WO2021027891A1 - 极速pcr反应检测系统及检测方法 - Google Patents

极速pcr反应检测系统及检测方法 Download PDF

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Publication number
WO2021027891A1
WO2021027891A1 PCT/CN2020/108969 CN2020108969W WO2021027891A1 WO 2021027891 A1 WO2021027891 A1 WO 2021027891A1 CN 2020108969 W CN2020108969 W CN 2020108969W WO 2021027891 A1 WO2021027891 A1 WO 2021027891A1
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Prior art keywords
temperature
reaction tube
transmission mechanism
module
tube fixing
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PCT/CN2020/108969
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English (en)
French (fr)
Inventor
周荣
刘文宽
李潇
王宪华
徐辉
周志超
高文娟
李蕾
廖小红
Original Assignee
广州市华南医学研究中心
广州呼研所医药科技有限公司
生物岛实验室
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Application filed by 广州市华南医学研究中心, 广州呼研所医药科技有限公司, 生物岛实验室 filed Critical 广州市华南医学研究中心
Priority to AU2020327511A priority Critical patent/AU2020327511B2/en
Priority to US17/634,799 priority patent/US20220274117A1/en
Priority to KR1020227007426A priority patent/KR20220041207A/ko
Priority to EP20852952.9A priority patent/EP4015620A4/en
Priority to JP2022509139A priority patent/JP7442875B2/ja
Priority to CA3147023A priority patent/CA3147023A1/en
Publication of WO2021027891A1 publication Critical patent/WO2021027891A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B01L7/5255—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones by moving sample containers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/185—Means for temperature control using fluid heat transfer medium using a liquid as fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1894—Cooling means; Cryo cooling
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50855—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using modular assemblies of strips or of individual wells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • G01N2035/00366—Several different temperatures used
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00445—Other cooling arrangements

Definitions

  • the invention relates to a PCR reaction detection system and a detection method, in particular, it can provide an extremely fast PCR reaction detection system and a detection method.
  • PCR Polymerase Chain Reaction
  • Patent application number CN201780033562.8 Fast thermal cycle for sample analysis and processing. It discloses a device for heat treatment of nucleic acid according to the thermal curve. The device uses a reciprocating device to realize that the reactor is in at least one bath During the middle period, the holding frame can perform relative reciprocating movement with at least one bath, and the heat conduction between the bath medium and the reactor is improved through vibration, thereby speeding up the heat cycle.
  • the structure of the device is complicated, and the bath medium needs to be replaced regularly, and the bath medium may overflow from the bath; the temperature setting of the bath of the instrument cannot meet the requirements of the rapid PCR for the temperature rise and fall rate. Therefore, how to increase the temperature rise and fall speed of the PCR reaction is an urgent problem to be solved by the extremely fast PCR instrument.
  • an extremely fast PCR reaction detection system includes a temperature control device, a transmission device, and a reaction tube fixing device;
  • the temperature control device includes at least two temperature control modules, and at least one of the temperature control modules is a high temperature module And at least one of the temperature control modules is a low temperature module; wherein the heating temperature of the high temperature module is a first predetermined temperature, the heating temperature of the low temperature module is a second predetermined temperature, and the first predetermined temperature is greater than the The second predetermined temperature;
  • the transmission device cooperates with the temperature control device and the reaction tube fixing device, so that the reaction tube on the reaction tube fixing device is switched between the high temperature module and the low temperature module .
  • the transmission device is connected to the temperature control device, and can drive the high temperature module and the low temperature module to move, so that the reaction tube on the reaction tube fixing device is between the high temperature module and the low temperature module. Switch between;
  • the transmission device is connected to the reaction tube fixing device, and drives the reaction tube fixing device to move, so that the reaction tube on the reaction tube fixing device is switched between the high temperature module and the low temperature module;
  • the transmission device includes a first transmission mechanism and a second transmission mechanism, the first transmission mechanism is connected to the temperature control device and can drive the temperature control device to move, and the second transmission mechanism is fixed to the reaction tube
  • the device is connected and can drive the reaction tube fixing device to move; the first transmission mechanism cooperates with the second transmission mechanism, so that the reaction tube on the reaction tube fixing device is between the high temperature module and the low temperature module Switch between.
  • the transmission device includes a first transmission mechanism and a second transmission mechanism.
  • the first transmission mechanism is connected to the temperature control device and can drive the temperature control device to move horizontally.
  • the second transmission mechanism is connected to the temperature control device.
  • the reaction tube fixing device is connected and can drive the reaction tube fixing device to move vertically; the reaction tube fixing device is mounted on the upper part of the temperature control device, and the first transmission mechanism cooperates with the second transmission mechanism to make the reaction
  • the reaction tube on the tube fixing device is switched between the high temperature module and the low temperature module.
  • the lower part of the temperature control device is connected with the first transmission mechanism, and the temperature control device moves back and forth under the action of the first transmission mechanism.
  • the first transmission mechanism includes a screw motor and two guide transmission shafts.
  • the two guide transmission shafts are fixed to the front of the screw motor, and the temperature control module can move back and forth along the guide transmission shaft.
  • the guide rail is arranged, It can keep the temperature control module balanced and move smoothly;
  • the second transmission mechanism drives the reaction tube fixing device to move up and down through the rotation of the eccentric wheel.
  • the reaction tube fixing device further includes a transverse support plate fixedly installed at the lower part of the reaction tube fixing device
  • the second transmission mechanism includes a stepping motor II and an eccentric wheel
  • the eccentric wheel is fixed on the stepping motor II
  • the eccentric wheel supports the upper lateral support plate
  • the stepping motor II drives the eccentric wheel to rotate
  • the reaction tube fixing device moves up and down by driving the lateral support plate to move up and down.
  • the reaction tube fixing device further includes vertical support rods and uprights, the vertical support rods are fixed on both sides of the reaction tube fixing position, the uprights pass through the vertical support rods vertically, and the uprights are slidably connected to the vertical support rods
  • a spring is sleeved on the top of the column to withstand the vertical support rod. The function of the column and the vertical support rod is to limit the up and down movement direction of the reaction tube fixing device, which can ensure the vertical movement of the reaction tube fixing device.
  • control system is a control circuit that performs real-time control and signal transmission on the temperature control device, the first transmission mechanism, the optical detection device, and the reaction tube fixing device
  • the reaction detection system further includes an optical detection device located in front of the temperature control device.
  • the optical detection device includes an optical reading head and a third transmission mechanism.
  • the optical reading head is mounted on the upper part of the third transmission mechanism and moves left and right under the driving of the third transmission mechanism.
  • the third transmission mechanism includes a stepping motor I, a guide rail, a belt, and a connecting piece.
  • the upper part of the connecting piece is fixed with the optical reading head, the middle part of the connecting piece is provided with a groove for sliding connection with the guide rail, and the lower part of the connecting piece is provided with the belt Connected splint.
  • the reaction tube fixing device includes a reaction tube fixing position and a gland, and the gland is used to cover the reaction tube fixing position.
  • the material of the gland is stainless steel, which is not easy to be deformed, can produce the same gland pressure for sample holes in different positions, and can make the reaction tube and temperature control module in different positions have good adhesion, so that the reaction tube
  • the uniform heating can also make the position of the system in the reaction tube relative to the detection hole more uniform, achieving good repeatability and consistency of the detection results.
  • the first preset temperature is greater than or equal to 100°C, and the second preset temperature is less than or equal to 55°C;
  • the range of the first preset temperature is 100-150°C
  • the range of the second preset temperature is 15-55°C.
  • the first preset temperature is 120°C; the second preset temperature is 47°C.
  • the temperature control device further includes a middle temperature control module, and the heating temperature range of the middle temperature module is between the first preset temperature and the second preset temperature.
  • the temperature control module includes a medium temperature module, a low temperature module, and a high temperature module in order from front to back, wherein a detection hole is opened on the side wall of the foremost medium temperature module, and the upper part of the medium temperature module, the low temperature module and the high temperature module are all provided with Sample hole.
  • the temperature control module can keep the temperature constant or change the temperature.
  • An extremely fast PCR detection method using the extremely fast PCR reaction detection system described above, includes the following steps:
  • reaction tube is fixed in the reaction tube fixing device, and the transmission device moves, so that the reaction tube is inserted into the high temperature module to perform a high temperature bath;
  • the transmission device moves, so that the reaction tube is inserted into the low-temperature module to cool down;
  • the transmission device includes a second transmission mechanism; wherein, step 2 also includes: moving the high temperature module to just below the reaction tube, the second transmission mechanism of the vertical transmission mechanism is lowered to the lowest point, and the reaction tube performs a high temperature warm bath; step 3 also includes: After the temperature of the reaction tube reaches the required temperature, the second transmission mechanism of the vertical transmission mechanism is reset, the low-temperature module is moved directly below the reaction tube, and the second transmission mechanism of the vertical transmission mechanism drops to the lowest point to cool down;
  • step 3 after the reaction tube is cooled, further includes the following steps: the second transmission mechanism is reset, and the middle temperature module is moved directly under the reaction tube for a certain time.
