Microfluidic chip for PCR and PCR method

Abstract

The present application relates to the technical field of microfluidics, and particularly relates to a microfluidic chip for PCR and a PCR method, comprising a hybridization and extension zone, a denaturation zone and a low-temperature overshoot zone; a reaction droplet passes through the low-temperature overshoot zone from the denaturation zone to the hybridization and extension zone; the working temperature of the low-temperature overshoot zone is lower than that of the hybridization and extension zone. In the PCR reaction process, the droplet can be quickly temperatureed in an overshoot mode by entering the low-temperature overshoot zone first, so as to reduce or eliminate the temperature stabilization time of the droplet after reaching the hybridization and extension zone, shorten the overall time of the PCR reaction, and improve the PCR efficiency.

Description

Technical Field

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

[0002] PCR (polymerase chain reaction) technology, based on multi-cycle temperature ramping, can achieve millions-fold amplification of target DNA fragments and is a fundamental methodology for nucleic acid testing. When using dielectric wetting technology for PCR, the reaction can be completed by driving the reaction droplets rapidly back and forth between several constant temperature zones, without the need for a heating block to change temperature. However, in actual reactions, once the droplets reach the designated temperature zone, there is a certain difference between the actual temperature of the droplets and the temperature in the designated zone, requiring a longer stabilization time to reach the set temperature. This results in the actual reaction time being longer than the theoretical time, ultimately increasing the time required for the PCR process.

[0003] Therefore, how to shorten the reaction time during PCR and improve 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 from the denaturation zone through the low-temperature overshoot 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 region;

[0009] The reaction droplets pass through the high-temperature overshoot zone and 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 includes 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-increasing driving channel and a unidirectional temperature-lowering driving channel;

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

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

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

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

[0021] A PCR method, implemented by using any of the above-described microfluidic chips, 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 annealing 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 droplet is transported from the annealing extension zone to the denaturation zone and stays for a fourth preset time for denaturation; wherein the operating temperature of the denaturation zone ranges from 110 set degrees to 120 degrees Celsius, inclusive, and the fourth preset time ranges from 0.4 seconds to 1.2 seconds, inclusive.

[0034] The PCR microfluidic chip provided by the present invention includes a annealing extension zone, a denaturation zone, and a low-temperature overshoot zone; 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. Using the microfluidic chip provided by the present invention, during the PCR reaction process, the droplets can undergo an overshoot-type rapid temperature change by first entering the low-temperature overshoot zone, thereby reducing or eliminating the temperature stabilization time after the droplets reach the annealing extension zone, shortening the overall PCR reaction time and improving 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0036] Figure 1 A top view of the structure of a specific embodiment of the PCR microfluidic chip provided by the present invention;

[0037] Figure 2 A cross-sectional view of a specific embodiment of the PCR microfluidic chip provided by the present invention;

[0038] Figure 3 A structure top view of one embodiment of the microfluidic chip for PCR provided by the present application;

[0039] Figure 4 A structure top view of one embodiment of the microfluidic chip for PCR provided by the present application;

[0040] Figure 5 A structure top view of one embodiment of the microfluidic chip for PCR provided by the present application;

[0041] Figure 6 A structure top view of one embodiment of the microfluidic chip for PCR provided by the present application;

[0042] Figure 7 A structure top view of one embodiment of the microfluidic chip for PCR provided by the present application;

[0043] Figure 8 A flow chart of one embodiment of the PCR method provided by the present application.

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

[0045] In order to make the personnel in the technical field better understand the present application scheme, the present application is further described in detail below in combination with the drawings and specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without making creative efforts belong to the scope of protection of the present application.

[0046] The core of the present application is to provide a microfluidic chip for PCR, a structure schematic diagram of one embodiment of which is shown in Figure 1 which is called embodiment one, including an annealing and extension zone 10, a denaturation zone 20, and a low-temperature overshoot zone 30.

[0047] The reaction droplet passes through the low-temperature overshoot zone 30 from the denaturation zone 20 to the annealing and extension zone 10.

