Micro-fluidic chip of PCR (Polymerase Chain Reaction) and PCR method

Abstract

The invention relates to the technical field of microfluidics, in particular to a PCR (polymerase chain reaction) micro-fluidic chip and a PCR method, and the PCR micro-fluidic chip comprises a renaturation extension region, a denaturation region and a low-temperature overshoot region, reaction liquid drops reach the renaturation extension region from the denaturation region through the low-temperature overshoot region; the working temperature of the low-temperature overshoot area is lower than that of the renaturation extension area. By using the micro-fluidic chip provided by the invention, in the PCR reaction process, liquid drops can be subjected to overshoot type rapid temperature change in a manner of firstly entering the low-temperature overshoot area, so that the temperature stabilization time after the liquid drops reach the renaturation extension area is reduced or eliminated, the overall time of the PCR reaction is shortened, and the PCR efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics, and in particular to a PCR microfluidics chip and a PCR method. Background Art

[0002] PCR (polymerase chain reaction) technology is based on multi-cycle temperature rise and fall control, which can achieve millions of times amplification of DNA target fragments and is the basic methodology for nucleic acid detection. When PCR is performed based on dielectric wetting technology, the reaction droplets can be driven to move back and forth quickly between several constant temperature zones, and the PCR reaction can be completed without changing the temperature of the heating block. However, in the actual reaction, when the droplets move to the specified temperature zone, there is a certain difference between the actual temperature of the droplets and the temperature of the specified temperature zone, and a longer stabilization time is required to reach the set temperature, resulting in the actual required reaction time being longer than the theoretical time, which ultimately leads to an increase in the time consumption of the PCR process.

[0003] Therefore, how to shorten the reaction time in the PCR process and improve the PCR efficiency is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] The purpose of the present invention is to provide a PCR microfluidic chip and a PCR method to solve the problems of long reaction time and low PCR efficiency in the prior art.

[0005] In order to solve the above technical problems, the present invention provides a PCR microfluidic chip, comprising a renaturation extension zone, a denaturation zone and a low-temperature overshoot zone;

[0006] The reaction droplets pass through the low-temperature overshoot zone from the denaturation zone to the annealing extension zone;

[0007] The operating temperature of the low-temperature overshoot zone is lower than the operating temperature of the annealing extension zone.

[0008] Optionally, the microfluidic chip further includes a high-temperature overshoot zone;

[0009] The reaction droplets pass through the high temperature overshoot zone to reach the denaturation zone;

[0010] The operating temperature of the high-temperature overshoot zone is higher than the operating temperature of the denaturation zone.

[0011] Optionally, in the microfluidic chip, the microfluidic chip comprises a bidirectional driving channel;

[0012] The bidirectional driving channel is used to drive the reaction droplets to reciprocate in the bidirectional driving channel;

[0013] The bidirectional driving channel connects the annealing extension zone, the denaturation zone and the low-temperature overshoot zone in series;

[0014] The annealing extension zone is arranged between the low-temperature overshoot zone and the denaturation zone.

[0015] Optionally, in the microfluidic chip, the low-temperature overshoot zone, the annealing extension zone, the denaturation zone and the high-temperature overshoot zone of the microfluidic chip are sequentially arranged along the bidirectional driving channel.

[0016] Optionally, in the microfluidic chip, the microfluidic chip includes a unidirectional temperature increase driving channel and a unidirectional temperature decrease driving channel;

[0017] The unidirectional temperature-increasing driving channel is used to drive the reaction droplets to move from the renaturation extension zone to the denaturation zone;

[0018] The one-way temperature-lowering driving channel is used to drive the reaction droplets to move from the denaturation zone to the renaturation extension zone;

[0019] The low-temperature overshoot zone is arranged on the unidirectional temperature reduction driving channel and is located between the denaturation zone and the renaturation extension zone.

[0020] Optionally, in the microfluidic chip, the high-temperature overshoot zone of the microfluidic chip is arranged on the unidirectional temperature rise driving channel and is located between the renaturation extension zone and the denaturation zone.

