CN110564610B - Double-temperature-zone PCR amplification device - Google Patents
Double-temperature-zone PCR amplification device Download PDFInfo
- Publication number
- CN110564610B CN110564610B CN201910976547.8A CN201910976547A CN110564610B CN 110564610 B CN110564610 B CN 110564610B CN 201910976547 A CN201910976547 A CN 201910976547A CN 110564610 B CN110564610 B CN 110564610B
- Authority
- CN
- China
- Prior art keywords
- plate
- guide
- temperature
- temperature control
- stepping motor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000012408 PCR amplification Methods 0.000 title claims abstract description 48
- 230000033001 locomotion Effects 0.000 claims abstract description 21
- 229910052751 metal Inorganic materials 0.000 claims description 20
- 239000002184 metal Substances 0.000 claims description 20
- 239000004065 semiconductor Substances 0.000 claims description 12
- 229910052782 aluminium Inorganic materials 0.000 claims description 8
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 230000009977 dual effect Effects 0.000 claims description 5
- 229910001369 Brass Inorganic materials 0.000 claims description 3
- 239000004677 Nylon Substances 0.000 claims description 3
- 230000009471 action Effects 0.000 claims description 3
- 239000010951 brass Substances 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 229920001778 nylon Polymers 0.000 claims description 3
- 239000004033 plastic Substances 0.000 claims description 3
- 230000005484 gravity Effects 0.000 claims description 2
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 2
- 230000001960 triggered effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 13
- 230000000630 rising effect Effects 0.000 abstract description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 56
- 230000003321 amplification Effects 0.000 description 17
- 238000003199 nucleic acid amplification method Methods 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 14
- 108020004707 nucleic acids Proteins 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 238000010586 diagram Methods 0.000 description 8
- 238000001962 electrophoresis Methods 0.000 description 8
- 241000193163 Clostridioides difficile Species 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 101150032575 tcdA gene Proteins 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940057995 liquid paraffin Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000017525 heat dissipation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a double-temperature-zone PCR amplification device, which comprises a bottom plate, a main control circuit board, a motion module assembly and a microfluidic PCR plate motion assembly, wherein two vertical plates are arranged on the bottom plate, horizontal guide rails are arranged at the top ends of the two vertical plates, the motion module assembly comprises a stepping motor, a horizontal guide fixed plate, a vertical fixed support and a stepping motor, the bottom ends of the guide fixed plates are respectively in sliding fit with the two guide rails through two guide seats, the fixed support and one end of the guide fixed plate, the stepping motor is fixedly arranged on the fixed support, the output shaft of the stepping motor faces downwards and is fixedly provided with a gear, racks meshed with the gear are fixedly arranged on the bottom plate, and two constant temperature control modules are arranged in the interval between the two vertical plates; the microfluidic PCR plate moving assembly comprises a fixed frame, wherein a microfluidic PCR plate fixed box is connected to the fixed frame, and a microfluidic PCR plate is arranged in the microfluidic PCR plate fixed box. The dual-temperature-zone PCR amplification device has the advantages of high temperature rising and falling speed and short detection time.
Description
Technical Field
The invention relates to the technical field of biological and medical detection instruments, in particular to a double-temperature-zone PCR amplification device.
Background
The PCR amplification device is an instrument for amplifying a specific DNA by PCR (Polymerase chain reaction ) technology, and is widely used in medical and biological laboratories, for example, for determining whether a genetic disease is present in a sample, for diagnosing infectious diseases, for gene replication, and for paternity test.
The PCR amplification unit is generally composed of a heat cover part, a heat cycle part, a transmission part, a control part, a power supply part, and the like. The PCR apparatus can be classified into the following three types according to the temperature rising medium at the time of DNA amplification:
(1) Temperature-changing aluminum block type PCR instrument: the heat source is made of resistance wire, conductive heat film and heat pump type Peltier semiconductor element, and is heated by aluminum block with concave hole, cooled by tap water, refrigerating compressor or semiconductor; the advantages are that: the temperature conduction is fast, and the amplification consistency of each tube is good; when the specifications of the reaction tubes are consistent no paraffin oil is required to be coated outside; the microcomputer can be used for regulating temperature conversion; the instrument refrigeration component can be cooled to 4 ℃ after amplification is completed, and the sample is stored overnight; disadvantages: the temperature of the reaction liquid in the tube is lagged compared with the temperature of the aluminum block; a special thin-wall heat-resistant reaction tube which is closely matched with the shape of the concave hole of the aluminum block is needed to be used; the heat capacity of the aluminum block is difficult to overcome rapidly during temperature changing; the compressor is slow in refrigeration start, heavy and long in lag time.
