PCR amplification device

Abstract

The application provides a PCR amplification device, comprising a heating module, wherein the heating module comprises a fixed base, a fixed sleeve is arranged above the fixed base, an open cavity is arranged on the fixed sleeve, two oppositely arranged side walls of the open cavity can be attached to the wall of a reaction container of a microdroplet chip accommodated in the open cavity, and two heating pieces are arranged on the outer sides of the two side walls away from the fixed sleeve respectively. In the application, the two oppositely arranged heating pieces can simultaneously heat the two opposite walls of the reaction container of the microdroplet chip in the fixed sleeve. Compared with the bottom heating mode of the reaction container in the prior art, the heating contact area is increased, and the PCR amplification efficiency is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of digital PCR analyzers, and particularly relates to a PCR amplification device. BACKGROUND

[0002] Digital PCR is the latest quantitative technology, and is a method of absolute quantification based on single-molecule PCR method for counting nucleic acid quantification. The method mainly adopts microfluidic or microdroplet method in the current hot research field of analytical chemistry, disperses a large amount of diluted nucleic acid solution into microreactors or microdroplets of a biochip, and the number of nucleic acid templates in each reactor is less than or equal to 1. After a PCR module amplification cycle, a reactor with 1 nucleic acid molecule template gives a fluorescent signal, and a reactor without a template has no fluorescent signal. According to the relative proportion and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.

[0003] The PCR amplification principle is composed of three basic reaction steps of denaturation, annealing (renaturation) and extension: ① denaturation: the template DNA is heated to 90-95 DEG C for a certain time, so that the template DNA double strand or the double-stranded DNA formed by PCR amplification is dissociated to become a single strand, so as to combine with the primer to prepare for the next reaction; ② annealing (renaturation): after the template DNA is heated and denatured into a single strand, the temperature is reduced to 50-60 DEG C, and the primer is combined with the complementary sequence of the template DNA single strand; ③ extension: the DNA template-primer combination is under the action of DNA polymerase at 70-75 DEG C, with dNTP as the reaction raw material, the target sequence as the template, and according to the base pairing and semi-conservative replication principle, a new semi-conservative replication chain complementary to the template DNA chain is synthesized. Repeating the above process can complete the PCR amplification.

[0004] The PCR module is an essential link in the process of nucleic acid quantification detection, and the high efficiency and stability of the PCR module and the amplification time directly affect the nucleic acid detection time. At present, most of the PCR modules on the market heat the sample bottom surface through a heating sheet, and the heating area is relatively small, so the temperature rising and falling speed is difficult to increase, and the amplification time is about 2 hours, which limits the speed of nucleic acid detection. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a PCR amplification device which can heat two side surfaces of the reaction container of the microdroplet chip at the same time and improve the amplification efficiency.

[0006] In order to solve the above problems, the present application provides a PCR amplification device, comprising a heating module, the heating module comprises a fixed base, the upper side of the fixed base is provided with a fixed sleeve, the fixed sleeve is provided with an open cavity, the two opposite side walls of the open cavity can be matched with the wall of the reaction container of the microdroplet chip accommodated in the open cavity, and the two side walls are respectively provided with heating pieces away from the outer side of the fixed sleeve.

[0007] In some embodiments, the heating module further comprises a heat insulation plate, the heat insulation plate is provided with a through hole matched with the opening of the open cavity; and / or, the side of the heating piece away from the fixed sleeve is provided with a heat dissipation fin.

[0008] In some embodiments, the PCR amplification device further comprises a crimping structure for crimping the microdroplet chip on the heat insulation plate and making the reaction container in close contact with the side wall.

[0009] In some embodiments, the crimping structure comprises a first transmission shaft and a second transmission shaft parallel to each other, the first transmission shaft is correspondingly arranged on the first side of the heating module, the second transmission shaft is correspondingly arranged on the second side of the heating module, the first transmission shaft and the second transmission shaft are respectively sleeved with a pressing block, and the rotation of the first transmission shaft and the second transmission shaft in a first rotation direction can drive the top end of the pressing block to rotate towards the microdroplet chip, and the top end of the pressing block finally contacts the upper surface of the microdroplet chip and exerts force towards the depth direction of the open cavity.

[0010] In some embodiments, the PCR amplification device further comprises a water tank assembly, the water tank assembly comprises a tank body and a tank cover covering the tank body, the first transmission shaft and the second transmission shaft are pivotally connected to the tank cover, and the heating module is connected to the tank cover.