[0021] A PCR method, the PCR method is implemented by any of the microfluidic chips described above, comprising:

[0022] transporting the reaction droplets from the denaturation zone to the low-temperature overshoot zone;

[0023] The reaction droplets stay in the low-temperature overshoot zone for a first preset time, and then the reaction droplets are transported from the low-temperature overshoot zone to the annealing and extension zone for annealing and extension;

[0024] The reaction droplets stay in the annealing and extension zone for a second preset time, and then the reaction droplets are transported from the annealing and extension zone to the denaturation zone for denaturation.

[0025] Optionally, in the PCR method, the microfluidic chip includes the bidirectional driving channel;

[0026] The step of transporting the reaction droplets from the denaturation zone to the low-temperature overshoot zone comprises:

[0027] The reaction droplets are transported along the bidirectional driving channel from the denaturation zone through the annealing extension zone to the low-temperature overshoot zone.

[0028] Optionally, in the PCR method, the microfluidic chip includes the bidirectional driving channel and the high-temperature overshoot zone;

[0029] The step of transporting the reaction droplets from the annealing and extension zone to the denaturation zone for denaturation comprises:

[0030] The reaction droplets are transported along the bidirectional driving channel from the renaturation extension zone through the denaturation zone to the high-temperature overshoot zone, and then transported from the high-temperature overshoot zone to the denaturation zone for denaturation.

[0031] Optionally, in the PCR method, the operating temperature of the denaturation zone is higher than the reaction temperature of the denaturation reaction;

[0032] The step of transporting the reaction droplets from the annealing and extension zone to the denaturation zone for denaturation comprises:

[0033] The reaction droplets are transported from the renaturation extension zone to the denaturation zone, and stay there for a fourth preset time for denaturation; wherein the operating temperature of the denaturation zone ranges from 110 set degrees to 120 degrees Celsius, including endpoint values, and the fourth preset time ranges from 0.4 seconds to 1.2 seconds, including endpoint values.

[0034] The microfluidic chip for PCR provided by the present invention includes a renaturation extension zone, a denaturation zone and a low-temperature overshoot zone; the reaction droplets pass from the denaturation zone through the low-temperature overshoot zone to the renaturation extension zone; the working temperature of the low-temperature overshoot zone is lower than the working temperature of the renaturation extension zone. Using the microfluidic chip provided by the present invention, during the PCR reaction, the droplets can be subjected to an overshoot-type rapid temperature change by first entering the low-temperature overshoot zone, so as to reduce or eliminate the temperature stabilization time after the droplets reach the renaturation extension zone, shorten the overall PCR reaction time, and improve the PCR efficiency. The present invention also provides a PCR method having the above-mentioned beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] FIG1 is a top view of the structure of a specific embodiment of a PCR microfluidic chip provided by the present invention;

[0037] FIG2 is a cross-sectional view of a specific embodiment of a PCR microfluidic chip provided by the present invention;

[0038] FIG3 is a top view of the structure of a specific embodiment of a PCR microfluidic chip provided by the present invention;

[0039] FIG4 is a top view of the structure of a specific embodiment of a PCR microfluidic chip provided by the present invention;

[0040] FIG5 is a top view of the structure of a specific embodiment of a PCR microfluidic chip provided by the present invention;

[0041] FIG6 is a top view of the structure of a specific embodiment of a microfluidic chip for PCR provided by the present invention;

[0042] FIG7 is a top view of the structure of a specific embodiment of a PCR microfluidic chip provided by the present invention;

[0043] FIG8 is a schematic flow chart of a specific implementation of the PCR method provided by the present invention.