(2) Water bath type PCR instrument: the instrument has 3 water baths with different temperatures, and a mechanical device is used for shifting and raising the temperature of the rack with the reaction tube so as to circulate the temperature; the advantages are that: water is a heat transfer medium, the temperature is easy to be constant, and the heat capacity is large; the shape of the reaction tube has no special requirement, and the temperature conversion is faster and the amplification effect is stable; the method has higher operation efficiency and good specificity of amplified products; disadvantages: the high-temperature bath is unstable, and the water surface is required to be covered by liquid paraffin; the time required for changing the temperature of the water bath is long, the operation of complex procedures (such as nested PCR) is not easy to implement, and the volume of the instrument is large; the room temperature affects the lower temperature limit.
(3) Temperature-variable flow PCR instrument: according to the dynamics principle of air flow, the temperature is raised and lowered by taking cold and hot air flow as a medium; the advantages are that: the temperature change is rapid, the amplification effect is good, and the method is suitable for micro and rapid PCR; the reactor is not limited by shape, and liquid paraffin is not required to be coated outside the tube; measuring the temperature of the liquid in the pipe as a temperature control basis, and displaying the temperature truly and reliably; the microcomputer is easy to set a complex temperature changing program; the portable instrument with lighter weight is easy to manufacture, and is suitable for outgoing operation; disadvantages: the room temperature is taken as the lower temperature limit, the low temperature is difficult to control, the dynamic requirement on the air flow is high, and the temperature of each pipe can be uniform by careful design.
It can be seen that the existing PCR device generally has the problems of low temperature rising and falling speed, difficult temperature control, slow liquid heat conduction and large equipment volume.
Disclosure of Invention
The invention aims to provide a double-temperature-zone PCR amplification device, which solves the problems in the prior art, improves the temperature rise and fall speed of the PCR device, shortens the detection time of the PCR amplification device and improves the sensitivity and consistency.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides a double-temperature-zone PCR amplification device which comprises a bottom plate, a main control circuit board, a motion module assembly and a microfluidic PCR plate motion assembly, wherein two vertical plates which are parallel to each other and are opposite to each other are arranged on the bottom plate at intervals, the top ends of the two vertical plates are the same in height and are provided with horizontal guide rails, the main control circuit board is fixedly arranged on the bottom plate, the motion module assembly comprises a stepping motor, a horizontal guide fixing plate, a vertical fixing support and a stepping motor, the bottom end of the guide fixing plate is fixedly provided with two guide seats, the two guide seats are respectively in sliding fit with the two guide rails, one guide seat is fixedly connected with one end of the guide fixing plate, one guide seat is positioned between the other guide seat and the fixing support, the stepping motor is fixedly arranged on the fixing support, the output shaft of the stepping motor is downwards and is fixedly provided with a gear, the bottom plate is fixedly provided with a rack which is parallel to the vertical plate and is meshed with the gear, the bottom end of the fixing support is fixedly provided with a baffle, the bottom plate is fixedly provided with two photoelectric switches along the length direction of the two photoelectric switches, and the photoelectric switches can be arranged at random; two constant temperature control modules are arranged in the interval between the two vertical plates, the two constant temperature control modules are respectively positioned at two ends of the interval, the two photoelectric switches are in one-to-one correspondence with the two constant temperature control modules, one constant temperature control module is a high temperature control module, and the other constant temperature control module is a low temperature control module;
The microfluidic PCR plate moving assembly comprises a fixed frame, wherein a microfluidic PCR plate fixed box is detachably connected to the fixed frame, and a microfluidic PCR plate is arranged in the microfluidic PCR plate fixed box; the fixing frame is connected with the guide fixing plate through a plurality of connecting components, the connecting components comprise a linear bearing arranged on the guide fixing plate and a guide shaft, the bottom end of the guide shaft is fixedly connected with the fixing frame, and the guide shaft is in sliding fit with the linear bearing; each vertical plate is provided with a sliding groove on the side wall facing the other vertical plate, the two sliding grooves are flat, and the two sliding grooves are in sliding fit with the fixed frame; the height of the bottom surface of the chute is fluctuated; the constant temperature control module, the stepping motor and the photoelectric switch are all electrically connected with the main control circuit board.
Preferably, the connecting assembly further comprises a spring sleeved on the guide shaft, the top end of the spring abuts against the bottom surface of the guide fixing plate, and the bottom end of the spring abuts against the top surface of the fixing frame.
Preferably, the bottom surface of the chute is high in the middle and low at both ends.