[0011] In some embodiments, the rotation of the first transmission shaft and the second transmission shaft in a second rotation direction can drive the bottom end of the pressing block to rotate towards the microdroplet chip, the bottom end of the pressing block finally contacts the lower surface of the heat insulation plate and exerts force towards one side of the microdroplet chip, and the first rotation direction is opposite to the second rotation direction.

[0012] In some embodiments, the crimping structure further comprises a bidirectional rotating motor, the bidirectional rotating motor can drive the first end of the first transmission shaft, and the second end of the first transmission shaft is drivingly connected to the second end of the second transmission shaft through a transmission device.

[0013] In some embodiments, the transmission device includes a first gear connected to a second end of the second transmission shaft, a second gear meshing with the first gear, a first pulley rotating coaxially with the second gear, a second pulley connected to a second end of the first transmission shaft, and a first belt tensioned by the first pulley and the second pulley.

[0014] In some embodiments, the output shaft of the bidirectional rotary motor is fitted with a third pulley, the first end of the first drive shaft is fitted with a fourth pulley, and a second belt is tensioned between the third pulley and the fourth pulley.

[0015] In some embodiments, the heating module has multiple heating modules, and multiple pressure blocks are sleeved on the first drive shaft on the first side of the multiple heating modules. The multiple pressure blocks are arranged one-to-one with the first side of the multiple heating modules. The multiple pressure blocks are sleeved on the second drive shaft on the second side of the multiple heating modules. The multiple pressure blocks are arranged one-to-one with the second side of the multiple heating modules.

[0016] The present invention provides a PCR amplification device in which two oppositely arranged heating plates can simultaneously heat the two opposite walls of the reaction container of the microdroplet chip in the fixing sleeve. Compared with the bottom heating method of the reaction container in the prior art, the heating contact area is increased, thereby improving the PCR amplification efficiency. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the disassembled structure of the heating module in the PCR amplification device according to an embodiment of the present invention (the figure shows the microdroplet chip);

[0018] Figure 2 for Figure 1 The heating module in the middle is shown in the cross-section after assembly;

[0019] Figure 3 A schematic diagram showing the state in which the top of the pressure block applies force to the upper surface of the microdroplet chip to press the microdroplet chip and the heating module together;

[0020] Figure 4 A schematic diagram showing the state in which the bottom end of the pressure block is applied to the lower surface of the heat insulation plate to lift the microdroplet chip and release it from the heating module;

[0021] Figure 5 A three-dimensional structural diagram of the pressure block in an unstressed state;

[0022] Figure 6 This is an axonometric view of the PCR amplification apparatus according to an embodiment of the present invention, viewed from one perspective;

[0023] Figure 7Figure 3 is a side view of the PCR amplification device of the embodiment of the present application, in another perspective;

[0024] Figure 8 Figure 4 is a top view of the PCR amplification device of the embodiment of the present application.

[0025] Reference signs are indicated as:

[0026] 1, heating module; 11, fixed base; 12, fixed sleeve; 121, open cavity; 13, heating sheet; 14, heat insulation plate; 141, through hole; 15, heat dissipation sheet; 31, first transmission shaft; 32, second transmission shaft; 33, pressing block; 34, bidirectional rotating motor; 351, first gear; 352, second gear; 353, first pulley; 354, second pulley; 355, first belt; 356, third pulley; 357, fourth pulley; 358, second belt; 41, box body; 42, box cover; 5, supporting leg; 100, micro-droplet chip. DETAILED DESCRIPTION

[0027] Reference is made in combination with Figures 1 to 8 According to the embodiment of the present application, a PCR amplification device is provided, which comprises a heating module 1, the heating module 1 comprises a fixed base 11, the fixed base 11 is provided with a fixed sleeve 12 above, the fixed sleeve 12 is configured with an open cavity 121, two opposite side walls of the open cavity 121 can be fitted with the wall of the reaction container (also called chip reaction cavity) of the micro-droplet chip 100 accommodated in the open cavity 121, two side walls are respectively provided with a heating sheet 13 away from the outer side of the fixed sleeve 12, the heating sheet 13 is specifically, for example, a semiconductor heating sheet, for example, a commonly used Peltier heating sheet, etc. In this technical solution, two opposite heating sheets 13 can simultaneously heat the two opposite walls of the reaction container of the micro-droplet chip 100 in the fixed sleeve 12. Compared with the bottom heating mode of the reaction container in the prior art, the heating contact area is increased, and the PCR amplification efficiency is improved. It can be understood that the material of the fixed sleeve 12 should be a heat-conducting material, such as aluminum alloy, etc.

[0028] In some embodiments, the heating module 1 further comprises a heat insulation plate 14, wherein a through hole 141 is formed on the heat insulation plate 14, and the opening cavity 121 is adapted to the opening of the through hole 141, and the reaction container can be inserted into the opening cavity 121 through the through hole 141, and the heat insulation plate 14 can ensure the heat utilization of the two heating pieces 13 below the heat insulation plate 14, and the heating heat is more concentrated; in some embodiments, the side of the heating piece 13 away from the fixing sleeve 12 is provided with a heat dissipation fin 15, especially when the heating piece 13 is a semiconductor heating piece, which can efficiently exchange heat of the cold end or the hot end of the heating piece 13, and ensure that the heating piece 13 can be more efficient whether it is a refrigeration or heating cycle.

[0029] The two heat dissipation fins 15, the heating piece 13 and the fixing sleeve 12 clamped between the two heating pieces 13 are bolted through four screws at four corners, and the clamping force is adjusted by adjusting the screwing of the screws, so as to ensure that the opening cavity wall of the fixing sleeve 12 is more closely contacted with the reaction container, and the temperature conduction efficiency is ensured.

[0030] In some embodiments, the PCR amplification device further comprises a crimping structure for crimping the micro-droplet chip 100 on the heat insulation plate 14, and closely contacting the reaction container with the side wall, so that the heat generated by the heating piece 13 can be more efficiently conducted to the sample in the reaction container.

[0031] Specifically refer to Figure 3As shown, the pressing structure comprises first transmission shaft 31 and second transmission shaft 32 which are parallel to each other, the first transmission shaft 31 is arranged on the first side of the heating module 1, the second transmission shaft 32 is arranged on the second side of the heating module 1, the first transmission shaft 31 and the second transmission shaft 32 are respectively sleeved with a pressing block 33, the rotation of the first transmission shaft 31 and the second transmission shaft 32 in the first rotation direction can drive the top end of the pressing block 33 to rotate towards the micro-droplet chip 100, and the top end of the pressing block 33 finally contacts the upper surface of the micro-droplet chip 100 and exerts force towards the depth direction of the opening cavity 121. Further, the rotation of the first transmission shaft 31 and the second transmission shaft 32 in the second rotation direction can drive the bottom end of the pressing block 33 to rotate towards the micro-droplet chip 100, and the bottom end of the pressing block 33 finally contacts the lower surface of the heat insulation plate 14 and exerts force towards one side of the micro-droplet chip 100, the first rotation direction is opposite to the second rotation direction. In this way, the pressing block 33 can not only exert downward pressure on the micro-droplet chip 100 to ensure the close contact of the reaction container and the fixing sleeve 12, thereby ensuring the rapid and efficient heat conduction of the heating sheet 13, but also can exert lifting force on the heat insulation plate 14 through the pressing block 33, so that the micro-droplet chip 100 on the heat insulation plate 14 is displaced upward, thereby reducing the frictional resistance when grabbing the micro-droplet chip 100.

[0032] In some embodiments, the PCR amplification device further comprises a water tank assembly, the water tank assembly comprises a tank body 41 and a tank cover 42 arranged above the tank body 41, the first transmission shaft 31 and the second transmission shaft 32 are pivotally connected to the tank cover 42, the heating module 1 is connected to the tank cover 42, and the tank body 41 has corresponding water inlet pipes and water outlet pipes for connecting with external cooling sources to quickly cool the reaction container when needed.

[0033] In some embodiments, the pressing structure further comprises a bidirectional rotating motor 34, the bidirectional rotating motor 34 is connected to the first end of the first transmission shaft 31, the second end of the first transmission shaft 31 is drivingly connected to the second end of the second transmission shaft 32 through a transmission device, that is, the bidirectional rotating motor 34 is used to generate the first rotation direction and the second rotation direction.

[0034] Specifically, the box cover 42 is provided with two fixed plates spaced apart from each other, and a through hole is formed in the fixed plates, so that the first transmission shaft 31 and the second transmission shaft 32 can be arranged on the fixed plates, and the pivoting of the first transmission shaft 31 and the second transmission shaft 32 can be ensured. In addition, a photoelectric barrier (not shown in the figure) can be arranged near the pressing block 33, so that the rotation angle of the pressing block 33 can be detected, and the pressing (first rotation direction) or lifting (second rotation direction) of the pressing block 33 can be ensured.