[0044] In the figure, it includes 01-upper substrate, 02-common electrode, 03-dielectric hydrophobic layer, 04-hydrophobic layer, 05-dielectric layer, 06-driving electrode, 07-lower substrate, 10-renaturation extension zone, 20-denaturation zone, 30-low temperature overshoot zone, 40-high temperature overshoot zone, 08-bidirectional driving channel, 09A-unidirectional cooling driving channel, 09B-unidirectional heating driving channel. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] The core of the present invention is to provide a microfluidic chip for PCR, a structural schematic diagram of a specific embodiment thereof is shown in FIG1 , which is referred to as specific embodiment 1, and includes a renaturation extension zone 10, a denaturation zone 20 and a low-temperature overshoot zone 30;

[0047] The reaction droplets pass from the denaturation zone 20 through the low-temperature overshoot zone 30 to the annealing extension zone 10;

[0048] The operating temperature of the low-temperature overshoot zone 30 is lower than the operating temperature of the annealing extension zone 10 .

[0049] The microfluidic chip used for rapid PCR usually includes an upper plate and a lower plate arranged relatively to each other, wherein a liquid control channel is formed between the upper plate and the lower plate. As a specific embodiment, the upper plate includes, from top to bottom, an upper substrate 01, a common electrode 02, and a dielectric hydrophobic layer 03 (of course, a separate dielectric layer 05 and a hydrophobic layer 04 can also be selected according to actual conditions, and the present invention is not limited thereto); the lower plate includes, from top to bottom, a hydrophobic layer 04, a dielectric layer 05, a driving electrode 06, and a lower substrate 07. In addition, the microfluidic chip also includes a low-temperature overshoot zone 30 and at least two reaction zones at different temperatures, namely, the denaturation zone 20 and the renaturation extension zone 10. The specific hierarchical structure can be referred to Figure 2.

[0050] Preferably, the reaction droplets are not lower than the working temperature corresponding to the annealing extension zone 10 throughout the whole process, that is, although the temperature of the low-temperature overshoot zone 30 is lower than the working temperature of the annealing extension zone 10, the reaction droplets will not wait until their own temperature is lower than the working temperature of the annealing extension zone 10 before leaving the low-temperature overshoot zone 30.

[0051] As a specific implementation, it also includes a high temperature overshoot region 40;

[0052] The reaction droplets pass through the high temperature overshoot zone 40 to reach the denaturation zone 20;

[0053] The operating temperature of the high-temperature overshoot zone 40 is higher than the operating temperature of the denaturation zone 20 .

[0054] In the preferred embodiment, a high-temperature overshoot zone 40 is further provided for the microfluidic chip. The reaction droplets first quickly pass through the high-temperature overshoot zone 40 having a temperature higher than that of the denaturation zone 20, so that their own temperature rises rapidly, so as to shorten the time they stay in the denaturation zone 20. Of course, it is preferred that the reaction droplets do not exceed the working temperature corresponding to the denaturation zone 20 throughout the entire process, that is, in the process of the reaction droplets passing through the high-temperature overshoot zone 40, the temperature of the droplets themselves will not exceed the working temperature of the denaturation zone 20, so as to avoid unexpected denaturation of the organic macromolecules in the reaction droplets.

[0055] Wherein, the microfluidic chip includes a bidirectional driving channel 08;

[0056] The bidirectional driving channel 08 is used to drive the reaction droplets to reciprocate in the bidirectional driving channel 08;

[0057] The bidirectional driving channel 08 connects the annealing extension zone 10, the denaturation zone 20 and the low temperature overshoot zone 30 in series;

[0058] The annealing extension zone 10 is disposed between the low-temperature overshoot zone 30 and the denaturation zone 20 .

[0059] Please refer to FIG3 , the bidirectional driving channel 08 is a linear driving channel. If there is no low-temperature overshoot zone 30, the reaction droplet should be directly transported from the denaturation zone 20 to the annealing extension zone 10, and then wait in the annealing extension zone 10 for the temperature to be reduced to the working temperature of the annealing extension zone 10. However, after the addition of the low-temperature overshoot zone 30, the reaction droplet needs to pass through the annealing extension zone 10 before reaching the low-temperature overshoot zone 30. After the temperature of the droplet is quickly lowered in the low-temperature overshoot zone 30, it returns to the annealing extension zone 10 for molecular annealing and extension. Such an arrangement can not only simplify and avoid the reaction droplet from being lowered in temperature by the low-temperature overshoot zone 30 when it goes from the annealing extension zone 10 to the denaturation zone 20 (that is, when the reaction droplet needs to be heated up), but also simplify the structure of the driving channel, and reduce the production difficulty and production cost of the microfluidic chip. FIG3 shows a top view of a microfluidic chip including multiple bidirectional driving channels 08, and other top views are similar, which will not be described later.