Preferably, the constant temperature control module comprises a heat conduction metal block, a temperature sensor, a semiconductor refrigerating sheet and a radiator, wherein the temperature sensor is fixedly sealed in a preformed hole of the heat conduction metal block by heat conduction glue, the semiconductor refrigerating sheet is arranged in a square groove of the radiator, the heat conduction metal block is arranged at the top of the radiator, the microfluidic PCR plate can be tightly attached to the top surface of the heat conduction metal block, and the temperature sensor is electrically connected with the main control circuit board through a signal line.
Preferably, the material of the heat conducting metal block is brass or aluminum, the size of the semiconductor refrigerating sheet is 40mm by 40mm, and the material of the radiator is aluminum oxide.
Preferably, the guide seat is connected with the guide fixing plate through a fixing screw, and the fixing screw is a plastic nylon screw; the fixed frame corresponds two the spout is provided with a gyro wheel respectively, the gyro wheel pass through the gyro wheel support with the direction fixed plate is connected, the gyro wheel can be in corresponding the spout is moved.
Preferably, the fixed box comprises an upper box and a lower box which are connected in a rotating way, two silica gel pads are arranged in the lower box, and two lock catches are arranged at the edge of the upper box.
Preferably, the bottoms of both the risers are provided with a void.
Compared with the prior art, the double-temperature-zone PCR amplification device has the following technical effects:
the double-temperature-zone PCR amplification device has the advantages of high sensitivity, good consistency, high temperature rise and fall speed and short detection time. The double-temperature-zone PCR amplification device reduces time loss and temperature fluctuation caused by temperature rise and reduction, greatly reduces power consumption, does not need a fan to carry out auxiliary heat dissipation, has a more compact structure, greatly shortens the PCR amplification time, improves the sensitivity and repeatability of PCR detection, and reduces the power consumption of the device.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a dual temperature zone PCR amplification device according to the present invention;
FIG. 2 is a schematic diagram showing a structure of a dual-temperature-zone PCR amplification device according to the present invention;
FIG. 3 is a schematic diagram showing the structure of a thermostatic control module in the dual-temperature-zone PCR amplification device of the present invention;
FIG. 4 is a schematic diagram showing a part of the structure of a dual-temperature-zone PCR amplification device according to the present invention;
FIG. 5 is a schematic diagram of the structure of a motion assembly of a microfluidic PCR plate in a dual-temperature-zone PCR amplification device of the present invention;
FIG. 6 is a schematic structural diagram of a microfluidic PCR plate cartridge in a dual-temperature-zone PCR amplification device of the present invention;
FIG. 7 is a diagram showing the comparison of electrophoresis results of the sensitivity detection of the dual-temperature-zone PCR amplification device of the present invention with that of a conventional PCR instrument, wherein a 1-100 bp DNA Marker is provided; the amplification products of the traditional PCR instrument for the sample to be tested are sequentially 20 ng/. Mu.L, 4 ng/. Mu.L, 0.8 ng/. Mu.L, 0.16 ng/. Mu.L and 0.032 ng/. Mu.L from 2 to 9; 1, 100bp DNA Marker; the amplification products of the double-temperature-zone PCR instrument of the sample to be detected are sequentially 20 ng/. Mu.L, 4 ng/. Mu.L, 0.8 ng/. Mu.L, 0.16 ng/. Mu.L and 0.032 ng/. Mu.L from the lower 2-9;
FIG. 8 is a diagram showing the comparison of electrophoresis results of the sensitivity detection of the dual-temperature-zone PCR amplification device and the conventional PCR instrument, wherein the left 5 channels are amplification products of the conventional PCR instrument, the right 5 channels are amplification products of the dual-temperature-zone PCR instrument, and the middle channel is a 100bp DNA Marker;
Wherein: the device comprises a base plate, a 2-main control circuit board, a 3-first vertical plate, a 4-first constant temperature control module, a 5-sliding groove, a 6-second vertical plate, a 7-microfluidic PCR plate movement assembly, an 8-microfluidic PCR plate fixing box, a 9-movement module assembly, a 10-roller, a 11-second constant temperature control module, a 12-baffle, a 13-gear, a 14-stepping motor, a 15-rack, a 16-photoelectric switch, a 17-radiator, a 18-fixing screw, a 19-semiconductor refrigerating sheet, a 20-temperature sensor, a 21-heat conducting metal block, a 22-linear bearing, a 23-guiding fixing plate, a 24-roller support, a 25-fixing frame, a 26-spring, a 27-guiding shaft, a 28-microfluidic PCR plate, a 29-upper box, a 30-first silica gel pad, a 31-lower box, a 32-first lock catch, a 33-second lock catch, a 34-second silica gel pad, a 35-fixing support, a 36-micro-pressing elastic bead lock catch, a 37-guide rail and a 38-guide seat.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by a person skilled in the art based on the embodiments of the invention without any inventive effort, are intended to fall within the scope of the invention.