  • the extremely fast PCR detection system of the present invention includes a high temperature module, a low temperature module, a traditional device, an optical detection device, and a reaction tube fixing device.
  • the reaction tube can be installed on the reaction tube fixing device, and press it with a gland after installation.
  • the reaction tube can move with the reaction tube fixing device in the experiment.
  • the high temperature module provides temperature control that exceeds the first preset temperature, which can quickly heat the reaction tube to the target temperature
  • the low temperature module provides temperature control of the second preset temperature, which can quickly cool the reaction tube to the target temperature.
  • the denaturation and annealing process of the normal amplification cycle adopts a pass-through temperature control. For pre-denaturation, a process that needs to be maintained at a temperature for a certain period of time is maintained by moving the temperature control module (high temperature module) up and down at a certain frequency to maintain a continuous temperature.
  • the reaction system moves smoothly and has a compact structure, which improves the movement speed of the instrument:
  • the PCR reaction tube and the temperature control device move relative to each other.
  • the first transmission mechanism drives the temperature control device to move horizontally.
  • the reaction tube fixing device is mounted on the upper part of the temperature control system.
  • the reaction tube fixing device is connected to the second transmission mechanism and can follow the first transmission mechanism.
  • the second transmission mechanism moves up and down, so as to realize the relative reciprocating movement of the reaction tube on the reaction tube fixing device between the temperature control modules.
  • the second transmission mechanism is not connected with the first transmission mechanism. Therefore, the first transmission mechanism does not Drive the reaction tube to move horizontally. Because the PCR reaction volume is small, the vibration of the reaction tube is reduced, the reaction liquid in the reaction tube can be prevented from hanging on the wall, the generation of bubbles in the reaction tube can be avoided, and the detection accuracy can be improved.
  • the temperature control module moves up and down at a certain frequency to maintain a continuous temperature.
  • the speed and flexibility of the movement are realized by using the motor to drive the eccentric wheel to rotate.
  • the eccentric wheel only needs to rotate a certain angle to achieve the effect of up and down movement in an instant. This way of up and down movement will be faster than belt transmission and thus can improve The speed and efficiency of the up and down movement can improve the accuracy of temperature control through this kind of movement method, but also speed up the movement speed of the instrument and shorten the detection time.
  • the temperature setting of the high temperature module and the low temperature module can increase the temperature rise and fall rate of the reaction liquid in the reaction tube.
  • the maximum temperature setting of the high-temperature module in the PCR machine is 99°C, while the temperature set by the high-temperature module of the present invention is more than 100°C. After many tests, it is proved that a relatively ideal heating rate can be reached at 120°C. At the same time, the present invention The detection instrument only needs to use a common reaction tube to meet the requirements. In the low temperature module of the present invention, the set temperature is lower than the target temperature setting of the experiment process, and the target temperature can be reached at the maximum speed, thereby increasing the temperature rise and fall speed.
  • the temperature of the low-temperature module was set to room temperature (25°C-30°C)
  • the temperature of the reaction tube was not stable during the annealing stage.
  • the wall of the reaction tube was quickly conducted to a lower temperature by the low-temperature module when the temperature dropped. State, even if the reaction tube leaves the low temperature module in the later stage, the temperature of the reaction tube is lower. After leaving the low temperature module, the low temperature continues to conduct from the reaction tube wall to the experimental system in the reaction tube, and the cooling trend does not slow down.
  • the low temperature module is set to 47 °C
  • the temperature is reduced After a period of time, the reaction tube is separated from the low temperature module, which can stabilize the annealing temperature, thereby improving the repeatability of PCR detection in terms of temperature control.
  • the upper part of the high-temperature module, medium-temperature module, and low-temperature module of the present invention is provided with a sample hole, and a detection hole is opened in the front part of the sample hole of the temperature control module, and the reaction tube is pressed against the sample through the gland on the sample hole In the hole, the reaction tube and the temperature control module can have a good fit, which can increase the temperature rise and fall rate of each sample, and also ensure the consistency of the temperature change of each sample tube, thereby improving the repetition of sample detection results Sex and consistency.
  • the material of the gland of the present invention is hard metal, preferably stainless steel.
  • the material of the gland will have a greater impact on the test results.
  • the material of the gland is aluminum metal strip, because the gland is easy to deform, the degree of compression above different sample holes is different, which affects the degree of fit between the reaction tube and the temperature control module, and will result in reaction tubes with different sample holes Uneven heating and uneven pressing force will also cause differences in the position of the system in the reaction tube relative to the detection hole. In extreme cases, the interface between the system and paraffin oil may be detected, which will eventually lead to poor repeatability of the instrument. By changing the material of the gland to stainless steel, this problem is solved very well.
  • the setting of the medium temperature module can significantly improve the amplification efficiency of PCR
  • the amplification efficiency does not improve. Observe the temperature control curve. Even if the low-temperature module provides a temperature of 47°C, the cooling rate is too fast. Simply provide a low-temperature temperature or remove the EP tube from The detachment of the low-temperature module (tested before) cannot slow down the rate of cooling. Therefore, if the annealing extension temperature needs to be maintained, a heat source needs to be used to raise the temperature of the EP tube after the annealing temperature is reached. The preferred way is to increase the extension temperature.
  • the extremely fast fluorescent PCR instrument of the present invention is simple to maintain and does not require replacement of heating medium.
  • the reaction tube is pressed into the sample detection hole through the gland, which can achieve a good fit between the reaction tube and the wall surface of the sample detection hole, replaces the conventional heat transfer method through the medium, and reduces the workload of instrument maintenance on the one hand And consumables, on the other hand, because the heat transfer process of the medium is omitted, the warm-up time of the instrument can be shortened, and the temperature of the temperature control module can be adjusted faster
  • Figure 1 Schematic diagram of the structure of the extremely fast PCR reaction detection system
  • FIG. 1 Schematic diagram of the structure of the optical detection device
  • Temperature control device 11, medium temperature module, 12, low temperature module, 13, high temperature module;
  • the first transmission mechanism 21, four-pole motor, 22, guide transmission shaft;
  • Reaction tube fixing device 51, horizontal support plate, 52, reaction tube fixing position, 53, gland, 54, vertical support, 55, column, 56, spring;
  • the FQPCR of the present invention is an extremely fast fluorescent PCR machine, and the QPCR is an ordinary fluorescent PCR machine.
  • an extremely fast PCR reaction detection system includes a temperature control system temperature control device, a transmission device and a reaction tube fixing device;
  • the temperature control device includes at least two temperature control modules, wherein at least one of the temperature control modules is a high temperature module and at least one of the temperature control modules is a low temperature module; wherein, the heating temperature of the high temperature module is the first A predetermined temperature, the heating temperature of the low temperature module is a second predetermined temperature, and the first predetermined temperature is greater than the second predetermined temperature;
  • the transmission device cooperates with the temperature control device and the reaction tube fixing device, so that the reaction tube on the reaction tube fixing device is switched between the high temperature module and the low temperature module.
  • the reaction tube can be installed on the reaction tube fixing device, and press it with a gland after installation.
  • the reaction tube can move with the reaction tube fixing device in the experiment.
  • the high temperature module provides temperature control of the first preset temperature, which can quickly raise the temperature of the reaction tube to the target temperature
  • the low temperature module provides temperature control of the second preset temperature, which can quickly cool the reaction tube to the target temperature.
  • the denaturation and annealing process of the normal amplification cycle adopts a pass-through temperature control. For pre-denaturation, a process that needs to be maintained at a temperature for a certain period of time is maintained by moving the temperature control module (high temperature module) up and down at a certain frequency to maintain a continuous temperature.
  • the minimum temperature of the first preset temperature is greater than the maximum temperature of the second preset temperature. You can choose according to your needs.
  • transmission device can be implemented in multiple ways, including but not limited to multi-axis robot operating arms, three-coordinate transmission equipment and other transmission equipment that can meet the above requirements.
  • the transmission device is connected to the temperature control device, and can drive the high temperature module and the low temperature module to move, so that the reaction tube on the reaction tube fixing device is in the high temperature module Switch with the low temperature module.
  • the transmission device can be a multi-axis robot operating arm or a three-coordinate transmission device.
  • the transmission device is connected to the reaction tube fixing device, and drives the reaction tube fixing device to move, so that the reaction tube on the reaction tube fixing device is between the high temperature module and the low temperature module. Switch between modules. In this way, the reaction tube on the reaction tube fixing device can be moved so that the reaction tube is inserted into the high temperature module or the low temperature module to provide the required reaction temperature for the reaction tube on the reaction tube fixing device.
  • the transmission device can be a multi-axis robot operating arm or a three-coordinate transmission device.
  • the transmission device includes a first transmission mechanism and a second transmission mechanism.
  • the first transmission mechanism is connected to the temperature control device and can drive the temperature control device to move.