[0060] In order to more intuitively study the temperature changes of PCR reaction droplets during their movement in the temperature range. 0.1 μg / mL sulforhodamine B solution can be added to the PCR reaction solution for temperature monitoring. Sulforhodamine B is a thermosensitive fluorescent dye. The higher the temperature, the lower the corresponding fluorescence intensity. By detecting its fluorescence intensity, the temperature change of the droplet can be characterized. Figure 4 compares the temperature change of the droplet after reaching the renaturation extension zone 10 from the denaturation zone 20 to the chip without the low-temperature overshoot zone 30 by monitoring the change of the fluorescence signal at the position of the renaturation extension zone 10. The results show that in the microfluidic chip without the low-temperature overshoot zone 30, after the PCR reaction liquid completes the reaction in the denaturation zone 20 and directly reaches the annealing extension zone 10, its fluorescence value gradually increases and takes about 8 seconds to stabilize (the line of the triangle node), indicating that the PCR reaction liquid of the chip needs 8 seconds to reach the specified temperature after moving from the denaturation zone 20 to the annealing extension zone 10; while in the microfluidic chip with the low-temperature overshoot zone 30 shown in FIG3 (the line of the circular node), after the PCR reaction liquid completes the reaction in the denaturation zone 20, it first passes through the annealing extension zone 10, then moves to the low-temperature overshoot zone 30, and finally returns to the annealing extension zone 10, the fluorescence value directly stabilizes. The total movement time of the PCR reaction droplet from the first arrival at the annealing extension zone 10 to the second arrival at the annealing extension zone 10 is only 2 seconds. It can be seen that the microfluidic chip with the low-temperature overshoot zone 30 can significantly shorten the droplet temperature stabilization time, thereby shortening the overall PCR reaction time. It should be noted that the moment when the fluorescence intensity is 500 in the figure indicates that no luminescent droplets are detected at the corresponding position of the renaturation extension zone 10 at this time, and the fluorescence intensity of 500 is the background light intensity (the time between the reaction droplets reaching the renaturation extension zone 10 twice is indicated by a dotted line).

[0061] Of course, other arrangements of the annealing extension zone 10, the denaturation zone 20 and the low-temperature overshoot zone 30 may also be adopted, such as setting the low-temperature overshoot zone 30 between the denaturation zone 20 and the annealing extension zone 10 (as shown in FIG5 ). Compared with the arrangement in the previous text, this arrangement will not lengthen the movement distance of the droplets, and will not increase the area of ​​the microfluidic chip. Of course, after such an arrangement, the reaction droplets will also pass through the low-temperature overshoot zone 30 with a lower temperature during the process of moving from the annealing extension zone 10 to the denaturation zone 20, that is, during the process of heating. Therefore, in order to maintain a higher PCR efficiency, the working temperature of the denaturation zone 20 can be appropriately increased to shorten the residence time of the reaction droplets in the denaturation zone 20.

[0062] Furthermore, the microfluidic chip is provided with the low-temperature overshoot zone 30, the annealing extension zone 10, the denaturation zone 20 and the high-temperature overshoot zone 40 of the microfluidic chip in sequence along the bidirectional driving channel 08.

[0063] Still referring to FIG. 3 , in this preferred embodiment, the high temperature overshoot zone 40 is also arranged on the bidirectional driving channel 08. Of course, in order to prevent the reaction droplets from being affected by the high temperature overshoot zone 40 during the cooling process, the high temperature overshoot zone 40 is arranged on the side of the denaturation zone 20 away from the annealing extension zone 10. This arrangement can also reduce the difficulty of chip design and production while ensuring a high PCR efficiency. Similarly, the high temperature overshoot zone 40 can also be arranged at other positions of the bidirectional driving channel 08 according to actual needs, and the present invention is not limited here.