The invention aims to provide a double-temperature-zone PCR amplification device, which solves the problems in the prior art, improves the temperature rise and fall speed of the PCR device, shortens the detection time of the PCR amplification device and improves the sensitivity and consistency.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.
As shown in fig. 1-6: the double-temperature-zone PCR amplification device comprises a bottom plate 1, a main control circuit board 2, a motion module assembly 9 and a microfluidic PCR plate 28 motion assembly 7. The main control circuit board 2 is fixedly arranged on the bottom plate 1, and the main control circuit board 2 is composed of a PLC module DVP28SA2110T and a temperature control module DVP02 TUN-S.
Two mutually parallel opposite vertical plates which are arranged at intervals are arranged on the bottom plate 1, namely a first vertical plate 3 and a second vertical plate 6, the top ends of the two vertical plates are the same in height and are provided with horizontal guide rails 37, the motion module assembly 9 comprises a stepping motor 14, a horizontal guide fixing plate 23, a vertical fixing support 35 and the stepping motor 14, two guide seats 38 are fixedly arranged at the bottom end of the guide fixing plate 23, the guide seats 38 are connected with the guide fixing plate 23 through fixing screws 18, and the fixing screws 18 are plastic nylon screws; the two guide seats 38 are respectively in sliding fit with the two guide rails 37, the fixed support 35 is fixedly connected with one end of the guide fixed plate 23, one guide seat 38 is positioned between the other guide seat 38 and the fixed support 35, the stepping motor 14 is fixedly arranged on the fixed support 35, the output shaft of the stepping motor 14 faces downwards and is fixedly provided with the gear 13, the bottom plate 1 is fixedly provided with the rack 15 which is parallel to the vertical plate and is meshed with the gear 13, the bottom end of the fixed support 35 is fixedly provided with the baffle 12, the bottom plate 1 is fixedly provided with two photoelectric switches 16 which are arranged along the length direction of the vertical plate, and the baffle 12 can block a column and trigger any photoelectric switch 16, preferably a groove type photoelectric switch;
two constant temperature control modules, namely a first constant temperature control module 4 and a second constant temperature control module 11, are arranged in the interval between the two vertical plates, and are respectively positioned at two ends of the interval, wherein one constant temperature control module is a high temperature control module, and the other constant temperature control module is a low temperature control module; each constant temperature control module comprises a heat conduction metal block 21, a temperature sensor 20, a semiconductor refrigerating sheet 19 and a radiator 17, wherein the temperature sensor 20 is fixedly sealed in a reserved hole of the heat conduction metal block 21 by heat conduction glue, the semiconductor refrigerating sheet 19 is arranged in a square groove of the radiator 17, the heat conduction metal block 21 is arranged at the top of the radiator 17, the microfluidic PCR plate 28 can be tightly attached to the top surface of the heat conduction metal block 21, and the temperature sensor 20 is electrically connected with the main control circuit board 2 through a signal wire. The heat conducting metal block 21 is made of brass or aluminum, so that the heat conducting performance is high, and the temperature rising and falling speed of the microfluidic PCR plate 28 is improved; the semiconductor refrigerating sheet 19 has a size of 40mm by 40mm, the radiator 17 is a section radiator and an insert radiator, and the radiator 17 is made of alumina.
The positions of the two photoelectric switches 16 are in one-to-one correspondence with the positions of the two constant temperature control modules, so that the movement positions of the stepping motor 14 are controlled through the setting positions of the photoelectric switches 16, and when the baffle 12 moves to the positions of the photoelectric switches 16 and the photoelectric switches 16 are blocked to trigger the photoelectric switches 16, the main control circuit board controls the stepping motor 14 to stop moving, so that the microfluidic PCR plate 28 can accurately stay on the surfaces of the heat conducting metal blocks 21 of the two constant temperature control modules.
