Optical signal, fluorescence signal have reacted real-time amplified production DNA content, it is possible thereby to be tried to achieve by calibration curve method original
DNA concentration, so as to reach the effect of relative quantification.
The temperature control system of existing quantitative real time PCR Instrument on the market is substantially the heating and cooling based on Peltier semiconductor,
With each circulation ceaselessly alternating temperature of PCR reactions, the speed of heating and cooling is slower, causes whole proliferation time long, and
Special radiator fan is needed, structure is bigger, and accuracy of temperature control is not also high.Shared together in addition, annealing and extending in the instrument of part
One temperature, it is only necessary to which two high temperature (94 DEG C) and low temperature (64 DEG C or so) can improve on the premise of its normal amplification is ensured
Amplification efficiency.Warming and cooling rate based on semiconductor heat booster is typically in 3 DEG C/s, that is to say, that the time of heating and cooling process is every
Individual circulation just needs 20s, and this just increases the whole amplification procedure time cycle significantly.Furthermore compared with air heating, contact
The easy temperature control of formula METAL HEATING PROCESS method, air heating warming and cooling rate is fast but temperature accuracy is poor, typically has several DEG C of error, in addition
Required space is big, causes instrument sample flux small.
Therefore, prior art has yet to be improved and developed.
The content of the invention
It is an object of the invention to provide a kind of fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone and side
Method, it is intended to solve in the prior art the problem of PCR detection system amplification rate is slow, and the operating time is long.
Technical scheme includes：
A kind of fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, it is characterised in that including heated at constant temperature
Device, sample motion guide rail device and fluorescence detection device；The constant temperature heating device, fluorescence detection device with it is described
Sample motion guide rail device connects；
Wherein described constant temperature heating device includes the flat-temperature zone of at least two different temperatures；The sample motion guide rail device
Sample application zone including motor magnet assembly, for placing sample and the guide rail for connecting sample application zone and flat-temperature zone, it is described
Sample application zone is polarity identical bipolar magnet block with being respectively arranged with opposite face on the motor magnet assembly, by bipolar
Property caused by magnet piece magnetic field repulsive force drive sample application zone moved along guide rail, toggled between the flat-temperature zone of different temperatures,
Carry out sample amplification.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, the sample motion guide rail dress
Putting also includes motor and the timing belt for the motion of drive motor magnet assembly, by motor driven timing belt, makes motor
Magnet assembly moves.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, it is bipolar in the sample application zone
Property magnet piece and the motor magnet assembly in bipolar magnet block use contactless setting.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, it is provided with the sample application zone
For placing the rack for test tube of PCR reaction tubes.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, the constant temperature of the different temperatures
Heat-insulated valve is provided between area.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, the constant temperature heating device bag
Include high-temperature constant warm area and cryogenic thermostat area；The temperature of the high-temperature constant warm area be 90 DEG C -95 DEG C in any temperature value, the low temperature
The temperature of flat-temperature zone is any temperature value in 55 DEG C -72 DEG C.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, the constant temperature heating device is adopted
Heated with heating film.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, the cryogenic thermostat area is set
There is hole position, the fluorescence detection device is fixedly installed at the hole position in cryogenic thermostat area, and single PCR reaction tubes are entered by hole position
Row detection in real time.
The fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, wherein, described fluorescence detection device
Including mono-colour laser and detector；Wherein described detector is photocell element.
A kind of fluorescent quantitative PCR detection method based on magnetomotive switching flat-temperature zone, wherein, comprise the following steps：
A, sample is placed on the PCR reaction tube rack for test tubes of sample application zone, and by sample motion guide rail device by sample
It is sent to high-temperature constant warm area and keeps 12-17s, carries out DNA cracking；
B, sample is sent to from high-temperature constant warm area by cryogenic thermostat area by sample motion guide rail device again and keeps 28-
32s, carry out DNA cloning；
C, when sample leaves cryogenic thermostat area, fluoroscopic examination is carried out by fluorescence detection device successively to sample；
D, repeat the above steps n times；N≤35 time.