  • the mechanism is connected with the reaction tube fixing device and can drive the reaction tube fixing device to move; the first transmission mechanism cooperates with the second transmission mechanism so that the reaction tube on the reaction tube fixing device is at the high temperature Switch between the module and the low-temperature module.
  • the first transmission mechanism is used to control the movement of the temperature control module so that the high temperature module and the low temperature module can correspond to the required reaction tubes; and then the second transmission mechanism is used to drive the reaction tube fixing device to move, so that the reaction tube fixing device reacts The tube is inserted into the required high temperature module or low temperature module to meet the temperature required for the PCR reaction.
  • the transmission device includes a first transmission mechanism and a second transmission mechanism.
  • the first transmission mechanism is connected to the temperature control device and can drive the temperature control device to move horizontally.
  • the second transmission mechanism is connected with the reaction tube fixing device and can drive the reaction tube fixing device to move vertically; the reaction tube fixing device is mounted on the upper part of the temperature control device, the first transmission mechanism and the second transmission mechanism Cooperate, so that the reaction tube on the reaction tube fixing device is switched between the high temperature module and the low temperature module.
  • the reaction tube fixing device can move up and down with the second transmission mechanism.
  • the temperature control device described in this embodiment includes two temperature control modules, as shown in FIG. 1, there are a low temperature module 12 and a high temperature module 13 from front to back; it can also be three temperature control modules, as shown in FIG. After the sign-in, they are the medium temperature module 11, the low temperature module 12, and the high temperature module 13 in sequence; the temperature control module can keep the temperature constant or change the temperature.
  • the high temperature module can be set to a temperature exceeding 100°C (that is, the first preset temperature can be set to be greater than or equal to 100°C), so that the reaction tube can be quickly heated to the denaturation temperature, and the low temperature module 12 can be set to a temperature lower than The temperature required for the annealing and extension allows the reaction tube to quickly cool down to the annealing temperature, such as lower than 55°C (that is, the second preset temperature can be set to be less than or equal to 55°C).
  • a detection hole 14 is opened on the side wall of the frontmost low-temperature module, and a sample hole 15 is provided on the upper part of the low-temperature module 12 and the high-temperature module 13.
  • a detection hole 14 is opened on the side wall of the frontmost low-temperature module, and a sample hole 15 is provided on the upper part of the low-temperature module 12 and the high-temperature module 13.
  • the first transmission mechanism in the present invention includes a screw motor 21 and two guide transmission shafts 22.
  • the two guide transmission shafts 22 are fixed to the front of the screw motor 21, and the temperature control module is along the guide transmission shaft. 22 Perform forward and backward movement.
  • the optical detection device of the present invention includes an optical reading head 31 and a third transmission mechanism 32.
  • the optical reading head is mounted on the upper part of the third transmission mechanism and moves left and right under the drive of the third transmission mechanism 32.
  • the third transmission mechanism 32 includes a stepping motor 321, a guide rail 322, a belt 323, a connecting piece 324, the upper part of the connecting piece 324 is fixed with the optical reading head 321, and the middle of the connecting piece 324 is provided with a groove 3241 that is slidably connected to the guide rail, A splint 3242 connected with the belt is arranged at the lower part of the connecting piece.
  • the second transmission mechanism of the present invention includes a stepper motor II 41 and an eccentric wheel 42.
  • the eccentric wheel 42 is fixed on the stepper motor II 41.
  • the lower part of the reaction tube fixing device 5 is fixedly installed with a transverse support plate 51 for transverse support.
  • the lower part of the plate 51 is supported by an eccentric wheel.
  • the stepping motor II41 drives the eccentric wheel 42 to rotate, and realizes the up and down movement of the reaction tube fixing device by driving the lateral support plate to move up and down;
  • the reaction tube fixing device 5 also includes a reaction tube fixing position 52, a pressure
  • the cover 53, the vertical support rod 54, the upright 55, the upper part of the reaction tube fixing position is provided with a gland, the vertical support rod 54 is fixed on both sides of the reaction tube fixing position 52, the upright passes through the vertical support rod 54 vertically,
  • the column 55 is slidably connected to the vertical support rod 54, and the top of the column is sleeved with a spring 56 and bears the vertical support rod 54.
  • the ultra-fast PCR reaction detection system of the present invention also includes a control system 6.
  • the control system has a control circuit inside, which can control the temperature control device, the first transmission mechanism, the optical detection device, and the reaction tube fixing device. Real-time control and signal transmission.
  • two fans are arranged above the control circuit.
  • the reaction tube is an EP tube.
  • An extremely fast PCR detection method using the extremely fast PCR reaction detection system described above, includes the following steps:
  • the second transmission mechanism is reset, the low-temperature module moves to just below the reaction tube, and the second transmission mechanism drops to the lowest point to cool down;
  • the second transmission mechanism is reset, and the medium temperature module is moved to just below the reaction tube for a certain period of time.
  • the above samples and reagents contain reaction components, which include at least one enzyme, nucleic acids and/or particles containing at least one nucleic acid, primers for PCR, Primers for isothermal amplification, primers for other nucleic acid amplification and processing, dNTPs, Mg2+, fluorescent dyes and probes, control DNA, control RNA, control cells, control microorganisms, and other nucleic acid amplification, processing and analysis required Reagents.
  • the aforementioned nucleic acid-containing particles include at least one cell virus, white blood cells and stromal cells, circulating tumor cells, and embryonic cells for instrument detection.
  • the above methods and equipment are used for polymerase chain reaction, reverse transcription-polymerase chain reaction, end-point PCR, ligase chain reaction, nucleic acid sequencing, or pre-amplification of changes in each polymerase chain reaction (PCR) or Template enrichment, isothermal amplification, linear amplification, library preparation for sequencing, bridge amplification for sequencing.
  • the above-mentioned changes in polymerase chain reaction include reverse transcription-PCR, real-time fluorescent quantitative polymerase chain amplification reaction and real-time fluorescent quantitative reverse transcription polymerase chain amplification reaction, reverse polymerase chain amplification reaction, and anchoring Polymerase chain amplification reaction, asymmetric polymerase chain amplification reaction, multiplex PCR, color complementary polymerase chain amplification reaction, immunopolymerase chain amplification reaction, nested polymerase chain amplification reaction, Template enrichment for pre-amplification or nucleic acid sequencing, ELISA-PCR.
  • the reaction system moves smoothly and has a compact structure, which improves the movement speed of the instrument:
  • the PCR reaction tube and the temperature control device move relative to each other.
  • the first transmission mechanism drives the temperature control device to move horizontally.
  • the reaction tube fixing device is mounted on the upper part of the temperature control system.
  • the reaction tube fixing device is connected to the second transmission mechanism and can follow the first transmission mechanism.
  • the second transmission mechanism moves up and down, so as to realize the relative reciprocating movement of the reaction tube on the reaction tube fixing device between the temperature control modules.
  • the second transmission mechanism is not connected with the first transmission mechanism. Therefore, the first transmission mechanism does not Drive the reaction tube to move horizontally. Because the PCR reaction volume is small, the vibration of the reaction tube is reduced, the reaction liquid in the reaction tube can be prevented from hanging on the wall, the generation of bubbles in the reaction tube can be avoided, and the detection accuracy can be improved.
  • the temperature control module moves up and down at a certain frequency to maintain a continuous temperature.
  • the speed and flexibility of the movement are realized by using the motor to drive the eccentric wheel to rotate.
  • the eccentric wheel only needs to rotate a certain angle to achieve the effect of up and down movement in an instant. This way of up and down movement will be faster than belt transmission and thus can improve The speed and efficiency of the up and down movement can improve the accuracy of temperature control through this kind of movement method, but also speed up the movement speed of the instrument and shorten the detection time.
  • the temperature setting of the high temperature module and the low temperature module can increase the temperature rise and fall rate of the reaction liquid in the reaction tube.
  • the maximum temperature setting of the high-temperature module in the PCR machine is 99°C, while the temperature set by the high-temperature module of the present invention is more than 100°C. After many tests, it is proved that a relatively ideal heating rate can be reached at 120°C. At the same time, the present invention The detection instrument only needs to use a common reaction tube to meet the requirements.
  • the set temperature is lower than the target temperature setting of the experiment process, and the target temperature can be reached at a faster speed, thereby increasing the temperature rise and fall speed.
  • Test conditions the first preset temperature is 120°C, and the second preset temperature is below 40°C.
  • Annealing 66°C, 1sec, testing at this temperature, after testing, the next round of process starts;
  • the temperature of the low-temperature module was set to room temperature (25°C-30°C)
  • the temperature of the reaction tube was not stable during the annealing stage.
  • the wall of the reaction tube was quickly conducted to a lower temperature by the low-temperature module when the temperature dropped. State, even if the reaction tube leaves the low temperature module in the later stage, the temperature of the reaction tube is lower. After leaving the low temperature module, the low temperature continues to conduct from the reaction tube wall to the experimental system in the reaction tube, and the cooling trend does not slow down.