The microfluidic PCR plate 28 moving assembly 7 comprises a fixed frame 25, a microfluidic PCR plate 28 fixed box 8 is detachably connected to the fixed frame 25, a push type miniature elastic latch lock 36 is arranged in the fixed frame 25, an arrow-shaped lock head is arranged at the upper part of the microfluidic PCR plate 28 fixed box 8, the lock head is plugged into the fixed frame 25 according to the arrow direction, and the lock head is automatically locked with the push type miniature elastic latch lock 36; the microfluidic PCR plate 28 is arranged in a fixed box 8 of the microfluidic PCR plate 28, the fixed box comprises an upper box 29 and a lower box 31 which are connected in a rotating way, two silica gel mats are arranged in the lower box 31, a first silica gel mat 30 and a second silica gel mat 34 are respectively arranged in the lower box 31, two lock catches are arranged at the edge of the upper box 29, a first lock catch 32 and a second lock catch 33 are respectively arranged in the edge of the upper box 29, and the microfluidic PCR plate 28 is arranged between the upper box 29 and the lower box 31; the fixed frame 25 is connected with the guide fixed plate 23 through a plurality of connecting components, each connecting component comprises a linear bearing 22 arranged on the guide fixed plate 23, a guide shaft 27 with the bottom end fixedly connected with the fixed frame 25 and a spring 26 sleeved on the guide shaft 27, the top end of the spring 26 is propped against the bottom surface of the guide fixed plate 23, the bottom end of the spring 26 is propped against the top surface of the fixed frame 25, and the guide shaft 27 is in sliding fit with the linear bearing 22; each riser faces to the lateral wall of the other riser and is provided with a chute 5, the two chutes 5 are leveled, the two chutes 5 are in sliding fit with a fixed frame 25, the fixed frame 25 is provided with a roller 10 corresponding to the two chutes 5 respectively, the roller 10 is connected with a guide fixed plate 23 through a roller bracket 24, and the roller 10 can move in the corresponding chute 5.
The height of the bottom surface of the chute 5 is fluctuated, in the embodiment, the middle of the height of the bottom surface of the chute 5 is high, and the two ends of the bottom surface are low, when the moving assembly 7 of the microfluidic PCR plate 28 moves to the two ends of the chute 5, the bottom surface of the chute is tightly attached to the heat-conducting metal block 21 in the temperature control module under the action of self gravity and the elastic force of the spring 26; the constant temperature control module, the stepping motor 14 and the photoelectric switch 16 are all electrically connected with the main control circuit board 2.
In this embodiment, the bottoms of both risers are provided with a void to save material for the risers and reduce the overall weight of the device.
The use process of the double-temperature-zone PCR amplification device of the embodiment is as follows:
Firstly, a microfluidic PCR plate 28 is placed in a microfluidic PCR plate 28 fixed box 8, then the microfluidic PCR fixed box is inserted into a fixed frame 25, the microfluidic PCR fixed box is automatically buckled by an elastic latch, a main control circuit board 2 is started to work through computer operation, a stepping motor 14 rotates, a gear 13 arranged on the shaft of the stepping motor 14 is matched with a rack 15 to do linear motion to drive a motion module assembly 9 to do linear motion, a roller 10 rolls along a motion track plane, a microfluidic PCR plate 28 motion assembly 7 moves up and down through compression and extension of a spring 26, heating and cooling are stopped on the surfaces of two constant temperature heating module heat conduction metal blocks 21 under the action of a photoelectric switch 16, the main control circuit board 2 controls the microfluidic PCR motion assembly to rapidly switch between two constant temperature control modules at 95 ℃ and 60 ℃, and data such as temperature, amplification time, circulation number and the like are transmitted to a computer to perform calculation processing, so that the whole PCR amplification process is completed.
The double-temperature-zone PCR amplification device is compared with the traditional PCR instrument in time: a pair of primers is designed aiming at clostridium difficile tcdA gene (amplification fragment length is 369 bp), and the primer sequences are as follows: the upstream primer tcdA1F:5'-AGATTCCTATATTTACATGACAATAT-3' (SEQ ID NO: 1), and the downstream primer tcdA1R:5'-GTATCAGGCATAAAGTAATATACTTT-3' (SEQ ID NO: 2). The preparation reaction system is as follows: premixExTaqHS12.5. Mu.L, nucleic-freewater 8.5.5. Mu.L, tcdA-F1. Mu.L, tcdA-R1. Mu.L. Clostridium difficile clinical sample cultures were nucleic acid extracted using the Qiagen nucleic acid extraction kit (cat# 51306) and diluted to a nucleic acid concentration of 20 ng/. Mu.L at standard as the test samples.