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

[0049] The microfluidic chip used for rapid PCR usually includes an upper plate and a lower plate arranged relative to each other, and a liquid control flow 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 annealing 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 entire 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 embodiment, it also includes a high temperature overshoot region 40;

[0052] The reaction droplets pass through the high-temperature overshoot zone 40 and 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 this 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 corresponding working temperature of 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 Figure 3 The bidirectional drive channel 08 is a linear drive channel. If the low-temperature overshoot zone 30 is not present, the reaction droplets should be transported directly from the denaturation zone 20 to the annealing and extension zone 10, and then wait in the annealing and extension zone 10 for the temperature to drop to the operating temperature of the annealing and extension zone 10. However, after the low-temperature overshoot zone 30 is added, the reaction droplets need to pass through the annealing and extension zone 10 before reaching the low-temperature overshoot zone 30. After the low-temperature overshoot zone 30 quickly lowers the temperature of the droplets, they return to the annealing and extension zone 10 for molecular annealing and extension. This arrangement can simplify and avoid the reaction droplets being lowered in temperature by the low-temperature overshoot zone 30 when traveling from the annealing and extension zone 10 to the denaturation zone 20 (i.e., when the reaction droplets need to be heated), and can also simplify the structure of the drive channel, reducing the difficulty and cost of producing microfluidic chips. Figure 3 A top view of a microfluidic chip including a plurality of bidirectional driving channels 08 is given in FIG. , and other top views are similar and will not be described later.

[0060] To more intuitively study the temperature changes of PCR reaction droplets as they move through temperature ranges, a 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 whose fluorescence intensity decreases with increasing temperature. By measuring its fluorescence intensity, the temperature changes of the droplets can be characterized. Figure 4 By monitoring the fluorescence signal changes at the annealing and extension zone 10, the temperature changes after the droplet reaches the annealing and extension zone 10 from the denaturation zone 20 were compared with those in a chip without the low-temperature overshoot zone 30. The results showed that in the microfluidic chip without the low-temperature overshoot zone 30, after the PCR reaction solution completed the reaction in the denaturation zone 20 and directly reached the annealing and extension zone 10, its fluorescence value gradually increased and took about 8 seconds to stabilize (the lines at the triangular nodes). This indicates that the PCR reaction solution in this chip takes 8 seconds to reach the specified temperature after moving from the denaturation zone 20 to the annealing and extension zone 10; Figure 3In the microfluidic chip shown with a low-temperature overshoot zone 30 (circular node lines), after the PCR reaction liquid completes the reaction in the denaturation zone 20, it first passes through the annealing and extension zone 10, then moves to the low-temperature overshoot zone 30, and finally returns to the annealing and extension zone 10, where the fluorescence value directly stabilizes. The total movement time of the PCR reaction droplet from the first arrival at the annealing and extension zone 10 to the second arrival at the annealing and extension zone 10 is only 2 seconds. This shows that the microfluidic chip with a 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 annealing and extension zone 10 at that time. The fluorescence intensity of 500 is the background light intensity (the time between the reaction droplet's two arrivals at the annealing and extension zone 10 is outlined by the 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 arranging the low-temperature overshoot zone 30 between the denaturation zone 20 and the annealing extension zone 10 (e.g., Figure 5 As shown in the figure), compared with the previous configuration, this configuration does not lengthen the movement distance of the droplets, nor does it increase the area of ​​the microfluidic chip. Of course, after such a configuration, 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 and extension zone 10 to the denaturation zone 20, that is, during the process of heating. Therefore, in order to maintain a high PCR efficiency, the operating 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 sequentially 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 along the bidirectional driving channel 08 .

[0063] Please refer to Figure 3 In this preferred embodiment, the high-temperature overshoot zone 40 is also located on the bidirectional drive channel 08. 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 located on the side of the denaturation zone 20 away from the annealing and extension zone 10. This arrangement also reduces the difficulty of chip design and production while ensuring high PCR efficiency. Similarly, the high-temperature overshoot zone 40 can also be located elsewhere on the bidirectional drive channel 08 as needed, and the present invention is not limited thereto.

[0064] In addition, you can refer to Figure 6When three-stage PCR is required, the annealing and extension zone 10 can be divided into independent annealing and extension zones. Using lateral heat conduction across the chip, the extension zone is positioned appropriately (at 72°C) between the annealing and denaturation zones 20, serving as a temporary reaction zone for the extension reaction. The detailed PCR process is as follows: 1. After denaturation in the denaturation zone 20, the droplets are driven through the annealing zone to the low-temperature overshoot zone 30 and then back to the annealing zone for annealing. 2. After annealing, the droplets are driven to the extension zone for extension, or after reaching the annealing zone, they are driven to the denaturation zone 20 and then back to the annealing zone to quickly reach the desired temperature for extension. 3. After extension, the droplets are driven to the denaturation zone 20 for denaturation. Repeat steps 1, 2, and 3 30-50 times to complete PCR amplification.