[0064] In addition, referring to FIG. 6, when three-stage PCR is required, the annealing extension zone 10 can be divided into an independent annealing zone and an extension zone, and the lateral heat conduction of the chip is used to find a suitable position (72°C), and the extension zone is set between the annealing zone and the denaturation zone 20, and the extension zone is used as a temporary reaction zone for extension reaction. The detailed PCR process is as follows: 1. After the droplet is denatured in the denaturation zone 20, the droplet is driven to pass through the annealing zone to reach the low-temperature overshoot zone 30 and then return to the annealing zone for annealing; 2. After annealing, the droplet is driven to reach the extension zone for extension, or the droplet is driven to reach the annealing zone and then move to the denaturation zone 20 and then return to the annealing zone to quickly reach the specified temperature for extension; 3. After extension, the droplet is driven to reach the denaturation zone 20 for denaturation. Repeat steps 1, 2, and 3 for 30-50 cycles to complete PCR amplification.

[0065] The microfluidic chip for PCR provided by the present invention comprises a annealing extension zone 10, a denaturation zone 20 and a low-temperature overshoot zone 30; the reaction droplets pass through the low-temperature overshoot zone 30 from the denaturation zone 20 to reach the annealing extension zone 10; the working temperature of the low-temperature overshoot zone 30 is lower than the working temperature of the annealing extension zone 10. Using the microfluidic chip provided by the present invention, during the PCR reaction process, the droplets can be subjected to overshoot-type rapid temperature change by first entering the low-temperature overshoot zone 30, so as to reduce or eliminate the temperature stabilization time after the droplets reach the annealing extension zone 10, shorten the overall PCR reaction time, and improve the PCR efficiency.

[0066] On the basis of the first embodiment, another microfluidic chip with a driving channel layout is further provided, and its corresponding structural schematic diagram is shown in FIG7 , which is called the second embodiment, comprising a renaturation extension zone 10, a denaturation zone 20 and a low-temperature overshoot zone 30;

[0067] The reaction droplets pass from the denaturation zone 20 through the low-temperature overshoot zone 30 to the annealing extension zone 10;

[0068] The operating temperature of the low temperature overshoot zone 30 is lower than the operating temperature of the annealing extension zone 10;

[0069] The microfluidic chip includes a unidirectional temperature increase driving channel 09B and a unidirectional temperature decrease driving channel 09A;

[0070] The one-way temperature rise driving channel 09B is used to drive the reaction droplets to move from the renaturation extension zone 10 to the denaturation zone 20;

[0071] The one-way cooling driving channel 09A is used to drive the reaction droplets to move from the denaturation zone 20 to the renaturation extension zone 10;

[0072] The low-temperature overshoot zone 30 is disposed on the unidirectional temperature-lowering driving channel 09A and is located between the denaturation zone 20 and the annealing extension zone 10 .

[0073] The difference between this specific embodiment and the above specific embodiment is that this specific embodiment provides another connection method of driving channels between different regions, and the regional structure is the same as that of the above specific embodiment, which will not be elaborated here.

[0074] Furthermore, the high-temperature overshoot zone 40 of the microfluidic chip is disposed on the unidirectional temperature rise driving channel 09B and is located between the renaturation extension zone 10 and the denaturation zone 20 .

[0075] In this specific embodiment, the heating process and the cooling process of the reaction droplets pass through different driving channels respectively. Referring to Figure 7, the low-temperature overshoot zone 30 and the high-temperature overshoot zone 40 are respectively set on corresponding different driving channels. While avoiding the high-temperature overshoot zone 40 from affecting the cooling process and avoiding the low-temperature overshoot zone 30 from affecting the heating process, the path that the reaction droplets need to pass through is further simplified, thereby simplifying the production process.

