A vertical microfluidic chip for nucleic acid amplification

Abstract

The present invention discloses a vertical microfluidic chip for nucleic acid amplification. The vertical microfluidic chip includes a body with chambers, the chambers including: a first amplification chamber, which is used to perform a first round of amplification on a nucleic acid extract; and a second amplification chamber, which is used to perform a second round of amplification on the product of the first round of amplification; the body is provided with a first liquid outlet channel connected to the first amplification chamber and a liquid flow channel connected to the second amplification chamber; the vertical microfluidic chip also includes: an amplification rotary piston, which can be rotatably arranged in the body around a horizontally extending rotation axis, the first amplification chamber is located above the amplification rotary piston, the second amplification chamber is located below the amplification rotary piston, and the amplification rotary piston is provided with an amplification connecting groove capable of connecting the first liquid outlet channel and the liquid flow channel of the second amplification chamber. The present invention can perform nucleic acid amplification, has a simple structure, and is relatively accurate in reagent quantification.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid detection and relates to a vertical microfluidic chip for nucleic acid amplification. Background Art

[0002] Microfluidics is a technology for precisely controlling and manipulating microscale fluids. Specifically, it integrates the basic operational units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, onto a microfluidic chip measuring a few square centimeters, automating the entire analysis process. Currently, microfluidic chips have been applied to the field of PCR nucleic acid amplification. For example, Chinese patent CN111760601A discloses a microfluidic chip with an integrated fluidic switching valve. The chip comprises a microfluidic chip body, reagent channels, a fluidic switching valve, a waste liquid storage chamber, a sample storage chamber, a first cleaning fluid storage chamber, a second cleaning fluid storage chamber, an amplification fluid storage chamber, and a nucleic acid extraction and amplification detection chamber, forming a fully integrated nucleic acid detection microfluidic chip. The fluidic switching valve switches the connection between different storage chambers and the nucleic acid extraction and amplification detection chamber. During nucleic acid extraction and amplification detection, the fluidic switching valve needs to be switched to different positions, connecting different reagent storage chambers to the nucleic acid extraction and amplification detection chamber. An external power source is connected to the pressure caps of the different storage chambers. Currently, most microfluidic chips used for nucleic acid testing are horizontal chips, which require a large number of components to connect to external power sources. They have complex structures and their detection accuracy needs to be further improved. Summary of the Invention

[0003] The object of the present invention is to provide a vertical microfluidic chip for nucleic acid amplification, which has a simple structure and relatively accurate reagent quantification.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A vertical microfluidic chip for nucleic acid amplification includes a body with a chamber, wherein the chamber includes:

[0006] a first amplification chamber, which is used to perform a first round of amplification on the extracted nucleic acid; and

[0007] a second amplification chamber, which is used to perform a second round of amplification on the product of the first round of amplification;

[0008] The body is provided with a first liquid outlet channel communicating with the first amplification chamber and a liquid flow channel communicating with the second amplification chamber;

[0009] The vertical microfluidic chip also includes:

[0010] An amplification rotary piston is rotatably arranged in the main body around a horizontally extending rotation axis, the first amplification chamber is located above the amplification rotary piston, the second amplification chamber is located below the amplification rotary piston, and the amplification rotary piston is provided with an amplification connecting groove capable of connecting the first liquid outlet channel and the liquid flow channel of the second amplification chamber.

[0011] Preferably, the amplification communicating groove is provided on the outer circumferential surface of the amplification rotary piston.

[0012] More preferably, the amplification connecting groove has a starting end and an ending end, and a central angle between the starting end and the ending end is greater than zero.

[0013] Furthermore, the central angle is 180 degrees.

[0014] More preferably, the amplification rotary piston includes a body and a sealing layer coated on the body, and the amplification communicating groove is opened on the sealing layer or opened on the body and passes through the sealing layer.

[0015] More preferably, there are multiple second amplification chambers, each of which is provided with a liquid flow channel, each of which corresponds to an amplification connecting groove, and multiple amplification connecting grooves are arranged in parallel along the circumferential direction of the amplification rotary piston.

[0016] More preferably, the starting ends of the amplification communicating grooves are spaced apart and arranged at the same circumferential position of the amplification rotary piston.

[0017] Furthermore, the lengths of the amplification connecting grooves are different from each other, and the terminal ends of any two adjacent amplification connections are spaced apart by a distance in the axial direction and the circumferential direction of the amplification rotary piston.

[0018] Preferably, the vertical microfluidic chip also includes an extraction rotary piston arranged above the first amplification chamber, and the extraction rotary piston can be rotatably arranged in the main body around a horizontally extending rotation axis. The main body is provided with a first liquid inlet channel connected to the first amplification chamber, and the extraction rotary piston is provided with a nucleic acid transfer groove that can be connected to the first liquid inlet channel.