  • the low temperature module is set to 47 °C
  • the temperature is reduced After a period of time, the reaction tube is separated from the low temperature module, which can stabilize the annealing temperature, thereby improving the repeatability of PCR detection in terms of temperature control.
  • test results of different sample test holes are consistent and repeatable.
  • the upper part of the high-temperature module, medium-temperature module, and low-temperature module of the present invention is provided with a sample hole, and a detection hole is opened in the front part of the sample hole of the temperature control module, and the reaction tube is pressed against the sample through the gland on the sample hole In the hole, the reaction tube and the temperature control module can have a good fit, which can increase the temperature rise and fall rate of each sample, and also ensure the consistency of the temperature change of each sample tube, thereby improving the repetition of sample detection results Sex and consistency.
  • the material of the gland of the present invention is hard metal, preferably stainless steel.
  • the material of the gland will have a greater impact on the test results.
  • the material of the gland is aluminum metal strip, because the gland is easy to deform, the degree of compression above different sample holes is different, which affects the degree of fit between the reaction tube and the temperature control module, and will result in reaction tubes with different sample holes Uneven heating and uneven pressing force will also cause differences in the position of the system in the reaction tube relative to the detection hole. In extreme cases, the interface between the system and paraffin oil may be detected, which will eventually lead to poor repeatability of the instrument. By changing the material of the gland to stainless steel, this problem is solved.
  • the specific detection process is as follows:
  • Control group 1 The gland used by the instrument is made of aluminum metal
  • the temperature control module is numbered from left to right as No. 1-8, and each of the 8 positions of No. 1-8 positions is tested once to investigate the repeatability of different hole positions.
  • prepare a 10X test system at one time divide it into 8 100ul transparent reaction tubes, each tube is divided into 20ul mixed system + 20ul paraffin oil, respectively placed in the 1-8 position for testing, temperature control process as follows:
  • the gland used in the instrument is made of stainless steel.
  • the temperature control module detection holes are numbered from left to right as No. 1-8. Tests were performed at No. 3, No. 5, and No. 7, and 3 rounds of repetition were carried out. A total of 9 curves were used to investigate the repeatability of different holes. .
  • the results show that the material of the gland has a very large impact on the repeatability of the detection results of different hole positions. Only the non-deformable metal material is used to change the detection holes in different positions on the temperature control module. When the reaction tube can be compressed, the reaction tube and the detection module have a good fit, and the repeatability can meet the detection requirements.
  • the specific detection process is as follows:
  • the temperature control module is numbered from left to right as No. 1-8, and 5 repeated tests are performed at the positions 2 and 5 to investigate the repeatability.
  • the temperature control process is as follows:
  • the setting of the temperature control module of the extremely fast PCR instrument is 120°C for the high temperature module and 47°C for the low temperature module.
  • test results are shown in Figures 8 and 9, the amplification curves of 5 repeated tests at each position of No. 2 and No. 5, it can be seen that the single well position test has good repeatability.
  • the detection instrument of the present invention has good repeatability in the same hole position.
  • the setting of the medium temperature module can significantly improve the amplification efficiency of PCR
  • the amplification efficiency does not improve. Observe the temperature control curve. Even if the low-temperature module provides a temperature of 47°C, the cooling rate is too fast. Simply provide a low-temperature temperature or remove the EP tube from The detachment of the low-temperature module cannot slow down the cooling rate. Therefore, if the annealing extension temperature needs to be maintained, a heat source must be used to heat the EP tube after the annealing temperature is reached. The preferred way is to add a medium temperature pool with extension temperature. After reaching the annealing temperature through the low-temperature bath, it enters the middle-temperature bath for maintenance and testing, and then enters the high-temperature bath for denaturation and melting.