The PCR amplification reaction was performed simultaneously using the double-temperature-zone PCR amplification apparatus of the present invention and a Bio-RadC1000PCR instrument (Bio-Rad Co.). Wherein, the volume of the reaction system of the double-temperature-zone PCR amplification device is 9 mu l, 1 mu l of sample to be detected is added respectively, and the amplification conditions are that the pre-start is performed for 8s at 95 ℃, 7s at 95 ℃ and 20s at 60 ℃ for 40 cycles; the volume of the reaction system of the Bio-RadC PCR instrument is 25. Mu.l, 2. Mu.l of the sample to be detected is added respectively, and the amplification conditions are that the PCR instrument is pre-started for 15min at 95 ℃, 30s at 94 ℃, 30s at 52 ℃,40 s at 72 ℃,35 cycles and 5min at 72 ℃. And the amplification time of the double-temperature-zone PCR amplification device is 20min, the amplification time of the Bio-RadC1000PCR instrument is 106min, and the amplification time of the double-temperature-zone PCR amplification device is far shorter than that of the Bio-RadC1000PCR instrument.
The double-temperature-zone PCR amplification device is compared with the electrophoresis result of the sensitivity detection of the traditional PCR instrument:
The clinical sample culture of Clostridium difficile was subjected to nucleic acid extraction using Qiagen nucleic acid extraction kit (cat# 51306), calibration and gradient dilution to nucleic acid concentrations of 20 ng/. Mu.L, 4 ng/. Mu.L, 0.8 ng/. Mu.L, 0.16 ng/. Mu.L, and 0.032 ng/. Mu.L, and the PCR amplification reaction was performed simultaneously using the double-temperature-zone PCR amplification apparatus and Bio-RadC1000PCR instrument (Bio-Rad Co.) of the present invention as a sample to be tested, and the reaction system and amplification conditions were the same as in example two.
All PCR amplified products were detected by electrophoresis on a 1.5% agarose gel, and the result of electrophoresis (see FIG. 7). As can be seen from the results shown in FIG. 7, the detection sensitivity of the dual-temperature-zone PCR amplification device and the Bio-RadC1000,1000 PCR instrument for the clostridium difficile tcdA gene were both 0.16 ng/. Mu.L, and the detection sensitivity of the dual-temperature-zone PCR amplification device was identical to that of the Bio-RadC1000,1000 PCR instrument.
Comparing the electrophoresis result of the double-temperature-zone PCR amplification device with that of the traditional PCR instrument for repeated detection:
The clinical sample culture of Clostridium difficile was subjected to nucleic acid extraction using Qiagen nucleic acid extraction kit (cat# 51306), calibration and dilution to a nucleic acid concentration of 20 ng/. Mu.L, and as a sample to be tested, PCR amplification reaction was simultaneously carried out using the double-temperature-zone PCR amplification apparatus of the present invention and a Bio-RadC1000PCR instrument (Bio-Rad Co.) and the sample was subjected to 5 repetitions, and the reaction system and amplification conditions were the same as those of example two. All PCR amplified products were detected by electrophoresis on a 1.5% agarose gel, and the result of electrophoresis (see FIG. 8). As can be seen from the results shown in FIG. 8, the double-temperature-zone PCR amplification device and the Bio-RadC-1000 PCR instrument were positive and basically consistent in brightness for 5 duplicate wells of the clostridium difficile tcdA gene, and the double-temperature-zone PCR amplification device was consistent in detection reproducibility with the Bio-RadC-1000 PCR instrument.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "top", "bottom", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The principles and embodiments of the present invention have been described in this specification with reference to specific examples, the description of which is only for the purpose of aiding in understanding the method of the present invention and its core ideas; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (5)
1. A double-temperature-zone PCR amplification device is characterized in that: the device comprises a bottom plate, a main control circuit board, a motion module assembly and a microfluidic PCR plate motion assembly, wherein two vertical plates which are parallel to each other, opposite to each other and arranged at intervals are arranged on the bottom plate, the top ends of the two vertical plates are the same in height and are provided with horizontal guide rails, the main control circuit board is fixedly arranged on the bottom plate, the motion module assembly comprises a stepping motor, a horizontal guide fixing plate, a vertical fixing support and a stepping motor, two guide seats are fixedly arranged at the bottom end of the guide fixing plate, the two guide seats are respectively in sliding fit with the two guide rails, the fixing support is fixedly connected with one end of the guide fixing plate, one guide seat is positioned between the other guide seat and the fixing support, the stepping motor is fixedly arranged on the fixing support, the output shaft of the stepping motor faces downwards and is fixedly provided with a gear, the bottom plate is fixedly