[0019] More preferably, the chamber further includes a buffer chamber for receiving the amplification product of the first amplification chamber, the buffer chamber is located between the extraction rotary piston and the amplification rotary piston, and the first liquid outlet channel can be connected to the amplification connecting groove through the buffer chamber.

[0020] More preferably, a second liquid inlet channel and a second liquid outlet channel connected to the buffer chamber are provided on the main body, and the extraction rotary piston is also provided with a pipetting connecting groove capable of connecting the first liquid outlet channel and the second liquid inlet channel, and the amplification connecting groove can connect the second liquid outlet channel and the liquid flow channel of the second amplification chamber.

[0021] Further, when the pipetting connecting groove connects the first liquid outlet channel and the second liquid inlet channel, the nucleic acid transfer groove and the first liquid inlet channel are connected.

[0022] Preferably, the chamber also includes a buffer chamber for receiving the amplification product of the first amplification chamber, the buffer chamber is located above the amplification rotary piston, the first liquid outlet channel can be connected to the amplification connecting groove through the buffer chamber, the buffer chamber is connected to a quantitative chamber opened in the main body through an air flow channel, and the vertical microfluidic chip also includes a quantitative piston slidably arranged in the quantitative chamber.

[0023] Preferably, an amplification reagent is provided in the first amplification chamber.

[0024] The present invention adopts the above solution and has the following advantages compared with the prior art:

[0025] The microfluidic chip for nucleic acid amplification of the present invention can perform PCR nested amplification and adopts a vertical structure to effectively eliminate the influence of bubbles, ensure accurate liquid intake, and achieve precise liquid quantification. The liquid pathway of the microfluidic chip is switched by rotating a piston, and the operation is convenient and controllable, and it is easy to automate. The structure is compact and simple, and it is easy to batch produce; there is no aerosol pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. 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.

[0027] Figure 1 is a three-dimensional schematic diagram of a vertical microfluidic chip according to an embodiment of the present invention;

[0028] Figure 2 A side view of a vertical microfluidic chip according to an embodiment of the present invention;

[0029] Figure 3 is an internal perspective view of a vertical microfluidic chip according to an embodiment of the present invention;

[0030] Figure 4a 、 Figure 4b and Figure 4c They are schematic diagrams of an extraction rotary piston at different viewing angles according to an embodiment of the present invention;

[0031] Figure 5 A cross-sectional view of an extraction rotary piston along its length direction according to an embodiment of the present invention;

[0032] Figure 6a and Figure 6b They are schematic diagrams of an amplified rotary piston at different viewing angles according to an embodiment of the present invention;

[0033] Figure 7 1 is a cross-sectional view of an amplified rotary piston along its length according to an embodiment of the present invention.

[0034] in,

[0035] 1. Main body; 11. Mixing piston; 12. Magnetic body; 13. Interface; 14. Dosing piston; 15. Cover;

[0036] 100, well; 101, sample chamber; 102, reagent chamber; 103, waste liquid chamber; 104, first amplification chamber; 105, buffer chamber; 106, quantitative chamber; 107, second amplification chamber; 108, liquid flow channel; 109, waste liquid channel; 110, first liquid inlet channel; 111, first liquid outlet channel; 112, second liquid inlet channel; 113, second liquid outlet channel;

[0037] 2. Extraction rotary piston; 21. Reagent connecting groove; 22. Waste liquid connecting groove; 23. Nucleic acid transfer groove; 24. Pipetting connecting groove; 200. Curved segment; 201. First body; 202. First sealing layer; 203. Connecting hole;

[0038] 3. Enlarged rotary piston; 31. Enlarged connecting groove; 300. Corner section; 301. Second body; 302. Second sealing layer; 303. Connecting hole. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is intended to help understand the present invention, but does not constitute a limitation of the present invention.

[0040] As used in this specification and claims, the terms "comprises" and "include" merely indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list; a method or apparatus may also include other steps or elements. The term "and / or" as used herein includes any combination of one or more of the associated listed items.

[0041] It should be noted that, unless otherwise specified, when a feature is referred to as being "fixed" or "connected" to another feature, it may be directly fixed or connected to the other feature or indirectly fixed or connected to the other feature. Furthermore, terms such as "upper," "lower," "left," and "right" used in this disclosure are intended solely to describe the relative positions of the components of the disclosure as shown in the accompanying drawings.

[0042] It should be further understood that in this disclosure, "plurality" refers to two or more, and other quantifiers are similar. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, and B exists alone.