  • the setting of the temperature control module of the extremely fast PCR instrument is 120°C for the high temperature module and 47°C for the low temperature module.
  • Pre-denaturation 90°C, 2min;
  • Figure 11 shows the QPCR instrument and FQPCR test for the amplification of 10 ⁇ 1 ⁇ 10 ⁇ 5 copies. From the point of view of the curve, there is no significant difference between the tests of the two instruments, and the Ct value spacing between samples of different concentrations is also the same.
  • Table 2 shows the amplified Ct values of the QPCR and FQPCR sensitivity tests.
  • Figure 12 shows the standard curve obtained from the sensitivity test of the two instruments and the gradient dilution test of the quantitative reference substance. It can be seen from the standard curve that the slopes of the two curves are equivalent, and the correlation coefficient R 2 is also greater than 0.99.
  • the amplification efficiency is calculated by the standard curve, and the amplification efficiency of the two instruments is similar (110%-115%).
  • the FQPCR instrument and the control QPCR instrument carried out the sensitivity test of 10 ⁇ 1 ⁇ 10 ⁇ 5copies concentration, and there was no significant difference in amplification efficiency and detection limit. Both instruments can detect 10copies concentration samples.
  • the FQPCR instrument and the control QPCR instrument carried out the sensitivity test of 10 ⁇ 1 ⁇ 10 ⁇ 5copies concentration, and there was no significant difference in amplification efficiency and detection limit.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, "a plurality of” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection. , Or integrated; it can be mechanically connected or electrically connected; it can be directly connected or indirectly connected through an intermediary, it can be the internal communication of two components or the interaction relationship between two components, unless otherwise specified The limit.
  • installed can be a fixed connection or a detachable connection. , Or integrated; it can be mechanically connected or electrically connected; it can be directly connected or indirectly connected through an intermediary, it can be the internal communication of two components or the interaction relationship between two components, unless otherwise specified The limit.
  • the specific meaning of the above-mentioned terms in the present invention can be understood according to specific circumstances.
  • the first feature “on” or “under” the second feature may be in direct contact with the first and second features, or the first and second features may be indirectly through an intermediary. contact.
  • the "above”, “above” and “above” of the first feature on the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the level of the first feature is higher than the second feature.
  • the “below”, “below” and “below” of the second feature of the first feature may mean that the first feature is directly below or obliquely below the second feature, or it simply means that the level of the first feature is smaller than the second feature.

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Abstract

本发明公开了一种极速PCR反应检测系统，包括温度控制装置、传动装置及反应管固定装置；温度控制装置至少包括2个温控模块，其中至少1个温控模块为高温模块和至少1个温控模块为低温模块；其中，高温模块的加热温度为第一预定温度，低温模块的加热温度为第二预定温度；传动装置与温度控制装置及反应管固定装置相配合，使得反应管固定装置上的反应管在高温模块与低温模块之间进行切换，该装置结构紧凑，高温模块和低温模块的温度设置，能提升反应管中反应液的升降温速率，同时，本发明极速荧光PCR反应检测系统检测结果重复性好，结果准确。

Description

极速PCR反应检测系统及检测方法 技术领域
本发明涉及一种PCR反应检测系统及检测方法，特别是能够提供极速PCR反应检测系统及检测方法。
背景技术
PCR(Polymerase Chain Reaction,聚合酶联反应)是分子生物学研究的重要手段，反应时间一直是PCR反应的限制因素，一个常规的PCR反应往往要一两个小时，对于临床特种应用来说，扩增时长限制了其应用。
专利申请号为CN201780033562.8的专利：用于样品分析与处理的快速热循环，公开了一种用于按照热曲线对核酸进行热处理的设备，该设备通过往复装置，实现反应器处于至少一个浴槽中时让握持架能与至少一个浴槽之间进行相对往复运动，并通过震动改进浴槽介质与反应器之间的热传导，从而加快热循环速度。
然而，该装置的结构复杂，并且需要定期更换浴槽介质，浴槽介质还有可能从浴槽中溢出；该仪器的浴槽的温度的设置，并不能满足极速PCR对于升降温速率的要求。因此，怎样提高PCR反应的升降温速度，是极速PCR仪亟待解决的问题。
发明内容
针对上述问题，一种极速PCR反应检测系统，包括温度控制装置、传动装置及反应管固定装置；所述温度控制装置至少包括2个温控模块，其中至少1个所述温控模块为高温模块和至少1个所述温控模块为低温模块；其中，所述高温模块的加热温度为第一预定温度，所述低温模块的加热温度为第二预定温度，所述第一预定温度大于所述第二预定温度；所述传动装置与所述温度控制装置及所述反应管固定装置相配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。
优选的，所述传动装置与所述温度控制装置连接，并能够驱动所述高温模块与所述低温模块移动，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换；
或所述传动装置与所述反应管固定装置连接，并驱动所述反应管固定装置移动，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换；
或所述传动装置包括第一传动机构及第二传动机构，所述第一传动机构与温度控制装置连接，并能够驱动所述温度控制装置移动，所述第二传动机构与所述反应管固定装置连接，并能够驱动所述反应管固定装置移动；所述第一传动机构与所述第二传动机构配合，使得所述反应管固定装置上反应管在所述高温模块与所述低温模块之间进行切换。
优选的，所述传动装置包括第一传动机构及第二传动机构，所述第一传动机构与温度控制装置连接，并能够驱动所述温度控制装置水平移动，所述第二传动机构与所述反应管固定装置连接，并能够驱动所述反应管固定装置垂直移动；所述反应管固定装置架在温度控制装置上部，所述第一传动机构与所述第二传动机构配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。
优选的，所述温度控制装置的下部与第一传动机构连接，在第一传动机构的作用下温度控制装置前后运动。
优选的，所述第一传动机构包括丝杆电机和2根导向传动轴，2根导向传动轴固定于丝杆电机前部，温控模块可沿导向传动轴进行前后运动，该导轨的设置，能够让温控模块保持平衡，进行平稳的移动；