provided with a baffle which is parallel to the vertical plates and is meshed with the gear, and the bottom end of the fixing support is fixedly provided with two photoelectric switches which are arranged along the length direction of the vertical plates, and the photoelectric switch can be triggered by any photoelectric switch column; two constant temperature control modules are arranged in the interval between the two vertical plates, the two constant temperature control modules are respectively positioned at two ends of the interval, the two photoelectric switches are in one-to-one correspondence with the two constant temperature control modules, one constant temperature control module is a high temperature control module, and the other constant temperature control module is a low temperature control module;
The microfluidic PCR plate moving assembly comprises a fixed frame, wherein a microfluidic PCR plate fixed box is detachably connected to the fixed frame, and a microfluidic PCR plate is arranged in the microfluidic PCR plate fixed box; the fixing frame is connected with the guide fixing plate through a plurality of connecting components, the connecting components comprise a linear bearing arranged on the guide fixing plate and a guide shaft, the bottom end of the guide shaft is fixedly connected with the fixing frame, and the guide shaft is in sliding fit with the linear bearing; each vertical plate is provided with a sliding groove on the side wall facing the other vertical plate, the two sliding grooves are flat, and the two sliding grooves are in sliding fit with the fixed frame; the height of the bottom surface of the chute is fluctuated; the constant temperature control module, the stepping motor and the photoelectric switch are electrically connected with the main control circuit board;
The connecting assembly further comprises a spring sleeved on the guide shaft, the top end of the spring is propped against the bottom surface of the guide fixing plate, and the bottom end of the spring is propped against the top surface of the fixing frame; the middle of the height of the bottom surface of the chute is high, and the two ends of the bottom surface of the chute are low; when the microfluidic PCR plate moving assembly moves to the two ends of the chute, the microfluidic PCR plate moving assembly is tightly attached to the heat conducting metal block in the constant temperature control module under the action of self gravity and the elastic force of the spring; the bottoms of the two vertical plates are provided with a hollow part.
2. The dual temperature zone PCR amplification device as set forth in claim 1, wherein: the constant temperature control module comprises a heat conduction metal block, a temperature sensor, a semiconductor refrigerating sheet and a radiator, wherein the temperature sensor is fixedly sealed in a preformed hole of the heat conduction metal block by heat conduction glue, the semiconductor refrigerating sheet is arranged in a square groove of the radiator, the heat conduction metal block is arranged at the top of the radiator, the microfluidic PCR plate can be tightly attached to the top surface of the heat conduction metal block, and the temperature sensor is electrically connected with the main control circuit board through a signal wire.
3. The dual temperature zone PCR amplification device as claimed in claim 2, wherein: the heat conducting metal block is made of brass or aluminum, the semiconductor refrigerating sheet is 40mm in size, and the radiator is made of aluminum oxide.
4. The dual temperature zone PCR amplification device as set forth in claim 1, wherein: the guide seat is connected with the guide fixing plate through a fixing screw, and the fixing screw is a plastic nylon screw; the fixed frame corresponds two the spout is provided with a gyro wheel respectively, the gyro wheel pass through the gyro wheel support with the direction fixed plate is connected, the gyro wheel can be in corresponding the spout is moved.
5. The dual temperature zone PCR amplification device as set forth in claim 1, wherein: the fixed box comprises an upper box and a lower box which are connected in a rotating way, two silica gel pads are arranged in the lower box, and two lock catches are arranged at the edge of the upper box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910976547.8A CN110564610B (en) | 2019-10-15 | 2019-10-15 | Double-temperature-zone PCR amplification device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910976547.8A CN110564610B (en) | 2019-10-15 | 2019-10-15 | Double-temperature-zone PCR amplification device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110564610A CN110564610A (en) | 2019-12-13 |
| CN110564610B true CN110564610B (en) | 2024-08-23 |
Family
ID=68784893
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910976547.8A Active CN110564610B (en) | 2019-10-15 | 2019-10-15 | Double-temperature-zone PCR amplification device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110564610B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111790462B (en) * | 2020-08-13 | 2024-01-09 | 河南默深智工医疗科技有限公司 | Heating device for microfluidic detection |