[0076] The present invention further provides a PCR method, a flow chart of a specific implementation method thereof is shown in FIG8 , which is referred to as specific implementation method three. The PCR method is implemented by a microfluidic chip as described in any one of the above, comprising:

[0077] S101 : transporting the reaction droplets from the denaturation zone 20 to the low-temperature overshoot zone 30 .

[0078] S102: The reaction droplets stay in the low-temperature overshoot zone 30 for a first preset time, and then the reaction droplets are transported from the low-temperature overshoot zone 30 to the annealing and extension zone 10 for annealing and extension.

[0079] S103: The reaction droplets stay in the annealing and extension zone 10 for a second preset time, and then the reaction droplets are transported from the annealing and extension zone 10 to the denaturation zone 20 for denaturation.

[0080] As a specific implementation, the microfluidic chip includes the bidirectional driving channel 08;

[0081] The step of transporting the reaction droplets from the denaturation zone 20 to the low-temperature overshoot zone 30 includes:

[0082] The reaction droplets are transported along the bidirectional driving channel 08 from the denaturation zone 20 through the annealing extension zone 10 to the low-temperature overshoot zone 30 .

[0083] This specific implementation method can refer to the above text. In order to avoid the reaction droplet being disturbed by the low-temperature overshoot zone 30 during the process of changing from the low-temperature state (the corresponding position is the renaturation extension zone 10) to the high-temperature state (corresponding to the denaturation zone 20), the low-temperature overshoot zone 30 cannot be directly set between the renaturation extension zone 10 and the denaturation zone 20. In order to simplify the chip structure and improve the production speed of the microfluidic chip, the driving channel of the reaction droplet is preferably linear. Therefore, the low-temperature overshoot zone 30 should be set at the end of the renaturation extension zone 10 away from the denaturation zone 20. Accordingly, when cooling is required, the reaction droplet first passes through the renaturation extension zone 10 to reach the low-temperature overshoot zone 30, and then returns to the renaturation extension zone 10 after rapid cooling.

[0084] Accordingly, the microfluidic chip includes the bidirectional driving channel 08 and the high-temperature overshoot region 40;

[0085] The step of transporting the reaction droplets from the annealing and extension zone 10 to the denaturation zone 20 for denaturation comprises:

[0086] The reaction droplets are transported along the bidirectional driving channel 08 from the renaturation extension zone 10 through the denaturation zone 20 to the high-temperature overshoot zone 40, and then transported from the high-temperature overshoot zone 40 to the denaturation zone 20 for denaturation.

[0087] As mentioned above, the high temperature overshoot zone 40 is arranged on the side of the denaturation zone 20 away from the annealing extension zone 10. This can avoid the interference of the high temperature overshoot zone 40 on the cooling process of the reaction droplets while ensuring the simplicity of the chip structure, thereby improving the PCR efficiency.

[0088] Preferably, the operating temperature of the denaturation zone 20 is higher than the reaction temperature of the denaturation reaction;

[0089] The step of transporting the reaction droplets from the annealing and extension zone 10 to the denaturation zone 20 for denaturation comprises:

[0090] The reaction droplet is transported from the renaturation extension zone 10 to the denaturation zone 20, and stays for a fourth preset time for denaturation; wherein the operating temperature of the denaturation zone 20 ranges from 110 set degrees to 120 degrees Celsius, including endpoint values, and the fourth preset time ranges from 0.4 seconds to 1.2 seconds, including endpoint values.

[0091] In this preferred embodiment, the working temperature of the denaturation zone 20 is further increased so that the working temperature is higher than the reaction temperature of the corresponding denaturation reaction. Within the appropriate temperature range, nucleic acid denaturation can be completed rapidly in a short time without the need for a long reaction time. This allows the reaction droplets to stay for a very short time after reaching the denaturation zone 20, without having to wait until the droplet temperature reaches the reaction temperature of the denaturation zone 20. The reaction temperature can reach the temperature required for the denaturation reaction, and then directly return from the denaturation zone 20 to the renaturation extension zone 10, further reducing the time consumption of PCR and improving efficiency.