或，第二传动机构通过偏心轮转动带动反应管固定装置上下运动。
优选的，所述反应管固定装置还包括反应管固定装置下部固定安装的横向支撑板，所述第二传动机构包括步进电机Ⅱ和偏心轮，所述偏心轮固定在步进电机Ⅱ上，偏心轮支撑位于上部的横向支撑板，步进电机Ⅱ带动偏心轮转动，通过带动横向支撑板上下运动实现反应管固定装置的上下运动。
优选的，所述反应管固定装置还包括竖向支撑杆、立柱，所述竖向支撑杆固定于反应管固定位两侧，立柱垂直穿过竖向支撑杆，立柱与竖向支撑杆滑动连接，所述立柱顶部套设弹簧并顶住竖向支撑杆，立柱与竖向支撑杆的作用在于限定反应管固定装置的上下运动方向，能保证反应管固定装置垂直方向上下运动。
优选的，还包括控制系统，所述控制系统为控制电路，对温度控制装置、第一传动机构、光学检测装置、反应管固定装置进行实时控制及信号传输
优选的，所述反应检测系统还包括光学检测装置，所述光学检测装置位于温度控制装置前部。
优选的，所述光学检测装置包括光学读头和第三传动机构，所述光学读头安装在第三传动机构上部，在第三传动机构的带动下进行左右移动。
优选的，所述第三传动机构包括步进电机Ⅰ、导轨、皮带、连接件，所述连接件上部固定光学读头，连接件中部设置与导轨滑动连接的凹槽，连接件下部设置与皮带连接的夹板。
优选的，所述反应管固定装置包括反应管固定位、压盖，所述压盖用于盖合反应管固定位。
优选的，所述压盖材质为不锈钢，不锈钢材质不易变形，能对不同位置样品孔都产生相同的压盖力度，能使不同位置反应管与温控模块的贴合度均良好，让反应管受热均匀，也能让反应管内体系相对于检测孔的位置都比较均一，达到检测结果重复性和一致性良好。
优选的，所述第一预设温度大于或等于100℃，所述第二预设温度小于或等于55℃；
优选的，所述第一预设温度的范围为100～150℃，所述第二预设温度的范围为15～55℃。
优选的，所述第一预设温度为120℃；所述第二预设温度为47℃。
优选的，所述温度控制装置还包括中温控模块，所述中温模块的加热温度范围为所述第一预设温度与所述第二预设温度之间。
优选的，所述温控模块的由前向后依次为中温模块、低温模块、高温模块，其中最前部中温模块侧壁开有检测孔，所述中温模块、低温模块、高温模块上部均设置有样品孔。
优选的，温控模块能保持温度恒定或改变温度。
一种极速PCR检测方法，应用如上所述极速PCR反应检测系统，包括如下操作步骤：
①准备：启动仪器预热，将待测样本和试剂装入反应管并混合；
②反应管固设于反应管固定装置，传动装置动作，使得反应管插入高温模块中，进行高温温浴；
③反应管温度达到所需温度后，传动装置动作，使得反应管插入低温模块中，进行降温；
④数据采集；
⑤重复第②～④步骤。
优选的，传动装置包括第二传动机构；其中，步骤②还包括：高温模块移动至反应管正下方，垂直传动机构第二传动机构下降至最低点，反应管进行高温温浴；步骤③还包括：反应管温度达到所需温度后，垂直传动机构第二传动机构复位，低温模块移动到反应管正下方，垂直传动机构第二传动机构下降至最低点，进行降温；
或/和，步骤③反应管降温之后，还包括如下步骤：第二传动机构复位，中温模块移动到反应管的正下方，保持一定时间。
工作原理：
本发明所述极速PCR检测系统，包括高温模块、低温模块、传统装置、光学检测装置、反应管固定装置。反应管可以安装在反应管固定装置上，安装后使用压盖压住，实验中反应管可以随反应管固定装置运动。高温模块提供超过第一预设温度的温控控制，可以使反应管快速升温至目标温度，低温模块提供第二预设温度的温度控制，可以使反应管快速降温至目标温度。正常扩增循环的变性及退火过程，采用路过型温度控制，对于预变性需要在一个温度保持一定时间的流程，通过在该温控模块(高温模块)以一定频率上下运动保持一个持续的温度。
有益效果：
1、反应系统移动平稳，结构紧凑，提升仪器的运动速度：
PCR反应管与温度控制装置进行相对运动，第一传动机构带动温度控制装置进行水平运动，所述反应管固定装置架在温控系统上部，反应管固定装置与第二传动机构相连并可随第二传动机构上下运动，从而实现反应管固定装置上的反应管在温控模块之间进行相对往复运动，第二传动机构与第一传动机构不连接，因此，第一传动机构运动时，不会带动反应管进行水平运动，由于PCR反应体积都较小，减少反应管的震动，能避免反应管中的反应液挂壁，能避免反应管中气泡的产生，从而提高检测的准确性。
本发明所述仪器，在使用时，为了PCR在预变性需要在一个温度保持一定时间的流程，采用的方式是在该温控模块以一定频率的上下运动来保持一个持续的温度，为保证上下运动的速度和灵活性，本发明采用电机带动偏心轮转动实现，偏心轮只需要转动一定的角度，就可以瞬间达到上下运动效果，该上下运动的方式，会比皮带传动更快，从而能够提升上下运动的速度和效率，通过该种运动方式，能提高温度控制的准确度同时也能加快仪器的运动速度，缩短检测时间。
2、高温模块和低温模块的温度设置，能提升反应管中反应液的升降温速率。
一般PCR仪中的高温模块温度设置最高为99℃，而本发明所述高温模块设置的温度为超过100℃，经过多次试验，证明在120℃能达到较为理想的升温速率，同时，本发明所述检测仪器，只需要使用普通反应管就可以达到要求。本发明所述低温模块，所设置的温度低于实验流程目标温度设置，可以以最大速度到达目标温度，进而提高升降温速度。
在试验过程中，发现当低温模块温度设置为室温(25℃-30℃)时，反应管温度在退火阶段没有稳定，分析原因，发现降温时，反应管壁迅速的被低温模块传导至较低温状态，即使反应管后期脱离低温模块，但反应管温度较低，脱离低温模块后低温继续从反应管壁向反应管内的实验体系传导，降温趋势没有减缓，当低温模块设置为47℃后，降温一段时间后使反应管脱离低温模块，能稳定住退火温度，从而能在温度控制方面提升PCR检测的重复性。
3、不同样品孔、相同样品孔检测结果一致性、重复性良好。
本发明所述高温模块、中温模块、低温模块，上部均设置有样品孔，在温控模块样品孔的前部，开设有检测孔，通过样品孔上的压盖，将反应管压紧在样品孔中，能让反应管与温控模块具有良好的贴合度，从而能提升每个样品的升降温速率，同时也能保证每个样品管的温度变化的一致，进而提升样品检测结果的重复性、一致性。
本发明所述压盖的材质为硬金属材质，优选为不锈钢材质。压盖材质的不同，会对检测结果造成较大的影响。当压盖的材质为铝质金属条时，由于压盖易变形，从而导致不同样品孔上方的压紧程度不同，影响反应管与温控模块的贴合度，会导致不同样品孔的反应管受热不均，同时，压盖力度不均，也会造成反应管内体系相对于检测孔位置差异，极端的情况可能检测到体系与石蜡油的交界面，最终导致仪器的重复性不佳。通过将压盖的材质更换为不锈钢材质，则很好的解决了这一问题。
4、中温模块的设置，能显著提升PCR的扩增效率
将低温池的温度从室温提高到47℃，扩增效率没有提高，观察温控曲线，即便低温模块提供47℃一个的温度，但降温速率过快，单纯提供一个低温温度、或者将EP管从低温模块脱离(之前测试过)，都不能减缓降温的速率，故如需在退火延伸温度加以保持，需要在到达退火温度后给一个热源使EP管升温，优选的方式是增加一个延伸温度的中温池，在EP管 通过低温池达到退火温度后进入中温池保持并检测，再进入高温池进行变性解链。试验表明，增加中温模块用于增加退火时间后，同样扩增效率用时缩短，增加退火延伸时间对提高扩增效率有效。
5、本发明所述极速荧光PCR仪与普通荧光PCR仪在扩增效率与检出限方面没有显著差异，检测结果重复性好，结果准确。
FQPCR仪与对照的QPCR仪进行10^1～10^5copies浓度的灵敏度测试，扩增效率及检出限没有显著差异，两台仪器均可以检出10copies浓度样本；从标曲可以看出，两条曲线的斜率相当，相关系数R2也都大于0.99。通过标准曲线计算扩增效率，两台仪器的扩增效率相近(110％～115％)之间。
6、本发明所述极速荧光PCR仪，温度控制模块位于第一传动机构的上方，相对于温控模块位于第一传动机构的下方，能让样品放置操作更加方便。
7、本发明所述极速荧光PCR仪，维护简单，不需要进行加热介质的更换。
本发明通过压盖将反应管压紧于样品检测孔中，能达到反应管与样品检测孔的壁面具有良好的贴合度，替代常规的通过介质传热方式，一方面减少仪器维护的工作量和耗材，另一方面，由于省略了介质的传热过程，能缩短仪器的预热时间，温控模块温度能更快调节
。
附图说明
图1极速PCR反应检测系统的结构示意图；
图2温控系统与第一传动机构的结构示意图；
图3光学检测装置结构示意图；
图4第二传动机构、反应管固定装置的结构示意图；
图5 PCR仪温度测试曲线；
图6铝制压盖不同检测位置的扩增曲线；
图7钢制压盖不同检测位置的扩增曲线；
图8 2号位的重复性测试的扩增曲线；
图9 5号位的重复性测试的扩增曲线；
图10 Ct值的正太分布及直方图；
图11 QPCR&FQPCR灵敏度测试扩增曲线(对数坐标轴)；
图12 QPCR&FQPCR标准曲线图。
其中：
1、温度控制装置，11、中温模块，12、低温模块，13、高温模块；
2、第一传动机构，21、四杆电机，22、导向传动轴；
3、光学检测装置，31、光学读头，32、第三传动机构，321，步进电机Ⅰ，322、导轨，323、皮带，324、连接件，3241、凹槽，3242、夹板；
4、第二传动机构，41、步进电机Ⅱ，42、偏心轮；
5、反应管固定装置，51、横向支撑板，52、反应管固定位，53、压盖，54、竖向支撑件，55、立柱，56、弹簧；
6、控制系统。
附图说明构成本申请的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及具体实施方式，对本发明进行进一步的详细说明。应当理解的是，此处所描述的具体实施方式仅用以解释本发明，并不限定本发明的保护范围。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式的目的，不是旨在于限制本发明。本文所使用的术语“及/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明所述FQPCR为极速荧光PCR仪，QPCR为普通荧光PCR仪。
如图1所示，一种极速PCR反应检测系统，包括温度控制系统温度控制装置、传动装置及反应管固定装置；
所述温度控制装置至少包括2个温控模块，其中至少1个所述温控模块为高温模块和至少1个所述温控模块为低温模块；其中，所述高温模块的加热温度为第一预定温度，所述低温模块的加热温度为第二预定温度，所述第一预定温度大于所述第二预定温度；
所述传动装置与所述温度控制装置及所述反应管固定装置相配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。
如此，反应管可以安装在反应管固定装置上，安装后使用压盖压住，实验中反应管可以随反应管固定装置运动。高温模块提供第一预设的温度的温控控制，可以使反应管快速升温至目标温度，低温模块提供第二预设温度的温度控制，可以使反应管快速降温至目标温度。正常扩增循环的变性及退火过程，采用路过型温度控制，对于预变性需要在一个温度保持一定时间的流程，通过在该温控模块(高温模块)以一定频率上下运动保持一个持续的温度。
需要说明的是，第一预设温度的最小温度大于第二预设温度的最大温度。具体可根据需要进行选择。
需要说明的是，“传动装置”的具体实现方式可以有多种，包括但不限于多轴机器人操作臂、三坐标传动设备等能够满足上述要求的传动设备。
具体地，一实施例中，所述传动装置与所述温度控制装置连接，并能够驱动所述高温模 块与所述低温模块移动，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。如此，可以通过传动装置高温模块及低温模块运动，为反应管固定装置上的反应管提供所需的反应温度。此时，该传动装置可为多轴机器人操作臂或三坐标传动设备。
或，一实施例中，所述传动装置与所述反应管固定装置连接，并驱动所述反应管固定装置移动，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。如此，可以通过反应管固定装置上的反应管运动，使得反应管插入到高温模块中或低温模块中，为反应管固定装置上的反应管提供所需的反应温度。此时，该传动装置可为多轴机器人操作臂或三坐标传动设备。
或，再一实施例中，所述传动装置包括第一传动机构及第二传动机构，所述第一传动机构与温度控制装置连接，并能够驱动所述温度控制装置移动，所述第二传动机构与所述反应管固定装置连接，并能够驱动所述反应管固定装置移动；所述第一传动机构与所述第二传动机构配合，使得所述反应管固定装置上反应管在所述高温模块与所述低温模块之间进行切换。如此，利用第一传动机构来控制温度控制模块运动，使得高温模块与低温模块能够对应所需的反应管；然后再通过第二传动机构来驱动反应管固定装置移动，使得反应管固定装置的反应管插入到所需的高温模块或低温模块中，以满足PCR反应所需的温度。
或，再另一实施例中，所述传动装置包括第一传动机构及第二传动机构，所述第一传动机构与温度控制装置连接，并能够驱动所述温度控制装置水平移动，所述第二传动机构与所述反应管固定装置连接，并能够驱动所述反应管固定装置垂直移动；所述反应管固定装置架在温度控制装置上部，所述第一传动机构与所述第二传动机构配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。反应管固定装置可随第二传动机构上下运动。
本实施例所述温度控制装置包括2个温控模块，如图1所示，有前到后依次为低温模块12、高温模块13；也可以是3个温控模块，如图2所示，由签到后依次为中温模块11、低温模块12、高温模块13；所述温控模块能保持温度恒定或改变温度。
本实施例中所述高温模块可设置温度超过100℃(即第一预设温度可以设置为大于或等于100℃)，使反应管可以快速升温至变性温度，低温模块12可以设置温度为低于退火延伸所要求的温度，使反应管快速降温达到退火温度，如低于55℃(即第二预设温度可以设置为小于或等于55℃)。
如图1所示，最前部低温模块侧壁开有检测孔14，所述低温模块12、高温模块13上部均设置有样品孔15。如图1所示，当温控模块为3个时，最前部的中温模块侧壁开有检测孔，中温模块11、低温模块12、高温模块13上部均设置有样品孔15。
如图2所示，本发明中所述第一传动机构包括丝杆电机21和2根导向传动轴22，2根导向传动轴22固定于丝杆电机21前部，温控模块沿导向传动轴22进行前后运动。
如图3所示，本发明所述光学检测装置包括光学读头31和第三传动机构32，所述光学读头安装在第三传动机构上部，在第三传动机构32的带动下进行左右移动；所述第三传动机构32包括步进电机Ⅰ321、导轨322、皮带323、连接件324，所述连接件324上部固定光学读头321，连接件324中部设置与导轨滑动连接的凹槽3241，连接件下部设置与皮带连接的夹板3242。
如图4所示，本发明所述第二传动机构包括步进电机Ⅱ41和偏心轮42，偏心轮42固定在步进电机Ⅱ41上，反应管固定装置5下部固定安装横向支撑板51，横向支撑板51下部由偏心轮支撑，步进电机Ⅱ41带动偏心轮42转动，通过带动横向支撑板上下运动实现反应管固定装置的上下运动；所述反应管固定装置5还包括反应管固定位52、压盖53、竖向支撑杆54、立柱55，所述反应管固定位上部设置压盖，所述竖向支撑杆54固定于反应管固定位52两侧，立柱垂直穿过竖向支撑杆54，立柱55与竖向支撑杆54滑动连接，所述立柱顶部套设弹簧56并顶住竖向支撑杆54。
如图1所示，本发明所述极速PCR反应检测系统还包括控制系统6，所述控制系统内部 有控制电路，可以对温度控制装置、第一传动机构、光学检测装置、反应管固定装置进行实时控制及信号传输。
为了让仪器具有较好的散热，在本发明仪器的后端，位于控制电路上方设置2个风扇。
反应管为EP管。
一种极速PCR检测方法，应用如上所述极速PCR反应检测系统，包括如下操作步骤：
①准备：启动仪器预热，将待测样本和试剂装入反应管并混合；
②高温模块移动至反应管正下方，第二传动机构下降至最低点，反应管进行高温温浴；
③反应管温度达到所需温度后，第二传动机构复位，低温模块移动到反应管正下方，第二传动机构下降至最低点，进行降温；
④第二传动机构复位，中温模块移动到反应管的正下方，保持一定时间。
⑤数据采集；
⑥重复第②～⑤步骤。
当使用上述方法和设备来进行核酸分析和处理时，上述样本和试剂包含了反应成分，此反应成分包括了至少一种酶、含有至少一种核酸的核酸和/或粒子、PCR所用的引物、等温扩增所用的引物、其它核酸扩增和处理的引物、dNTP、Mg2+、荧光染料和探针、控制DNA、控制RNA、控制细胞、控制微生物，和其它核酸扩增、处理和分析所需的试剂。上述含有核酸的粒子包含至少一个细胞病毒、白血球和基质细胞、循环肿瘤细胞、胚胎细胞仪器检测。
上述方法和设备被用于聚合酶链式反应、逆转录-聚合酶链式反应、终点PCR、连接酶链式反应、核酸测序或各聚合酶链式反应(PCR)的变化的预扩增或模板富集、等温扩增、线性扩增、用于测序的文库制备、用于测序的桥扩增。上述聚合酶链式反应的变化包括逆转录-PCR、实时荧光定量聚合酶链式扩增反应和实时荧光定量逆转录聚合酶链式扩增反应、反向聚合酶链式扩增反应、锚定聚合酶链式扩增反应、不对称聚合酶链式扩增反应、多重PCR、色彩互补聚合酶链式扩增反应、免疫聚合酶链式扩增反应、巢式聚合酶链式扩增反应、预扩增或核酸测序的模板富集、ELISA-PCR。
试验检测：
1、反应系统移动平稳，结构紧凑，提升仪器的运动速度：
PCR反应管与温度控制装置进行相对运动，第一传动机构带动温度控制装置进行水平运动，所述反应管固定装置架在温控系统上部，反应管固定装置与第二传动机构相连并可随第二传动机构上下运动，从而实现反应管固定装置上的反应管在温控模块之间进行相对往复运动，第二传动机构与第一传动机构不连接，因此，第一传动机构运动时，不会带动反应管进行水平运动，由于PCR反应体积都较小，减少反应管的震动，能避免反应管中的反应液挂壁，能避免反应管中气泡的产生，从而提高检测的准确性。
本发明所述仪器，在使用时，为了PCR在预变性需要在一个温度保持一定时间的流程，采用的方式是在该温控模块以一定频率的上下运动来保持一个持续的温度，为保证上下运动的速度和灵活性，本发明采用电机带动偏心轮转动实现，偏心轮只需要转动一定的角度，就可以瞬间达到上下运动效果，该上下运动的方式，会比皮带传动更快，从而能够提升上下运动的速度和效率，通过该种运动方式，能提高温度控制的准确度同时也能加快仪器的运动速度，缩短检测时间。
2、高温模块和低温模块的温度设置，能提升反应管中反应液的升降温速率。
一般PCR仪中的高温模块温度设置最高为99℃，而本发明所述高温模块设置的温度为超过100℃，经过多次试验，证明在120℃能达到较为理想的升温速率，同时，本发明所述检测仪器，只需要使用普通反应管就可以达到要求。
本发明所述低温模块，所设置的温度低于实验流程目标温度设置，可以以较快的速度到达目标温度，进而提高升降温速度。
测试条件：第一预设温度为120℃，第二预设温度为40度以下。
扩增条件设置：
变性：88℃，1sec；
退火：66℃，1sec，该温度下检测，检测后开始下一轮流程；
Figure PCTCN2020108969-appb-000001
对本实验进行温度曲线的记录，如图5所示。
经过分析，该温度模块的设置，最大升温速度达到10℃/S，降温达到了8℃/S，平均升级降温速度会在7℃/S以上。对于实际工作65-90℃，一次升降温大约需要7秒，加上解链1秒和延展1秒以及检测1秒，合计1个循环10秒，40循环为440秒，大约6.6分钟，再计入2分钟预变性时间，合计约为8.6分钟完成1次PCR。
在试验过程中，发现当低温模块温度设置为室温(25℃-30℃)时，反应管温度在退火阶段没有稳定，分析原因，发现降温时，反应管壁迅速的被低温模块传导至较低温状态，即使反应管后期脱离低温模块，但反应管温度较低，脱离低温模块后低温继续从反应管壁向反应管内的实验体系传导，降温趋势没有减缓，当低温模块设置为47℃后，降温一段时间后使反应管脱离低温模块，能稳定住退火温度，从而能在温度控制方面提升PCR检测的重复性。
3、不同样品检测孔的检测结果一致性、重复性良好。
本发明所述高温模块、中温模块、低温模块，上部均设置有样品孔，在温控模块样品孔的前部，开设有检测孔，通过样品孔上的压盖，将反应管压紧在样品孔中，能让反应管与温控模块具有良好的贴合度，从而能提升每个样品的升降温速率，同时也能保证每个样品管的温度变化的一致，进而提升样品检测结果的重复性、一致性。
本发明所述压盖的材质为硬金属材质，优选为不锈钢材质。压盖材质的不同，会对检测结果造成较大的影响。当压盖的材质为铝质金属条时，由于压盖易变形，从而导致不同样品孔上方的压紧程度不同，影响反应管与温控模块的贴合度，会导致不同样品孔的反应管受热不均，同时，压盖力度不均，也会造成反应管内体系相对于检测孔位置差异，极端的情况可能检测到体系与石蜡油的交界面，最终导致仪器的重复性不佳。通过将压盖的材质更换为不锈钢材质，则很好的解决了这一问题。
具体检测过程如下：
对照组1：仪器所使用的压盖为铝质金属材质
温度控制模块从左到右依次编号为1号-8号，在1号位-8号位8个位置各做1次试验，考察不同孔位重复性情况。测试前一次性配制10X测试体系，分装于8个100ul的透明反应管中，每管分装20ul混合后体系+20ul石蜡油，分别放置于1号位-8号位进行测试，温度控制流程如下：
设置扩增条件：
预变性：88℃，2min；
变性：88℃，1sec；
退火：64℃，1sec，该温度下检测，检测后开始下一轮流程；
Figure PCTCN2020108969-appb-000002
检测结果如图6所示，图6中可以看到，8号位的重复性还可以，3号位的重复性不佳。
实验组：仪器所使用的压盖为不锈钢材质。
温度控制模块检测孔从左到右依次编号为1号-8号，在3号位、5号位、7号位测试，进行了3轮重复，共9条曲线，考察不同孔位重复性情况。
设置扩增条件：
预变性：88℃，2min；
变性：88℃，1sec；
退火：64℃，1sec，该温度下检测，检测后开始下一轮流程；
Figure PCTCN2020108969-appb-000003
测试前一次性配制10X测试体系，分装于9个100ul的透明反应管中，每管分装20ul混合后体系+20ul石蜡油，分别放置于在3号位、5号位、7号位测试。检测结果如图7所示。
由图6、7所示，结果表明，压盖的材质对不同孔位检测结果的重复性有非常大的影响，只有使用不易变形的金属材质，将温控模块上不同位置的检测孔中的反应管都能压紧的情况下，反应管与检测模块才有良好的贴合度，重复性才能满足检测要求。
4、同一孔位重复性良好
具体检测过程如下：
温度控制模块从左到右依次编号为1号-8号，在2号位和5号位两个位置各做5次重复试验，考察重复性情况。测试前一次性配制10X测试体系，分装于10个100ul的透明反应管中，每管分装20ul混合后体系+20ul石蜡油，放置于冰箱中(2-8℃)保存，每次实验前从冰箱取出2个反应管分别放置于2号位和5号位进行测试，温度控制流程如下：
本次检测，极速PCR仪温控模块的设置为：高温模块120℃，低温模块47℃。
设置扩增条件：
预变性：88℃，2min；
变性：88℃，1sec；
退火：64℃，1sec，该温度下检测，检测后开始下一轮流程；
Figure PCTCN2020108969-appb-000004
检测结果如图8、9所示，2号位及5号位每个位置5次重复测试的扩增曲线，可以看出，单独孔位测试具有较好的重复性。
具体试验数据见下表：
表1 2号位和5号位Ct值重复性数据
Figure PCTCN2020108969-appb-000005
从上表的Ct值分析情况来看，2号位和5号位的极差都不超过0.4，所有的10个Ct值一起分析，整个极差也不超过0.4。将上表中所述2号位及5号位各5次重复测试的Ct值做正态分布及直方图，从图10中可以看出，10个Ct值有9个与均值差在±0.1范围内，只有1个测试的Ct值与均值差异超出0.2。
如上数据说明，本发明所述检测仪器在同一孔位重复性良好。
5、中温模块的设置，能显著提升PCR的扩增效率
将低温池的温度从室温提高到47℃，扩增效率没有提高，观察温控曲线，即便低温模块提供47℃一个的温度，但降温速率过快，单纯提供一个低温温度、或者将EP管从低温模块脱离，都不能减缓降温的速率，故如需在退火延伸温度加以保持，需要在到达退火温度后给一个热源使EP管升温，优选的方式是增加一个延伸温度的中温池，在EP管通过低温池达到退火温度后进入中温池保持并检测，再进入高温池进行变性解链。
6、本发明所述极速荧光PCR仪与普通荧光PCR仪在扩增效率与检出限方面没有显著差异，检测结果重复性好，结果准确。
本次检测，极速PCR仪温控模块的设置为：高温模块120℃，低温模块47℃。
样品处理：使用本实验极速荧光PCR仪及标准QPCR仪进行不同浓度梯度10^1～10^5拷贝扩增实验，考察灵敏度和检测范围；
实验流程为：
预变性：90℃，2min；
变性：90℃，1sec；
退火：60，7sec，该温度下检测，检测后开始下一轮流程；
Figure PCTCN2020108969-appb-000006
图11为对照用的QPCR仪及FQPCR测试10^1～10^5拷贝的扩增情况。从曲线上来看，两台仪器的测试没有显著差异，不同浓度样本之间的Ct值间距也相当。
表2 QPCR及FQPCR灵敏度测试的扩增Ct值
Figure PCTCN2020108969-appb-000007
表2为QPCR及FQPCR灵敏度测试的扩增Ct值，图12为两台仪器进行灵敏度测试将定量对照品梯度稀释测试得到的标准曲线。从标曲可以看出，两条曲线的斜率相当，相关系数R 2也都大于0.99。通过标准曲线计算扩增效率，两台仪器的扩增效率相近(110％～115％)之间。
FQPCR仪与对照的QPCR仪进行10^1～10^5copies浓度的灵敏度测试，扩增效率及检出限没有显著差异，两台仪器均可以检出10copies浓度样本。
FQPCR仪与对照的QPCR仪进行10^1～10^5copies浓度的灵敏度测试，扩增效率及检出限没有显著差异。
需要说明的是，“某体”、“某部”可以为对应“构件”的一部分，即“某体”、“某部”与该“构件的其他部分”一体成型制造；也可以与“构件的其他部分”可分离的一个独立的构件，即“某体”、“某部”可以独立制造，再与“构件的其他部分”组合成一个整体。本申请对上述“某体”、“某部”的表达，仅是其中一个实施例，为了方便阅读，而不是对本申请的保护的范围的限制，只要包含了上述特征且作用相同应当理解为是本申请等同的技术方案。
需要说明的是，本申请“单元”、“组件”、“机构”、“装置”所包含的构件亦可灵活进行组合，即可根据实际需要进行模块化生产，以方便进行模块化组装。本申请对上述构件的划分，仅是其中一个实施例，为了方便阅读，而不是对本申请的保护的范围的限制，只要包含了上述构件且作用相同应当理解是本申请等同的技术方案。
在本发明的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”、“顺时针”、“逆时针”、“轴向”、“径向”、“周向”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本发明中，除非另有明确的规定和限定，第一特征在第二特征“上”或“下”可以是第一和第二特征直接接触，或第一和第二特征通过中间媒介间接接触。而且，第一特征在第二特征“之上”、“上方”和“上面”可是第一特征在第二特征正上方或斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”可以是第一特征在第二特征正下方或斜下方，或仅仅表示第一特征水平高度小于第二特征。
需要说明的是，当元件被称为“固定于”、“设置于”、“固设于”或“安设于”另一个元件，它可以直接在另一个元件上或者也可以存在居中的元件。当一个元件被认为是“连接”另一个元件，它可以是直接连接到另一个元件或者可能同时存在居中元件。进一步地，当一个元件被认为是“固定传动连接”另一个元件，二者可以是可拆卸连接方式的固定，也可以不可拆卸连接的固定，能够实现动力传递即可，如套接、卡接、一体成型固定、焊接等，在现有技术中可以实现，在此不再累赘。当元件与另一个元件相互垂直或近似垂直是指二者的理想状态是垂直，但是因制造及装配的影响，可以存在一定的垂直误差。本文所使用的术语“垂直的”、“水平的”、“左”、“右”以及类似的表述只是为了说明的目的，并不表示是唯一的实施方式。
以上实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种极速PCR反应检测系统，其特征在于，包括温度控制装置、传动装置及反应管固定装置；
    所述温度控制装置至少包括2个温控模块，其中至少1个所述温控模块为高温模块和至少1个所述温控模块为低温模块；其中，所述高温模块的加热温度为第一预定温度，所述低温模块的加热温度为第二预定温度，所述第一预定温度大于所述第二预定温度；
    所述传动装置与所述温度控制装置及所述反应管固定装置相配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。
  2. 根据权利要求1所述极速PCR反应检测系统，其特征在于，所述传动装置与所述温度控制装置连接，并能够驱动所述高温模块与所述低温模块移动，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换；
    或所述传动装置与所述反应管固定装置连接，并驱动所述反应管固定装置移动，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换；
    或所述传动装置包括第一传动机构及第二传动机构，所述第一传动机构与温度控制装置连接，并能够驱动所述温度控制装置移动，所述第二传动机构与所述反应管固定装置连接，并能够驱动所述反应管固定装置移动；所述第一传动机构与所述第二传动机构配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。
  3. 根据权利要求1所述极速PCR反应检测系统，其特征在于，所述传动装置包括第一传动机构及第二传动机构，所述第一传动机构与温度控制装置连接，并能够驱动所述温度控制装置水平移动，所述第二传动机构与所述反应管固定装置连接，并能够驱动所述反应管固定装置垂直移动；所述反应管固定装置架在温度控制装置上部，所述第一传动机构与所述第二传动机构配合，使得所述反应管固定装置上的反应管在所述高温模块与所述低温模块之间进行切换。
  4. 根据权利要求3所述极速PCR反应检测系统，其特征在于，所述温度控制装置的下部与第一传动机构连接，在第一传动机构的作用下温度控制装置前后运动；
    优选的，所述第一传动机构包括丝杆电机和2根导向传动轴，2根导向传动轴固定于丝杆电机前部，温控模块可沿导向传动轴进行前后运动；
    或，第二传动机构通过偏心轮转动带动反应管固定装置上下运动。
  5. 根据权利要求3所述的极速PCR反应检测系统，其特征在于，所述反应管固定装置还包括横向支撑板，所述横向支撑板固定安装在所述反应管固定装置的下部；所述第二传动机构包括步进电机Ⅱ和偏心轮，所述偏心轮固定在步进电机Ⅱ上，偏心轮支撑位于上部的横向支撑板，步进电机Ⅱ带动偏心轮转动，通过带动横向支撑板上下运动实现反应管固定装置的上下运动；
    优选的，所述反应管固定装置还包括竖向支撑杆、立柱，所述竖向支撑杆固定于反应管固定位两侧，立柱垂直穿过竖向支撑杆，立柱与竖向支撑杆滑动连接，所述立柱顶部套设弹 簧并顶住竖向支撑杆。
  6. 根据权利要求3所述的极速PCR反应检测系统，其特征在于，还包括控制系统，所述控制系统为控制电路，对温度控制装置、第一传动机构、光学检测装置、反应管固定装置进行实时控制及信号传输。
  7. 根据权利要求1所述极速PCR反应检测系统，其特征在于，所述反应检测系统还包括光学检测装置，所述光学检测装置位于温度控制装置前部；
    优选的，所述光学检测装置包括光学读头和第三传动机构，所述光学读头安装在第三传动机构上部，在第三传动机构的带动下进行左右移动；
    优选的，所述第三传动机构包括步进电机Ⅰ、导轨、皮带、连接件，所述连接件上部固定光学读头，连接件中部设置与导轨滑动连接的凹槽，连接件下部设置与皮带连接的夹板；或/和所述反应管固定装置包括反应管固定位、压盖，所述压盖用于盖合反应管固定位；
    优选的，所述压盖材质为不锈钢。
  8. 根据权利要求1至7任一所述极速PCR反应检测系统，其特征在于，所述第一预设温度大于或等于100℃，所述第二预设温度小于或等于55℃；
    优选的，所述第一预设温度的范围为100～150℃，所述第二预设温度的范围为15～55℃；
    优选的，所述温度控制装置还包括中温模块，所述中温模块的加热温度范围为所述第一预设温度与所述第二预设温度之间。
    优选的，所述温控模块由前向后依次为中温模块、低温模块、高温模块；
    优选的，所述最前部中温模块侧壁开有检测孔，所述中温模块、低温模块、高温模块上部均设置有样品孔；
    优选的，温控模块能保持温度恒定或改变温度。
  9. 一种极速PCR检测方法，其特征在于，包括如下操作步骤：
    ① 准备：启动仪器预热，将待测样本和试剂装入反应管并混合；
    ② 反应管固设于反应管固定装置，传动装置动作，使得反应管插入高温模块中，进行高温温浴；
    ③ 反应管温度达到所需温度后，传动装置动作，使得反应管插入低温模块中，进行降温；
    ④ 数据采集；
    ⑤ 重复第②～④步骤。
  10. 根据权利要求9所述的极速PCR检测方法，其特征在于，所述传动装置包括第二传动机构；其中，步骤②还包括：高温模块移动至反应管正下方，垂直传动机构第二传动机构下降至最低点，反应管进行高温温浴；步骤③还包括：反应管温度达到所需温度后，垂直传动机构第二传动机构复位，低温模块移动到反应管正下方，垂直传动机构第二传动机构下降至最低点，进行降温；
    或/和步骤③反应管降温之后，还包括如下步骤：第二传动机构复位，中温模块移动到反 应管的正下方。
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