| CN112280670A (en) * | 2020-10-14 | 2021-01-29 | 北京普若博升生物科技有限公司 | Polymerase chain reaction amplification device |
| CN112251348B (en) * | 2020-10-23 | 2024-03-26 | 中国科学院合肥物质科学研究院 | PCR circulation temperature partition control device |
| CN114456923A (en) * | 2022-01-28 | 2022-05-10 | 北京源景泰科生物科技有限公司 | Movable double-temperature-zone heating module |
| CN114958580B (en) * | 2022-05-31 | 2023-01-13 | 山东省高精生物诊断分析产业技术研究院有限公司 | A hot lid structure for full-automatic nucleic acid analytic system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174386A (en) * | 2011-02-18 | 2011-09-07 | 张家林 | Temperature control unit of narrow and long thermal cycle block and thermal cycler |
| CN105772125A (en) * | 2016-04-23 | 2016-07-20 | 北京化工大学 | 3D printing-based microfluidic chip fixture experiment platform |
| CN210974696U (en) * | 2019-10-15 | 2020-07-10 | 杭州比芯诊断技术有限公司 | Double-temperature-zone PCR amplification device |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102009044431A1 (en) * | 2009-11-05 | 2011-06-22 | FRIZ Biochem Gesellschaft für Bioanalytik mbH, 82061 | Device for carrying out a PCR |
| KR101368463B1 (en) * | 2010-04-23 | 2014-03-03 | 나노바이오시스 주식회사 | Device for amplify nucleic acid comprising two heating block |
| CN108342312B (en) * | 2017-01-24 | 2020-09-18 | 北京万泰生物药业股份有限公司 | Convection PCR amplification detection system and convection PCR amplification detection method |
| KR102081480B1 (en) * | 2017-01-25 | 2020-04-24 | 주식회사 유진셀 | Device for high-speed amplification of nucleic acid molecules |
| CN107384777A (en) * | 2017-09-04 | 2017-11-24 | 上海科源电子科技有限公司 | A kind of multi-temperature zone gene amplification device |
| CN107574120B (en) * | 2017-10-27 | 2020-11-27 | 深圳大学 | Fluorescent quantitative PCR detection system and method based on magnetic power switching constant-temperature area |
| CN209178400U (en) * | 2018-11-07 | 2019-07-30 | 莫纳（苏州）生物科技有限公司 | A kind of PCR instrument with sealer function |
| CN109609608B (en) * | 2019-01-17 | 2021-01-01 | 浙江大学 | Linear rapid double-temperature PCR amplification automatic control device and control method |
| CN110205242B (en) * | 2019-06-18 | 2024-04-26 | 苏州锐讯生物科技有限公司 | Microfluidic chip assembly for rapidly realizing digital PCR (polymerase chain reaction) and application thereof |
-
2019
- 2019-10-15 CN CN201910976547.8A patent/CN110564610B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174386A (en) * | 2011-02-18 | 2011-09-07 | 张家林 | Temperature control unit of narrow and long thermal cycle block and thermal cycler |
| CN105772125A (en) * | 2016-04-23 | 2016-07-20 | 北京化工大学 | 3D printing-based microfluidic chip fixture experiment platform |
| CN210974696U (en) * | 2019-10-15 | 2020-07-10 | 杭州比芯诊断技术有限公司 | Double-temperature-zone PCR amplification device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110564610A (en) | 2019-12-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110564610B (en) | Double-temperature-zone PCR amplification device | |
| WO2021027891A1 (en) | Rapid pcr reaction testing system, and testing method | |
| CN210974696U (en) | Double-temperature-zone PCR amplification device | |
| CN102046291B (en) | Thermal control system and method for chemical and biochemical reactions | |
| CN1329740C (en) | Heater-equipped pusher, electronic component handling apparatus, and temperature control method for electronic component | |
| CN110205242A (en) | Microfluidic chip assembly for rapidly realizing digital PCR reaction and application thereof | |
| CN203474810U (en) | Polymerase chain reaction device | |
| CN111013688B (en) | qPCR module and modularized qPCR device | |
| CN106442454A (en) | Quick gene amplification detection device and quick gene amplification detection method based on fluorescent quantitation | |
| CN102967494B (en) | Molten salt sample preparation device, prepared molten salt sample and preparation and crystallization method thereof | |
| CN104263634A (en) | Flow polymerase chain reaction (PCR) circulating heating apparatus based on capillaries and heating method | |
| CN206232730U (en) | The module thermal balance device of PCR gene amplification instrument | |
| CN107384777A (en) | A kind of multi-temperature zone gene amplification device | |
| CN215574767U (en) | Cooling device of thermal deformation and Vicat softening point temperature measuring instrument | |
| CN215599052U (en) | Protection heat flat plate heat conduction instrument for measuring medium temperature | |
| CN210945609U (en) | Extremely fast PCR reaction detection device | |
| WO2023104218A2 (en) | Pcr thermal cycling apparatus and control method | |
| CN116515614A (en) | Real-time fluorescence PCR analyzer | |
| CN207946382U (en) | A kind of determining instrument of thermal coefficient and specific heat capacity | |
| CN205562441U (en) | Dimension card softening temperature survey test instrument | |
| CN114181819B (en) | PCR detection device | |
| CN208594301U (en) | Digital pcr detector | |
| CN210528943U (en) | Linear scanning PCR instrument | |
| CN115340946B (en) | Amplification device for nucleic acid detection and detection method based on amplification device | |
| CN220590078U (en) | Vertical double-side heat conduction cold and hot table for measuring thermal strain of chip section |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |