CN113755316B - Switchable incubation module, PCR amplification detector - Google Patents
Switchable incubation module, PCR amplification detector Download PDFInfo
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- CN113755316B CN113755316B CN202111176405.7A CN202111176405A CN113755316B CN 113755316 B CN113755316 B CN 113755316B CN 202111176405 A CN202111176405 A CN 202111176405A CN 113755316 B CN113755316 B CN 113755316B
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- 238000011534 incubation Methods 0.000 title claims abstract description 42
- 238000012408 PCR amplification Methods 0.000 title claims abstract description 16
- 238000010438 heat treatment Methods 0.000 claims abstract description 292
- 238000006243 chemical reaction Methods 0.000 claims abstract description 63
- 230000007246 mechanism Effects 0.000 claims description 13
- 238000001917 fluorescence detection Methods 0.000 claims description 8
- 238000010792 warming Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 18
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
- B01L7/5255—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones by moving sample containers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
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Abstract
本发明涉及一种可切换的温育模块，包括用于加温微流控芯片的温育本体，温育本体包括：若干加热模块，用于加热微流控芯片上的反应腔；其中，至少有两加热模块上的加热温度不同；旋转驱动结构，用以驱使微流控芯片与加热模块相对转动；每一加热模块上均设置有相同数量的加热区，若干加热模块上的加热区与微流控芯片同心设置；开启温育时，在旋转驱动结构的驱使下，微流控芯片在不同加热温度的加热模块的加热区依序转动，以切换微流控芯片的反应腔在温育时的加热温度。本发明还涉及一种PCR扩增检测仪。通过可切换的温育模块，以快捷提供微流控芯片上的反应腔扩增反应所需温度。
The present invention relates to a switchable incubation module, comprising an incubation body for heating a microfluidic chip, wherein the incubation body comprises: a plurality of heating modules for heating a reaction chamber on a microfluidic chip; wherein at least two heating modules have different heating temperatures; a rotary drive structure for driving the microfluidic chip and the heating module to rotate relative to each other; each heating module is provided with the same number of heating zones, and the heating zones on a plurality of heating modules are arranged concentrically with the microfluidic chip; when incubation is started, driven by the rotary drive structure, the heating zones of the heating modules at different heating temperatures of the microfluidic chip rotate in sequence to switch the heating temperature of the reaction chamber of the microfluidic chip during incubation. The present invention also relates to a PCR amplification detector. Through the switchable incubation module, the temperature required for the amplification reaction of the reaction chamber on the microfluidic chip can be quickly provided.
Description
[ Field of technology ]
The invention relates to the technical field of fluorescence detection, in particular to a switchable incubation module and a PCR amplification detector.
[ Background Art ]
The substance for nucleic acid detection is a viral nucleic acid. Nucleic acid testing is the finding of the presence or absence of foreign invading viral nucleic acid in a patient's respiratory specimen, blood or stool to determine if it is infected with a virus. Thus, once positive for nucleic acid, the presence of virus in the patient is demonstrated. All organisms contain nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and in the case of novel coronaviruses, are viruses that contain only RNA, and specific RNA sequences in the virus are markers that distinguish the virus from other pathogens. In clinical laboratory testing, if a specific nucleic acid sequence of a novel coronavirus is detected in a sample of a patient, it should be suggested that the patient may be infected with the novel coronavirus.
The most common method of detecting virus-specific sequences is fluorescent quantitative PCR (polymerase chain reaction). The method is a method for measuring the total amount of products after each polymerase chain reaction cycle by using fluorescent chemical substances in DNA amplification reaction, and a method for quantitatively analyzing specific DNA sequences in a sample to be measured by an internal reference method or an external reference method. The fluorescence quantitative PCR instrument can monitor that the cycle number (Ct value) at which fluorescence reaches a preset threshold is related to the virus nucleic acid concentration, and the higher the virus nucleic acid concentration is, the smaller the Ct value is.
In the traditional nucleic acid detection flow, the processing steps are more and the time is long (in the traditional PCR amplification step, a single temperature control chamber is used for heating and cooling, a heating plate is used for controlling the temperature change of a PCR reaction cavity,
The temperature control chip is easy to heat, but the temperature of the chip is reduced only by natural convection, so that the temperature conversion can be achieved only by consuming a long time, the heating plate is high in cost and easy to damage, the requirement on PCR sensitivity is high, the chip is easy to be interfered by environment, at least 3 negative pressure chambers are needed for traditional nucleic acid detection, and meanwhile, extra equipment such as professionals and centrifuges are equipped, the construction cost is high, hospitals below the second level are difficult to develop independently, and the centralized distribution situation is presented.
Accordingly, there is a need for an improvement over the prior art to overcome the deficiencies described in the prior art.
[ Invention ]
In view of the shortcomings of the prior art, it is an object of the present invention to provide a switchable incubation module comprising an incubation body for warming a microfluidic chip, the incubation body comprising:
The heating modules are used for heating the reaction cavities on the microfluidic chip, wherein the heating temperatures of at least two heating modules are different;
the rotary driving structure is used for driving the microfluidic chip and the heating module to rotate relatively;
And when the micro-fluidic chip is started to incubate, the micro-fluidic chip is driven by a rotary driving structure to sequentially rotate in the heating areas of the heating modules with different heating temperatures so as to switch the heating temperature of the reaction cavity of the micro-fluidic chip during incubation.
Preferably, an insulating layer is also included to maintain the temperature within the incubation body.
Preferably, the heating areas on the heating modules are equidistant from the central axis of the heating module.
Preferably, the distances between the heating areas on the different heating modules and the central axis of the heating module are different, and after the microfluidic chip and the heating module are driven to rotate relatively, the reaction liquid to be heated on the microfluidic chip moves under the action of centrifugal force to contact the heating modules with different heating temperatures.
Preferably, the device further comprises a motion moving mechanism, wherein the motion moving mechanism is connected with the heating modules, and the motion moving mechanism is controlled to alternately control different heating modules to be contacted with the reaction cavities on the microfluidic chip.
Preferably, gaps exist between adjacent heating zones of the heating modules, wherein the heating zone on one heating module passes through the gaps between the heating zones of the other heating module to contact the reaction cavity on the microfluidic chip.
Preferably, the at least one heating module comprises a first heating body, a first temperature control heating plate and a first heat insulation fixing block, wherein the first heating body and the first temperature control heating plate are fixed through the first heat insulation fixing block, a plurality of first extending columns extend along the outer contour of the first heating body towards the direction close to the central axis of the first heating body, and a first heating column is further arranged on the first extending column, which is close to the side where the microfluidic chip is located, and is controlled to move to a reaction cavity on the microfluidic chip so as to provide proper reaction temperature.
Preferably, the rotary driving structure is used for driving the micro-fluidic chip to rotate.
Preferably, the rotation driving mechanism is used for driving a plurality of heating modules to rotate.
The invention also relates to a PCR amplification detector which is characterized by comprising a reaction module, a switchable incubation module and a fluorescence detection module which are sequentially arranged from top to bottom.
Compared with the prior art, the invention has the beneficial effects that:
The invention relates to a switchable incubation module, which comprises an incubation body for heating a microfluidic chip, wherein the incubation body comprises a plurality of heating modules for heating reaction chambers on the microfluidic chip, wherein at least two heating modules are different in heating temperature, a rotary driving structure is used for driving the microfluidic chip and the heating modules to rotate relatively, each heating module is provided with the same number of heating areas, the heating areas on the plurality of heating modules are arranged concentrically with the microfluidic chip, and when the incubation is started, the microfluidic chip is driven by the rotary driving structure to sequentially rotate in the heating areas of the heating modules with different heating temperatures so as to switch the heating temperature of the reaction chambers of the microfluidic chip during incubation. The invention also relates to a PCR amplification detector. The temperature required by the amplification reaction of the reaction cavity on the microfluidic chip is rapidly provided by the switchable incubation module.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings. Specific embodiments of the present invention are given in detail by the following examples and the accompanying drawings.
[ Description of the drawings ]
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
FIG. 1 is a schematic structural view of an incubation module of the present invention;
FIG. 2 is a schematic diagram of a plurality of heating modules according to the present invention;
FIG. 3 is a schematic diagram of a moving mechanism according to the present invention;
FIG. 4 is a schematic view of the structure of a different heating body according to the present invention;
FIG. 5 is a schematic diagram of a second heating module according to the present invention;
FIG. 6 is a schematic diagram of a third heating module according to the present invention;
FIG. 7 is a schematic view of a first heating module according to the present invention;
FIG. 8 is a schematic view of a rotary driving structure according to the present invention;
FIG. 9 is a schematic diagram of a fluorescence detection module according to the present invention.
Reference numerals illustrate:
100. a PCR amplification detector;
101. The device comprises a motion moving mechanism, 1011, a bracket, 1012, a round shaft, 1013, a micro push rod, 1014, a vertical plate, 1021, a third fixed plate, 1022, a second fixed plate, 1023, a first fixed plate, 1024, a guide rod, 1025, a temperature control module fixed plate, 1026 and a bearing;
102. Incubation module, 110, first heating module, 111, first extension column, 112, first heating column, 113, first temperature control heating plate, 114, first heat insulation fixing block, 115, first heating body;
120. The second heating module, 121, a heat conduction column, 122, a second temperature control heating plate, 123, a heat insulation fixing column, 124, a second heating body;
130. the heating device comprises a third heating module, a second extension column, a second heating column, a third temperature control heating plate, a third heat insulation fixing block, a third heating body and a third heating body, wherein the third heating module comprises a first extension column, a second extension column, a third heat insulation fixing block and a third heat insulation fixing block;
103. the rotary driving structure comprises 1031, a servo motor, 1032, a servo motor vertical plate, 1033, a servo motor fixing plate, 1034, a chip fixing bracket, 1035 and a turntable;
104. the device comprises a fluorescence detection module 1041, a light splitting sheet 1042, a light emitter 1043 and an imaging detector.
[ Detailed description ] of the invention
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses a device for practicing the invention. In the drawings, the shape and size may be exaggerated for clarity, and the same reference numerals will be used throughout the drawings to designate the same or similar components. In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc. are based on the orientation or positional relationship shown in the drawings. In particular, "height" corresponds to the top-to-bottom dimension, "width" corresponds to the left-to-right dimension, and "depth" corresponds to the front-to-back dimension. These relative terms are for convenience of description and are not generally intended to require a particular orientation. Terms (e.g., "connected" and "attached") referring to an attachment, coupling, etc., refer to a relationship wherein these structures are directly or indirectly secured or attached to one another through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below. It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
As shown in fig. 1-9, the present invention relates to a switchable incubation module 102 comprising an incubation body for warming a microfluidic chip, the incubation body comprising:
The heating modules are used for heating the reaction cavities on the microfluidic chip, wherein the heating temperatures of at least two heating modules are different;
the rotary driving structure 103 is used for driving the microfluidic chip and the heating module to rotate relatively;
And when the micro-fluidic chip is started to incubate, under the driving of the rotary driving structure, the micro-fluidic chip rotates sequentially in the heating areas of the heating modules with different heating temperatures so as to switch the heating temperature of the reaction cavity of the micro-fluidic chip during incubation. The number of heating modules is related to the number of different temperature values required for the microfluidic chip to react. When the microfluidic chip is used in a PCR amplification reaction, three different temperatures are typically required, where the incubation module 102 includes three heating modules, as shown in fig. 2 and 4, where the incubation module 102 includes a plurality of temperature change zones, each temperature change zone including a heating zone on each heating module, such as a temperature change zone including a heating zone on the first heating module 110, a heating zone on the second heating module 120, and a heating zone on the third heating module 130.
It should be understood that the incubation body is an incubation device, typically a sealed structure, etc., that is common in microfluidic chip warming processes.
In some embodiments, the heating module further includes a heating body, where the heating body may be configured in any shape, and only the heating area on the heating body needs to be ensured to heat the reaction cavity on the microfluidic chip. It should be understood that the microfluidic chip may be provided in any shape. Preferably, the heating body is annular, at this time, the microfluidic chip may also be disk-shaped, and the two cooperate to ensure that the heating region can be fully contacted with the reaction chamber.
In some embodiments, a thermal insulation layer is also included to maintain the temperature within the incubation body, preferably around the periphery of the microfluidic chip to provide a better thermal environment for the microfluidic chip.
In some embodiments, the heating zones on the plurality of heating modules are equidistant from a central axis of the heating modules. That is, as shown in fig. 2, several heating modules are located on the same circumference, and at this time, as the microfluidic chip rotates, the heating zones on different heating modules may contact the reaction chambers on the microfluidic chip to provide different heating temperatures.
In some embodiments, the distances between the heating areas on different heating modules and the central axis of the heating modules are different, namely, the different heating modules are not located on the same circumference, after the microfluidic chip and the heating modules are driven to rotate relatively, the reaction liquid to be heated on the microfluidic chip moves under the action of centrifugal force to contact the heating modules with different heating temperatures, for example, in a specific embodiment, the heating areas on different heating modules are located at different positions with the same radius, the temperatures of the heating areas on different heating modules are set according to the reaction requirements, after the microfluidic chip rotates relatively, the liquid to be heated in the microfluidic chip moves away from the center of the microfluidic chip under the action of centrifugal force, and the reaction liquid to be heated on the microfluidic chip contacts different heating modules successively in the moving process to realize heating with different temperatures.
When the micro-fluidic chip and the heating module are driven to rotate relatively, in some embodiments, the rotary driving structure is used for driving the micro-fluidic chip to rotate, and at this time, the rotary driving structure can be a turntable, and the turntable drives the micro-fluidic chip to rotate. In other embodiments, the rotary drive mechanism is used to drive a plurality of heating modules to rotate, and in this case, the rotary drive mechanism directly acts on different heating modules.
In some embodiments, the micro-fluidic chip further comprises a motion moving mechanism 101, wherein the motion moving mechanism 101 is connected with the heating modules, and the different heating modules are controlled to be in contact with the reaction cavities on the micro-fluidic chip in turn by controlling the motion moving mechanism. In some embodiments, the motion moving mechanism 101 comprises a fixed plate, a guide rod and a push rod, wherein one end of the guide rod is connected with the fixed plate, the other end of the guide rod is connected with the heating module, and the push rod pushes the fixed plate to move so as to drive the heating module to move towards the reaction cavity on the microfluidic chip. Specifically, the first heating module 110, the second heating module 120, and the third heating module 130 are respectively fixed to the first fixing plate 1023, the second fixing plate 1022, and the third fixing plate 1021 through guide rods 1024, and the motion control in the height direction can be implemented in the bearing 1026 through three independently operated push rods such as micro push rods 1013. As shown in fig. 1-4, the device is composed of a bracket 1011, a circular shaft 1012, micro push rods 1013 and a vertical plate 1014, wherein two brackets 1011 fixed on the two vertical plates are used for fixing the circular shaft 1012, and three micro push rods 1013 are fixed on the circular shaft 1012, so that control of different directions (three-dimensional directions) can be independently realized.
Furthermore, gaps exist between adjacent heating zones of the heating modules, wherein the heating zone on one heating module passes through the gaps between the heating zones of the other heating module to contact the reaction cavity on the microfluidic chip. I.e. if the first heating module 110 is the heating module closest to the microfluidic chip, the heat conducting pillars 121 on the second heating module 120 pass through the gaps between the first heating pillars 112 on the first heating module and the gaps between the second heating pillars 132 on the third heating module to approach the microfluidic chip, and similarly, the second heating pillars 132 on the third heating module 130 pass through the gaps between the pairs of heating pillars on the first heating module 110 to approach the microfluidic chip.
In some embodiments, when the heating body of the heating module is configured to be annular and includes three heating modules, as shown in fig. 4-7, the heating module may specifically include a first heating module 110, a second heating module 120 and a third heating module 130, where the three heating modules may be simultaneously disposed below the microfluidic chip or simultaneously disposed above the microfluidic chip, as shown in fig. 4, where the three heating modules are disposed below the microfluidic chip, the first heating module 110, the second heating module 120 and the third heating module 130 are mounted one by one below the microfluidic chip from top to bottom, or are separately disposed on both sides of the microfluidic chip. When the three heating modules are located at the same side of the microfluidic chip, the three heating modules have different diameters, that is, the first heating module 110, the second heating module 120, and the third heating module 130 are sleeved with each other and have the same center of circle, so as to ensure that they do not interfere with each other in the movement process. When the three heating modules are positioned on different sides of the microfluidic chip, the calibers of the heating modules positioned on the same side are ensured not to interfere with each other. It should be understood that when the heating body is annular, the heating module can heat all the reaction cavities on the microfluidic chip at one time, so that the method is quick and time-saving. When the heating module is not annular and three heating modules are provided, the heating module may be provided in any shape at this time, and in some embodiments, the heating module is different from the microfluidic chip in shape, but the heating module may sufficiently contact the reaction chamber on the microfluidic chip. In other embodiments, a heating module only corresponds to a partial region on the microfluidic chip, if the microfluidic chip includes N (N may be any positive integer) reaction chambers, that is, corresponds to 16 fluxes, and a heating module corresponds to one or more reaction chambers on the microfluidic chip, where only a staggered arrangement of a plurality of heating modules is required, so that the purpose of the staggered arrangement is to ensure that the three heating modules do not interfere with each other when moving along the height direction of the PCR amplification analyzer. When three heating modules are included, the three heating modules are arranged in a staggered manner. It should be noted that if the heating modules are rotatable, the target heating modules can be rotated to the position without interference without restricting the staggered distribution of the three heating modules, and then the target heating modules move along the height direction of the PCR amplification analyzer, and at this time, the heating modules only heat part of the reaction chambers at one time, but the heating modules can heat all the reaction chambers on the microfluidic chip along with the relative movement of the microfluidic chip and the heating modules.
In some embodiments, as shown in fig. 7, the first heating module 110 includes a first heating body 115, where the first heating body 115 is a temperature-controlled heat conducting plate, and the temperature-controlled heat conducting plate extends along its outer contour toward a direction close to its center to form a plurality of first extension columns 111, the first extension columns 111 are uniformly or unevenly distributed along the temperature-controlled heat conducting plate, and a first heating column 112 is further disposed on the first extension columns 111 and is located on a side of the first extension columns 111 adjacent to the microfluidic chip, and the first heating column 112 is denoted as a heating zone on the first heating module 110, that is, the first heating column 112 on the first heating module 110 is controlled to move to a reaction chamber on the microfluidic chip so as to provide a suitable reaction temperature. The first heating body 115 and the first temperature control heating plate 113 are fixed by a first heat insulation fixing block 114.
In some embodiments, as shown in fig. 6, the second heating module 120 includes a second heating body 124, where the second heating body 124 is a temperature-controlled heat conducting plate, a plurality of heat conducting columns 121 are disposed on a side of the second heating body 124 near the microfluidic chip, the heat conducting columns 121 are detachably connected or non-detachably connected to the second heating body 124, and the second heating body 124 and the second temperature-controlled heating plate 122 are fixed by heat insulation fixing columns 123.
In some embodiments, as shown in fig. 5, the third heating module 130 includes a third heating body 135, where the third heating body 135 is a temperature-controlled heat conducting plate, the third heating body 135 extends along its peripheral outline to a position far away from the central axis of the third heating body 135 to form a plurality of second extending columns 131, the second extending columns 131 are uniformly or unevenly distributed along the third heating body 135, and a second heating column 132 is further disposed on the second extending columns 131 and located on the side of the second extending columns 131, where the second heating column 132 is denoted as a heating area on the third heating module 130, that is, the second heating column 132 on the third heating module 130 is controlled to move to a reaction cavity on the microfluidic chip so as to provide a suitable reaction temperature. The third heating body 135 and the third temperature control heating plate 133 are fixed by a third heat insulation fixing block 134.
It should be understood that the first, second, third do not refer to the importance of an object, but merely serve to distinguish between different objects.
When two or more heating modules are included, the heating modules may be provided as one or more of the first heating module 110, the second heating module 120, and the third heating module 130.
When a plurality of extending columns are arranged on one heating module, gaps exist between two adjacent extending columns for accommodating a heating zone on other heating modules. Specifically, when the heating module is the first heating module 110, the temperature-control heat-conducting plate extends along the outer contour thereof to a direction close to the central axis thereof to form a plurality of first extending columns 111, and a gap is formed between two adjacent first extending columns 111 for accommodating the heat-conducting columns 121 on the second heating module 120 and the second heating columns 132 on the third heating module 130, and a variable temperature area is formed between two adjacent first extending columns 111, wherein the area includes the first heating column 112 corresponding to the first temperature, the heat-conducting column 121 corresponding to the second temperature and the second heating column 132 corresponding to the third temperature. Further, gaps exist between heating areas on different heating modules, and the gaps are 1cm-5cm, so that the temperature between the heating modules is not affected. Specifically, in each variable temperature region, a gap of 1cm to 5cm exists between the first heating column 112 and the heat conduction column 121, a gap of 1cm to 5cm exists between the heat conduction column 121 and the second heating column 132, and a gap of 1cm to 5cm exists between the second heating column 132 and the next first heating column 112.
Example two
A PCR amplification detector 100 includes a reaction module, a switchable incubation module as in embodiment one, and a fluorescence detection module arranged in sequence from top to bottom.
The reaction module comprises a microfluidic chip, and a plurality of reaction cavities can be arranged on the microfluidic chip for simultaneously carrying out a plurality of reactions. In some embodiments, there are 16 PCR reaction chambers on the microfluidic chip, corresponding to 16 fluxes.
As shown in fig. 8, the rotation driving structure 103 in the incubation module comprises a turntable 1035, and the turntable 1035 further comprises at least one accommodating area, and the heating column passes through the accommodating area to contact the reaction cavity on the microfluidic chip. In addition, the rotary driving structure 103 further includes a servomotor 1031, a servomotor riser 1032, a servomotor fixing plate 1033, and a chip fixing bracket 1034. The servo motor 1031 is fixed to the temperature control module fixing plate 1025 through a servo motor fixing plate 1033 and a servo motor vertical plate 1032, and a chip fixing bracket 1034 is fixed to the servo motor 1031 for controlling the turntable 1035 to rotate.
The fluorescence detection module 104, as shown in fig. 9, is composed of a light splitting sheet 1041, a light emitter 1042, and an imaging detector 1043. When 16 PCR reaction chambers are arranged on the microfluidic chip, the first heating module 110, the second heating module 120 and the third heating module 130 are alternately pushed to contact with the PCR reaction chambers on the microfluidic chip under the action of the micro push rod 1013, and finally, the temperature alternation control of the PCR reaction chambers is realized. After the PCR reaction is finished, the light emitter 1042 is started, the light reaches the PCR reaction cavity through the light splitting sheet 1041, and then is reflected to the light splitting sheet 1041 and refracted to finally enter the imaging detector 1043 to realize fluorescence detection. Specific implementation application 1 Mycoplasma Pneumoniae (MP) nucleic acid detection (PCR fluorescence method)
The throat swab sample (35 years old male sample) was added with 1mL of sterile physiological saline, after shaking thoroughly, the liquid was sucked into a centrifuge tube, and centrifuged at 12,000rpm for 5 minutes, 100 μl of the precipitate and 50 μl of the nucleic acid extract were added to the reservoir of the microfluidic chip by manual operation, and the microfluidic chip was controlled to rotate at 2,000rpm (acceleration of 6,000 rpm/s) for 7 minutes. After nucleic acid extraction was completed, 50 μl of supernatant was transferred to the PCR reaction chamber by manipulating the microfluidic chip at 1,500rpm (acceleration of 3,000 rpm/s) for 10 seconds. The PCR amplification step was performed at 50℃for 2 minutes and 1 cycle, at 95℃for 10 minutes and 1 cycle, and at 55℃for 45 seconds and 45 cycles. Fluorescence was reported as FAM (excitation wavelength 494nm, emission wavelength 522 nm), ct value was measured as 40, and the result showed a positive sample.
Specific implementation application 2. Verifying the temperature accuracy of the PCR temperature control Module
The experimental parameters are that the PCR amplification temperature is controlled to be 50 ℃ (module A), 75 ℃ (module B) and 95 ℃ (module C), and the cycle is 50 ℃ -95 ℃ -75 ℃, and 40 cycles are tested in total. Experimental analysis temperature measurements of the PCR reaction chambers were performed using a multiplex thermometer. The experimental objective is that the temperature of the PCR reaction chamber must be maintained at a set value + -0.5 deg.C. The experimental results show that the average temperature of the PCR reaction cavity can reach 95+/-0.25 ℃ in 92 seconds, the average temperature of the PCR reaction cavity can reach 75+/-0.31 ℃ in 85 seconds, and the average temperature of the PCR reaction cavity can reach 50+/-0.41 ℃ in 105 seconds. The above results show that the present invention can complete the PCR amplification step in a short time.
The fixing area is described in a progressive manner in each embodiment, and the same and similar parts of each embodiment are referred to each other, and each embodiment mainly describes differences from other embodiments. In particular, for system embodiments, since they are substantially similar to method embodiments, the description is relatively simple, as relevant to see a section of the description of method embodiments.
Claims (5)
1. A switchable incubation module comprising an incubation body for warming a microfluidic chip, the incubation body comprising:
the device comprises a microfluidic chip, a plurality of heating modules, a plurality of control modules and a plurality of control modules, wherein the plurality of heating modules are used for heating a reaction cavity on the microfluidic chip, and at least two heating modules are different in heating temperature;
the rotary driving structure is used for driving the microfluidic chip and the heating module to rotate relatively;
The heating modules are respectively provided with the same number of heating areas, and the heating areas on the heating modules are concentrically arranged with the microfluidic chip; the heating areas on the same heating module are equal to the distance between the central axes of the heating modules, the distances between the heating areas on different heating modules and the central axes of the heating modules are different, when the temperature is started, under the driving of a rotary driving structure, the microfluidic chip sequentially rotates in the heating areas of the heating modules with different heating temperatures so as to switch the heating temperature of the reaction cavity of the microfluidic chip during the temperature, the rotary driving structure comprises a turntable, at least one accommodating area is further arranged on the turntable, a heating column passes through the accommodating area and contacts the reaction cavity on the microfluidic chip, the rotary driving structure further comprises a servo motor, a servo motor vertical plate, a servo motor fixing plate and a chip fixing bracket, the servo motor is fixed on the temperature control module fixing plate through the servo motor fixing plate, and the chip fixing bracket is fixed on the servo motor and is used for controlling the turntable to rotate;
The micro-push rod pushes the fixed plate to move so as to drive the heating module to move towards the reaction cavity on the micro-fluidic chip, and a plurality of heating modules are respectively fixed on the fixed plates through the guide rods and realize movement control in the height direction through the micro-push rods which independently operate;
The motion moving mechanism further comprises a support, a round shaft and vertical plates, wherein the two vertical plates are oppositely arranged, the two supports are fixed on two sides of the two vertical plates to form a supporting structure, the round shaft is fixed between the two oppositely arranged supports, one ends of the miniature push rods are fixed on the round shaft, and the other ends of the miniature push rods are fixed on the fixed plates to independently realize motion control in different directions.
2. The switchable incubation module of claim 1 further comprising an insulating layer to maintain a temperature within the incubation body.
3. The switchable incubation module of claim 1 wherein gaps exist between adjacent ones of the heating modules, wherein a heating zone on one heating module passes through the gaps between heating zones of another heating module to contact a reaction chamber on a microfluidic chip.
4. The switchable incubation module of claim 1 or 3, wherein at least one of the heating modules comprises a first heating body, a first temperature-controlled heating plate and a first heat-insulating fixing block, wherein the first heating body and the first temperature-controlled heating plate are fixed through the first heat-insulating fixing block, a plurality of first extending columns extend along the outer contour of the first heating body towards the direction close to the central axis of the first heating body, and a first heating column is further arranged on the first extending column and is close to the side of the microfluidic chip, and the first heating column is controlled to move to a reaction cavity on the microfluidic chip so as to provide a proper reaction temperature.
5. A PCR amplification detector comprising a reaction module, a switchable incubation module according to any one of claims 1 to 4, and a fluorescence detection module arranged in sequence from top to bottom.
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| KR20130024223A (en) * | 2011-08-31 | 2013-03-08 | 한국과학기술원 | Rotary real-time pcr capable of sensing fluorescent material using light emitter with multiple wave-length and sensor |
| KR101513273B1 (en) * | 2012-08-30 | 2015-04-22 | 주식회사 메디센서 | A rotary type PCR machine and a PCR chip |
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| JPWO2018143469A1 (en) * | 2017-02-06 | 2019-12-12 | 国立大学法人大阪大学 | Gene amplification system, flow channel chip, rotation drive mechanism, and gene amplification method |
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