Beneficial effect：Fluorescence quantitative PCR detection system of the present invention based on magnetomotive switching flat-temperature zone, by adding
Sample area with setting bipolar magnet block, and bipolar magnet block and motor in sample application zone respectively on the motor magnet assembly
Bipolar magnet block on magnet assembly is parallel and contactless setting, while the two magnetic phase on that opposite face
Together.By caused by bipolar magnet block magnetic field repulsive force drive sample application zone moved along guide rail, the flat-temperature zone of different temperatures it
Between toggle, carry out sample amplification.The present invention makes to be placed with adding for PCR reaction tubes by using magnet homopolar-repulsion principle
Sample area toggles motion between different flat-temperature zones, greatly reduces the DNA cloning time, effectively increases detection effect
Rate.
Brief description of the drawings
Fig. 1 is the structural representation for the fluorescence quantitative PCR detection system that the present invention switches flat-temperature zone based on magnetomotive.
Fig. 2 is the front view for the fluorescence quantitative PCR detection system that the present invention switches flat-temperature zone based on magnetomotive.
Fig. 3 is sample application zone and motor magnetic in fluorescence quantitative PCR detection system of the present invention based on magnetomotive switching flat-temperature zone
The assembling schematic diagram of bipolar magnet block in iron assembly.
1st, installation riser is incubated, 2, valve lower baffle plate, 3, stepper motor, 4,15 tooth MXL synchronous pulleys, 5, loading slot assembling
Body, 6, high-temperature heating groove assembly, 7, timing belt, 8, gene neck assembly, 9, valve upper cover plate, 10, incubate the total bottom plate of groove,
11st, guide rail, 12, optocoupler, 13, low-temperature heat groove assembly, 14, motor support plate, 15, idler shaft supporting plate, 16, guide supporting
Plate, 17, idler shaft, 18 valve assemblies, 19 motor magnet assemblies, 20, valve driving machine board assembly, 21, bipolar magnet
Block.
Embodiment
Below with reference to accompanying drawing to it is of the present invention based on magnetomotive switch flat-temperature zone fluorescence quantitative PCR detection system and
The embodiment of method is described in detail as follows：
As shown in Figures 1 and 2, the present invention provides a kind of fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone
System, it includes constant temperature heating device, sample motion guide rail device and fluorescence detection device；The constant temperature heating device, fluorescence
Detection means is connected with the sample motion guide rail device.By sample motion guide rail device, sample is driven in heated at constant temperature
Heating and thermal insulation is carried out in device, and is detected by fluorescence detection device.In embodiments of the present invention, the heated at constant temperature dress
Putting includes the flat-temperature zone of at least two different temperatures；Different flat-temperature zones is set, meets temperature need different during DNA cloning
Ask, and it is separate by heat-insulated valve between each flat-temperature zone, without manually waiting same temperature province heating cooling,
Greatly reduce the whole amplification procedure cycle.
Specifically, the sample motion guide rail device includes motor magnet assembly 19, the sample application zone for placing sample
(labeling position in such as figure：Loading slot assembly 5) and for connecting sample application zone and flat-temperature zone (labeling position in such as figure：High temperature
Heating tank assembly 6 and low-temperature heat groove assembly 13) guide rail 11, on the sample application zone and the motor magnet assembly 19
It is polarity identical bipolar magnet block to be respectively arranged with opposite face, is driven and added by magnetic field repulsive force caused by bipolar magnet block
Sample area moves along guide rail, is toggled between the flat-temperature zone of different temperatures, carries out sample amplification.In other words, add described
Sample area with setting bipolar magnet block, and bipolar magnet block and electricity in sample application zone respectively on the motor magnet assembly 19
Bipolar magnet block on machine magnet assembly 19 is parallel and contactless setting, while the two magnetic on that opposite face
Property is identical.For example, bipolar magnet block in the side towards bipolar magnet block in motor magnet assembly 19 is S in sample application zone
Pole, and it is also S poles that motor magnet, which turns in part bipolar magnet block in the side towards bipolar magnet block in sample application zone, the two
Between to be arranged in parallel, and magnetic homopolar-repulsion principle can be passed through and produce magnetic force.So carried out in motor magnet assembly 19
During motion, sample application zone (connection is arranged on guide rail) can also be driven to be moved on guide rail, and match somebody with somebody by motor magnet cartridge
Body 19 toggles motion, drives sample application zone to be toggled between different flat-temperature zones, improves amplification efficiency.
As shown in figure 3, wherein icon 8 is gene neck assembly, rack for test tube as of the present invention.The sample application zone
In bipolar magnet block and the motor magnet assembly 19 in bipolar magnet block use contactless setting, and two
Person is to be arranged in parallel.In preferred embodiment, bipolar magnet block is provided with left and right sides of the sample application zone front end, equally
Ground, in the left and right sides of the front end of motor magnet assembly 19, distribution is provided with parallel and non-with sample application zone bipolar magnet block
The bipolar magnet block of contact, such as can be 45 ° or 135 ° settings.Contactless setting can use non-magnetic dividing plate to carry out
Barrier, simultaneously because the presence in magnetic field, the bipolar magnet of bipolar magnet block on motor magnet assembly 19 to sample rack
Block 21 has magnetic field repulses power, so as to drive sample rack side-to-side movement on guide rail.Heretofore described bipolar magnet block is
Refer to the neodymium-iron-boron iron block that both ends are respectively N and S poles, its two sides magnetic polarity is opposite.According to magnetic field, identical charges repel each other, there is a natural attraction between the sexes
The S poles of principle, the N poles of magnet and other one block of magnet attract each other, and magnetic field repulsive force be present between both N poles (or S poles), this
Inventive embodiments make use of magnetic field repulsive force to drive the rack for test tube in sample application zone to carry out moving back and forth switching.
More specifically, the sample motion guide rail device also includes motor and transported for drive motor magnet assembly 19
Dynamic timing belt 7, by motor driven timing belt 7, move motor magnet assembly 19.The motor is preferably stepper motor
3, the timing belt 7 takes turns operating to move by 15 tooth MXL timing belts 7, and the motor magnet assembly 19 is arranged on the sample
The outside of this motion guide rail device, the wheel operating of 15 tooth MXL timing belts 7 is driven by stepper motor 3, moves timing belt 7, and make
Motor magnet assembly 19 on timing belt 7 is moved, while is led by the use of same polarity magnetic field repulsive force as power, drive
Moved sample application zone on rail.
In preferred embodiment, the rack for test tube for placing PCR reaction tubes is provided with the sample application zone.For example, the examination
Pipe support can place the PCR reaction tubes or eight independent PCR reaction tubes of eight unions.
In addition, the constant temperature heating device includes 2 flat-temperature zones：High-temperature constant warm area and cryogenic thermostat area, wherein high-temperature constant
The temperature that warm area is set can be any temperature value between 90 DEG C -95 DEG C；Cryogenic thermostat area set temperature can be 55 DEG C-
Any temperature value between 72 DEG C.When specifically being detected, the high-temperature constant warm area or cryogenic thermostat area are with fixed temperature
Come what is heated, its high temperature is optimal with 94 DEG C, and low temperature is optimal with 64 DEG C.Two flat-temperature zones are all-sealed structure, the two
Between it is separate by heat-insulated valve.In existing DNA cloning technology, annealing and extension share a temperature, therefore only need
64 DEG C of flat-temperature zones can meet to anneal and extend demand；Other 94 DEG C can meet Deformation Demands.In addition, in the cryogenic thermostat
Area is provided with hole position, and the fluorescence detection device is fixedly installed at the hole position in cryogenic thermostat area, anti-to single PCR by hole position
Should pipe detected in real time.
Preferably, the constant temperature heating device is heated using heating film, i.e., described flat-temperature zone respectively has one layer to add up and down
Hot plate, it is for instance possible to use PI heating film contacts are heated, flat-temperature zone uses heating film independent control temperature, precision height
Up to ± 0.1 DEG C.Heat conduction can be carried out by metal in addition, such as by metal aluminum blocks, transfer heat to PCR reaction tubes, make
It is rapidly achieved reaction temperature, and heat conduction efficiency is high, substantially increases the speed of service and the detection of fluorescence quantitative PCR detection system
Efficiency.Specifically, the cuvette groove on the rack for test tube can be used made by metallic aluminium, and is internally provided with temperature in cuvette groove
Sensor is spent, constant temperature is controlled using pid algorithm, realizes that reaction type is heated, effectively improves temperature control precision.
In addition, described fluorescence detection device includes mono-colour laser and detector.Specifically, the fluorescence detection device
Detected using fluorescence co-focusing method, it is mainly made up of spectroscope, optical filter, convex lens and mono-colour laser, fixed
Single PCR reaction tubes are detected in real time at the hole position of the lower right side in cryogenic thermostat area, and by hole position.Wherein, it is described
The excitation wavelength of monochromatic long wavelength laser is 488nm, and the detector is photocell element, and detection wavelength of fluorescence is 520nm-
565nm, preferably 540nm.
The present invention also provides a kind of fluorescent quantitative PCR detection method based on magnetomotive switching flat-temperature zone in addition, and it includes
Following steps：
A, sample is placed on the PCR reaction tube rack for test tubes of sample application zone, and by sample motion guide rail device by sample
It is sent to high-temperature constant warm area and keeps 12-17s, carries out DNA cracking；
B, sample is sent to from high-temperature constant warm area by cryogenic thermostat area by the device of sample motion guide rail 11 again and keeps 28-
32s, carry out DNA cloning；
C, when sample leaves cryogenic thermostat area, fluoroscopic examination is carried out by fluorescence detection device successively to sample；
D, repeat the above steps n times；N≤35 time.
1-3 below in conjunction with the accompanying drawings, switch the fluorescent quantitation of flat-temperature zone based on magnetomotive to the present invention by specific embodiment
The specific implementation process of PCR detection system and method is illustrated：
First stage：In sample application zone, after sample is added into PCR reaction tubes and capping, it is put into loading slot assembly 5.
In flat-temperature zone, the bottom of heating tank assembly 6 is heated to 94 DEG C by PI films and keeps constant temperature (high-temperature constant warm area), heating tank
The bottom of assembly 13 is heated to 64 DEG C by PI films and keeps constant temperature (cryogenic thermostat area), simultaneously closes off valve assembly 18, beats
Valve opening door motor board assembly 20.Motor magnet assembly 19 is moved to high-temperature constant warm area under the drive of stepper motor 3, leads to
Cross homopolar-repulsion principle and produce external magnetic field power, motor magnet assembly 19 is driven loading slot assembly 5 on guide rail 11
Moved, the shutoff valve door under the driving of stepper motor of valve driving machine board assembly 20 so that loading slot assembly 5 is in high temperature
15s is kept in flat-temperature zone.
Second stage：Valve assembly 18 is opened, loading slot assembly 5 moves in the external magnetic field of motor magnet assembly 19
Under power effect, the top of heating tank assembly 13 is moved to, closes valve assembly 18 so that loading slot assembly 5 is permanent in low temperature
30s is kept in warm area.
Phase III：Valve assembly 18 is opened, loading slot assembly 5 moves in the external magnetic field of motor magnet assembly 19
Under power effect, cryogenic thermostat area is left, in the process, eight PCR reaction tubes of loading slot assembly 5 pass sequentially through fluorescence inspection
Survey device and carry out fluorescence signal acquisition.
Fourth stage：Carry out 40 circulations of second and third stage.By above-mentioned circulation, can detect in real time in PCR reaction tubes
Amplified production DNA content, and storage original DNA concentration can be calculated by standard curve, so as to reach the purpose of quantitative detection.
In summary, the homopolar-repulsion principle of the invention by using magnet, it is bipolar using being equipped with timing belt
Property magnet piece motor magnet assembly, drive and be equally equipped with the sample application zone of bipolar magnet block by guide rail in different constant temperature
Motion is toggled between area, substantially reduces proliferation time, and temperature control is more accurate, improves PCR detection effect
Rate, the present invention have high practicality and wide market application foreground.
It should be appreciated that the above-mentioned description for specific embodiment is more detailed, therefore can not be considered to this
The limitation of invention patent protection scope, scope of patent protection of the invention should be determined by the appended claims.
Claims (10)
1. a kind of fluorescence quantitative PCR detection system based on magnetomotive switching flat-temperature zone, it is characterised in that filled including heated at constant temperature
Put, sample motion guide rail device and fluorescence detection device；The constant temperature heating device, fluorescence detection device with the sample
This motion guide rail device connects；
Wherein described constant temperature heating device includes the flat-temperature zone of at least two different temperatures；The sample motion guide rail device includes
Motor magnet assembly, the sample application zone for placing sample and the guide rail for connecting sample application zone and flat-temperature zone, the sample-adding
Area is polarity identical bipolar magnet block with being respectively arranged with opposite face on the motor magnet assembly, passes through bipolarity magnetic
Magnetic field repulsive force caused by iron block drives sample application zone to be moved along guide rail, is toggled between the flat-temperature zone of different temperatures, carries out
Sample expands.
2. the fluorescence quantitative PCR detection system according to claim 1 based on magnetomotive switching flat-temperature zone, it is characterised in that
The sample motion guide rail device also includes motor and the timing belt for the motion of drive motor magnet assembly, passes through motor
Timing belt is driven, moves motor magnet assembly.
3. the fluorescence quantitative PCR detection system according to claim 1 based on magnetomotive switching flat-temperature zone, it is characterised in that
Bipolar magnet block in the sample application zone is set with the bipolar magnet block in the motor magnet assembly using contactless
Put.
4. the fluorescence quantitative PCR detection system according to claim 1 based on magnetomotive switching flat-temperature zone, it is characterised in that
The rack for test tube for placing PCR reaction tubes is provided with the sample application zone.
5. the fluorescence quantitative PCR detection system according to claim 4 based on magnetomotive switching flat-temperature zone, it is characterised in that
Heat-insulated valve is provided between the flat-temperature zone of the different temperatures.
6. the fluorescence quantitative PCR detection system according to claim 5 based on magnetomotive switching flat-temperature zone, it is characterised in that
The constant temperature heating device includes high-temperature constant warm area and cryogenic thermostat area；The temperature of the high-temperature constant warm area is in 90 DEG C -95 DEG C
Any temperature value, the temperature in the cryogenic thermostat area is any temperature value in 55 DEG C -72 DEG C.
7. the fluorescence quantitative PCR detection system according to claim 5 based on magnetomotive switching flat-temperature zone, it is characterised in that
The constant temperature heating device is heated using heating film.
8. the fluorescence quantitative PCR detection system according to claim 6 based on magnetomotive switching flat-temperature zone, it is characterised in that
The cryogenic thermostat area is provided with hole position, and the fluorescence detection device is fixedly installed at the hole position in cryogenic thermostat area, passes through hole
Single PCR reaction tubes are detected position in real time.
9. the fluorescence quantitative PCR detection system according to claim 8 based on magnetomotive switching flat-temperature zone, it is characterised in that
Described fluorescence detection device includes mono-colour laser and detector；Wherein described detector is photocell element.
10. a kind of fluorescent quantitative PCR detection method based on magnetomotive switching flat-temperature zone, it is characterised in that comprise the following steps：
A, sample is placed on the PCR reaction tube rack for test tubes of sample application zone, and transmitted sample by sample motion guide rail device
To high temperature flat-temperature zone and 12-17s is kept, carries out DNA cracking；
B, sample is sent to from high-temperature constant warm area by cryogenic thermostat area by sample motion guide rail device again and keeps 28-32s, entered
Row DNA cloning；
C, when sample leaves cryogenic thermostat area, fluoroscopic examination is carried out by fluorescence detection device successively to sample；
D, repeat the above steps n times；N≤35 time.
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