[0065] The PCR microfluidic chip provided by the present invention includes a annealing and extension zone 10, a denaturation zone 20, and a low-temperature overshoot zone 30. Reaction droplets pass from the denaturation zone 20 through the low-temperature overshoot zone 30 to the annealing and extension zone 10. The operating temperature of the low-temperature overshoot zone 30 is lower than that of the annealing and extension zone 10. Using the microfluidic chip provided by the present invention, during the PCR reaction, droplets can undergo a rapid overshoot-type temperature change by first entering the low-temperature overshoot zone 30. This reduces or eliminates the temperature stabilization time after the droplets reach the annealing and extension zone 10, shortening the overall PCR reaction time and improving PCR efficiency.

[0066] On the basis of the specific embodiment 1, another microfluidic chip with a driving channel layout is further provided, and its corresponding structural schematic diagram is shown as follows: Figure 7 As shown, it is called the second embodiment, which includes a annealing 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 rising driving channel 09B is used to drive the reaction droplets to move from the annealing 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 annealing 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 the driving channels between different regions. The regional structure is the same as that of the above specific embodiment and will not be elaborated here.

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

[0075] In this embodiment, the temperature rise process and the temperature fall process of the reaction droplet are respectively passed through different driving channels, which can be referred 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 affecting the cooling process and avoiding the low-temperature overshoot zone 30 affecting the heating process, it also further simplifies the path that the reaction droplets need to pass through and simplifies the production process.

[0076] The present invention also provides a PCR method, a schematic diagram of a specific embodiment of the method is shown in FIG. Figure 8 As shown, it is referred to as specific embodiment three, the PCR method is implemented by the microfluidic chip as described in any one of the above, including:

[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 embodiment, 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 embodiment 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 annealing 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 annealing 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 annealing extension zone 10 away from the denaturation zone 20. Accordingly, when cooling is required, the reaction droplet first passes through the annealing extension zone 10 to reach the low-temperature overshoot zone 30, and then returns to the annealing 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 includes:

[0086] The reaction droplets are transported along the bidirectional driving channel 08 from the annealing 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 set on the side of the denaturation zone 20 away from the annealing extension zone 10. This can ensure the simplicity of the chip structure while avoiding the interference of the high-temperature overshoot zone 40 on the cooling process of the reaction droplets, thereby improving 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 includes:

[0090] The reaction droplet is transported from the annealing 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 in the denaturation zone 20 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 then reach the temperature required for the denaturation reaction, and then directly return from the denaturation zone 20 to the annealing extension zone 10, further reducing the time consumption of PCR and improving efficiency.

[0092] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from the other embodiments. Reference can be made to the descriptions of the identical or similar parts between the various embodiments. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the descriptions are relatively simple, and the relevant parts can be referred to the descriptions of the methods.

[0093] It should be noted that, in this specification, relational terms such as first and second, etc. are used only 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 "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising 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 sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device comprising the element.

[0094] The above describes in detail the PCR microfluidic chip and PCR method provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is intended only to facilitate understanding of the method and core concept of the present invention. It should be noted that, for those skilled in the art, various improvements and modifications may be made to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A microfluidic chip for PCR, characterized in that: It includes annealing 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, wherein It also includes high temperature overshoot areas; The reaction droplets pass through the high-temperature overshoot zone and 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, wherein 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, wherein 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, wherein The microfluidic chip includes a unidirectional temperature-increasing driving channel and a unidirectional temperature-lowering driving channel; The unidirectional temperature-increasing driving channel is used to drive the reaction droplets to move from the annealing and extension zone to the denaturation zone; The one-way cooling driving channel is used to drive the reaction droplets to move from the denaturation zone to the annealing extension zone; The low-temperature overshoot zone is arranged on the unidirectional temperature-lowering driving channel and is located between the denaturation zone and the annealing 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 annealing 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, wherein 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, wherein 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 annealing 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, wherein 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 droplet is transported from the annealing extension zone to the denaturation zone and stays for a fourth preset time for denaturation; wherein the operating temperature of the denaturation zone ranges from 110 set degrees to 120 degrees Celsius, inclusive, and the fourth preset time ranges from 0.4 seconds to 1.2 seconds, inclusive.

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