[0035] In some embodiments, the transmission device comprises a first gear 351 connected to the second end of the first transmission shaft 31, a second gear 352 meshing with the first gear 351, a first belt pulley 353 coaxially rotating with the second gear 352, a second belt pulley 354 connected to the second end of the second transmission shaft 32, and a first belt 355 tensioned and connected between the first belt pulley 353 and the second belt pulley 354, so that when the first transmission shaft 31 is driven to rotate, the second transmission shaft 32 can be synchronously rotated in the opposite direction, so that the rotation directions of the pressing blocks 33 respectively sleeved on the two transmission shafts are opposite, and the pressing and lifting of the micro-droplet chip 100 are realized.

[0036] In some embodiments, the output shaft of the bidirectional rotating motor 34 is sleeved with a third belt pulley 356, the first end of the first transmission shaft 31 is sleeved with a fourth belt pulley 357, and a second belt 358 is tensioned and connected between the third belt pulley 356 and the fourth belt pulley 357. In this way, the position of the bidirectional rotating motor 34 can be flexibly selected. In a specific embodiment, the bottom of the box body 41 is arranged in the air through four legs 5, and the bottom of the box body 41 is provided with a corresponding mounting plate, and the bidirectional rotating motor 34 is mounted on the mounting plate.

[0037] In some embodiments, the heating module 1 has a plurality of the first transmission shaft 31 on the first side of the plurality of the heating module 1, and a plurality of the pressing blocks 33 are sleeved on the first transmission shaft 31. The plurality of the pressing blocks 33 are arranged one by one corresponding to the first side of the plurality of the heating module 1. A plurality of the second transmission shaft 32 are arranged on the second side of the plurality of the heating module 1, and a plurality of the pressing blocks 33 are sleeved on the second transmission shaft 32. The plurality of the pressing blocks 33 are arranged one by one corresponding to the second side of the plurality of the heating module 1. Through the arrangement of the plurality of the heating module 1, the efficiency of PCR amplification can be further improved.

[0038] The use process of the PCR amplification device of the present application is described below.

[0039] As Figures 1-8When one or more micro-droplet chips 100 are inserted into each heating module 1, the bidirectional rotating motor 34 receives a signal to start rotating, the third belt pulley 356 on the motor shaft drives the second belt 358 to start rotating, the first transmission shaft 31 is driven by the torque transmitted by the fourth belt pulley 357 to start rotating, then drives the other end of the second belt pulley 354 to rotate, the first belt 355 starts to rotate, the second gear 352 rotates, and through the meshing transmission, the first gear 351 on the second transmission shaft 32 starts to rotate in the opposite direction at the same ratio, driving the second transmission shaft 32 to rotate in the opposite direction, realizing the synchronous pressing of the top ends of all the pressing blocks 33 on the first transmission shaft 31 and the second transmission shaft 32 to the micro-droplet chip 100, and the degree of pressing is controlled by adjusting the distance between the photoelectric barrier and the photoelectric switch and the pulse number of the bidirectional rotating motor 34.

[0040] After the micro-droplet chip 100 is pressed, the PCR amplification process is started, and through circuit control, the heating sheet 13 on the side close to the fixed sleeve 12 starts to heat, the temperature of the inside of the heating sheet 13 rises, the fixed sleeve 12 starts to heat by heat conduction, the heated part of the micro-droplet chip (i.e. the two side walls of the reaction container) is rapidly heated, the internal sample liquid is also heated, the temperature rises rapidly, and finally the liquid temperature is kept constant near the set temperature value through the PID algorithm, and after a period of time, the circuit control is adjusted, the heating sheet 13 starts to rapidly reverse heat, the side close to the fixed sleeve 12 starts to cool, and the cooling liquid in the water tank also starts to circulate, accelerating the cooling process, the fixed sleeve 12 starts to cool by heat conduction, and the heated part of the micro-droplet chip also cools, and the sample in the micro-droplet chip 100 starts to rapidly cool, and after reaching the set temperature value, it is kept constant for a period of time, and the above process is repeated for multiple times to complete the PCR amplification. In the present application, the contact area of the heating sheet 13 and the fixed sleeve 12, the fixed sleeve 12 and the heated part of the micro-droplet chip is large and tight, so the heating speed is much faster than the original bottom heating method, greatly reducing the temperature rising and falling time, reducing the overall PCR amplification time, and achieving the goal of rapid PCR.

[0041] After the amplification is completed, because the micro-droplet chip 100 is tightly pressed into the fixed sleeve 12, the frictional resistance on both sides is relatively large, in order to facilitate grabbing, it is necessary to lift the micro-droplet chip 100 to a certain height to reduce the frictional resistance and facilitate grabbing. Therefore, after receiving a signal, the bidirectional rotating motor 34 starts to rotate in the opposite direction, the first transmission shaft 31 and the second transmission shaft 32 also rotate, the pressing blocks 33 start to move in the opposite direction synchronously, gradually lifting from the top end, and the bottom lifting position (i.e. the bottom end of the pressing block 33) starts to press the heat insulation plate 14, the bidirectional rotating motor 34 continues to rotate, the heat insulation plate 14 drives the micro-droplet chip 100 to be slowly lifted, and when the micro-droplet chip 100 is lifted to a certain height, the bidirectional rotating motor 34 stops rotating, and the micro-droplet chip 100 is positioned at a set position to facilitate grabbing.

[0042] Those skilled in the art can easily understand that the above-mentioned advantageous modes can be freely combined and superimposed without conflict.

[0043] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application. The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A PCR amplification device, characterized by, The application relates to a heating module (1) which comprises a fixed base (11), a fixed sleeve (12) arranged above the fixed base (11), an open cavity (121) formed in the fixed sleeve (12), two opposite side walls of the open cavity (121) capable of being attached to the wall of a reaction container of a micro-droplet chip (100) accommodated in the open cavity (121), and a heating sheet (13) arranged on the outer side of each of the two side walls away from the fixed sleeve (12); the heating sheet (13) is a semiconductor heating sheet. The heating module (1) further comprises a heat insulation plate (14) which is provided with a through hole (141) corresponding to the opening of the open cavity (121); and / or the heating sheet (13) is provided with a heat dissipation sheet (15) on the side away from the fixed sleeve (12). The application further relates to a pressing structure which is used for pressing the micro-droplet chip (100) on the heat insulation plate (14) and making the reaction container tightly contact with the side walls. The pressing structure comprises a first transmission shaft (31) and a second transmission shaft (32) which are parallel to each other, the first transmission shaft (31) is arranged on the first side of the heating module (1), the second transmission shaft (32) is arranged on the second side of the heating module (1), a pressing block (33) is sleeved on the first transmission shaft (31) and the second transmission shaft (32), the rotation of the first transmission shaft (31) and the second transmission shaft (32) in a first rotation direction can drive the top end of the pressing block (33) to rotate towards the micro-droplet chip (100), and the top end of the pressing block (33) finally contacts the upper surface of the micro-droplet chip (100) and exerts force on the depth direction of the open cavity (121).

2. The PCR amplification device of claim 1, wherein, The application further relates to a water tank assembly which comprises a tank body (41) and a tank cover (42) arranged above the tank body (41), the first transmission shaft (31) and the second transmission shaft (32) are pivotally connected to the tank cover (42), and the heating module (1) is connected to the tank cover (42).

3. The PCR amplification device of claim 2, wherein, The rotation of the first transmission shaft (31) and the second transmission shaft (32) in a second rotation direction can drive the bottom end of the pressing block (33) to rotate towards the micro-droplet chip (100), the bottom end of the pressing block (33) finally contacts the lower surface of the heat insulation plate (14) and exerts force on the side of the micro-droplet chip (100), and the first rotation direction is opposite to the second rotation direction.

4. The PCR amplification device according to any one of claims 1 to 3, characterized in that, The pressing structure further comprises a bidirectional rotation motor (34) which can drive the first end of the first transmission shaft (31), and the second end of the first transmission shaft (31) is drivingly connected to the second end of the second transmission shaft (32) through a transmission device.

5. The PCR amplification device of claim 4, wherein, The transmission device comprises a first gear (351) connected to the second end of the second transmission shaft (32), a second gear (352) meshingly connected with the first gear (351), a first belt pulley (353) coaxially rotating with the second gear (352), a second belt pulley (354) connected with the second end of the first transmission shaft (31), and a first belt (355) tautly connected by the first belt pulley (353) and the second belt pulley (354).

6. The PCR amplification device of claim 5, wherein, The output shaft of the bidirectional rotating motor (34) is sleeved with a third belt pulley (356), the first end of the first transmission shaft (31) is sleeved with a fourth belt pulley (357), and the third belt pulley (356) and the fourth belt pulley (357) are tautly connected with a second belt (358) therebetween.

7. The PCR amplification device of claim 1, wherein, The heating module (1) has a plurality of, a plurality of the first transmission shaft (31) on the first side of the heating module (1) is sleeved with a plurality of the pressing block (33), a plurality of the pressing block (33) and a plurality of the first side of the heating module (1) are arranged one by one, a plurality of the second transmission shaft (32) on the second side of the heating module (1) is sleeved with a plurality of the pressing block (33), and a plurality of the pressing block (33) and a plurality of the second side of the heating module (1) are arranged one by one.

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