[0092] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0093] It should be noted that, in this specification, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprises a ..." does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0094] The microfluidic chip and PCR method of the PCR provided by the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A PCR microfluidic chip, characterized in that: It includes renaturation extension zone, denaturation zone and low temperature overshoot zone; The reaction droplets pass from the denaturation zone through the low-temperature overshoot zone to the annealing extension zone; The operating temperature of the low-temperature overshoot zone is lower than the operating temperature of the annealing extension zone.

2. The microfluidic chip according to claim 1, characterized in that: It also includes high temperature overshoot areas; The reaction droplets pass through the high temperature overshoot zone to reach the denaturation zone; The operating temperature of the high-temperature overshoot zone is higher than the operating temperature of the denaturation zone.

3. The microfluidic chip according to claim 1, characterized in that: The microfluidic chip includes a bidirectional driving channel; The bidirectional driving channel is used to drive the reaction droplets to reciprocate in the bidirectional driving channel; The bidirectional driving channel connects the annealing extension zone, the denaturation zone and the low-temperature overshoot zone in series; The annealing extension zone is arranged between the low-temperature overshoot zone and the denaturation zone.

4. The microfluidic chip according to claim 3, characterized in that: The microfluidic chip is sequentially provided with the low-temperature overshoot zone, the annealing extension zone, the denaturation zone and the high-temperature overshoot zone of the microfluidic chip along the bidirectional driving channel.

5. The microfluidic chip according to claim 1, characterized in that: The microfluidic chip includes a unidirectional temperature increase driving channel and a unidirectional temperature decrease driving channel; The unidirectional temperature-increasing driving channel is used to drive the reaction droplets to move from the renaturation extension zone to the denaturation zone; The one-way temperature-lowering driving channel is used to drive the reaction droplets to move from the denaturation zone to the renaturation extension zone; The low-temperature overshoot zone is arranged on the unidirectional temperature reduction driving channel and is located between the denaturation zone and the renaturation extension zone.

6. The microfluidic chip according to claim 5, characterized in that: The high-temperature overshoot zone of the microfluidic chip is arranged on the unidirectional temperature-increasing driving channel and is located between the renaturation extension zone and the denaturation zone.

7. A PCR method, characterized in that: The PCR method is implemented by the microfluidic chip according to any one of claims 1 to 6, comprising: transporting the reaction droplets from the denaturation zone to the low-temperature overshoot zone; The reaction droplets stay in the low-temperature overshoot zone for a first preset time, and then the reaction droplets are transported from the low-temperature overshoot zone to the annealing and extension zone for annealing and extension; The reaction droplets stay in the annealing and extension zone for a second preset time, and then the reaction droplets are transported from the annealing and extension zone to the denaturation zone for denaturation.

8. The PCR method according to claim 7, characterized in that The microfluidic chip includes the bidirectional driving channel; The step of transporting the reaction droplets from the denaturation zone to the low-temperature overshoot zone comprises: The reaction droplets are transported along the bidirectional driving channel from the denaturation zone through the annealing extension zone to the low-temperature overshoot zone.

9. The PCR method according to claim 7, characterized in that The microfluidic chip includes the bidirectional driving channel and the high-temperature overshoot zone; The step of transporting the reaction droplets from the annealing and extension zone to the denaturation zone for denaturation comprises: The reaction droplets are transported along the bidirectional driving channel from the renaturation extension zone through the denaturation zone to the high-temperature overshoot zone, and then transported from the high-temperature overshoot zone to the denaturation zone for denaturation.

10. The PCR method according to claim 7, characterized in that The operating temperature of the denaturation zone is higher than the reaction temperature of the denaturation reaction; The step of transporting the reaction droplets from the annealing and extension zone to the denaturation zone for denaturation comprises: The reaction droplets are transported from the renaturation extension zone to the denaturation zone, and stay there for a fourth preset time for denaturation; wherein the operating temperature of the denaturation zone ranges from 110 set degrees to 120 degrees Celsius, including endpoint values, and the fourth preset time ranges from 0.4 seconds to 1.2 seconds, including endpoint values.

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