[0043] It will be further understood that the terms "first," "second," and the like are used to describe various types of information, but such information should not be limited to these terms. These terms are used solely to distinguish information of the same type from one another and do not indicate a particular order or level of importance. In fact, the terms "first," "second," and the like are fully interchangeable. For example, first information could be referred to as second information, and similarly, second information could be referred to as first information without departing from the scope of this disclosure.

[0044] Reference Figures 1 to 7 As shown, according to one embodiment of the present invention, a vertical microfluidic chip for nucleic acid amplification includes a main body 1 with a chamber, and the main body 1 is in the shape of a vertically arranged plate. The vertical microfluidic chip also includes an extraction rotary piston 2 and an amplification rotary piston 3, which are cylindrical in shape. The extraction rotary piston 2 is rotatably inserted into the main body 1, and the amplification rotary piston 3 is located directly below the extraction rotary piston 2 and is rotatably inserted into the main body 1. The extraction rotary piston 2 and the amplification rotary piston 3 can rotate around a horizontally extending rotation axis respectively, and the rotation axes of the two are parallel to each other and are located in the same vertical plane, that is, the rotation axes of the two are respectively along Figure 3 The vertical microfluidic chip is divided into three functional areas by the extraction rotary piston 2 and the amplification rotary piston 3. The extraction rotary piston 2 and the part above it are the nucleic acid extraction and purification area, where the nucleic acid sample is extracted and purified; the part between the extraction rotary piston 2 and the amplification rotary piston 3 is the first amplification area, where the piston undergoes the first round of PCR amplification after extraction and purification; the part below the amplification rotary piston 3 is the second amplification area, where the amplified product after the first round of PCR amplification undergoes the second round of PCR amplification, and its fluorescence signal is measured by the fluorescence detection device of the PCR instrument in this area.

[0045] like Figure 3As shown, the chamber of the nucleic acid extraction and purification zone (i.e., the chamber located above the extraction rotary piston 2) includes a sample chamber 101, a reagent chamber 102, and a waste liquid chamber 103, which are arranged in parallel at the upper portion of the body 1. The reagent chambers 102 are multiple and are arranged in parallel between the sample chamber 101 on the left and the waste liquid chamber 103 on the right. That is, the sample chamber 101, the multiple reagent chambers 102, and the waste liquid chamber 103 are arranged in an axially spaced arrangement along the extraction rotary piston 2. The sample chamber 101 and the reagent chamber 102 each have a hole 100 located at the upper end portion of the body 1 (preferably, the upper end surface of the body 1). These holes 100 allow sample addition or reagent filling into these chambers. These holes 100 also allow communication with the outside atmosphere to facilitate pipetting. When the vertical microfluidic chip is not in use, the holes 100 at the upper end of the body 1 are sealed by a sealing film; when in use, the sealing film is removed; after the nucleic acid sample is added to the sample chamber 101, the holes 100 on the sample chamber 101 are sealed by the removable cover 15. The sample chamber 101 is used to hold the nucleic acid sample; the reagent chamber 102 is used to store reagents required for nucleic acid extraction, such as lysate, rinse solution, eluent, etc.; the waste liquid chamber 103 is used to store waste liquid generated during the nucleic acid extraction and purification process. Specifically in this embodiment, the number of reagent chambers 102 is five, and from left to right, lysate, protease, rinse solution, rinse solution and eluent are stored in sequence. Correspondingly, the multiple reagent chambers 102 include a first reagent chamber for storing lysate, a second reagent chamber for storing protease, a third reagent chamber and a fourth reagent chamber for storing rinse solution, and a fifth reagent chamber for storing eluent.

[0046] The above chambers are connected or disconnected through the microchannels provided on the extraction rotary piston 2. Figure 3 As shown, the body 1 further defines a liquid flow channel 108, which communicates with the sample chamber 101 and each reagent chamber 102. The body 1 also defines a waste liquid channel 109, which communicates with the waste liquid chamber 103. The extraction rotary piston 2 selectively connects two of the liquid flow channels 108 and waste liquid channels 109, while disconnecting the others. As the extraction rotary piston 2 rotates a certain angle, the extraction rotary piston 2 connects the other two of the liquid flow channels 108 and waste liquid channels 109, while disconnecting the others. Specifically, two of the chambers are connected via a connecting groove provided on the outer surface of the extraction rotary piston 2.

[0047] For the layout of the connecting groove on the rotary piston 2, refer to Figures 4a to 4c As shown. Figures 4a to 4cAs shown, the extraction rotary piston 2 is provided with a reagent communication groove 21 that can connect the liquid flow channel 108 of the sample chamber 101 with the liquid flow channel 108 of the reagent chamber 102. The extraction rotary piston 2 is also provided with a waste liquid communication groove 22 that can connect the liquid flow channel 108 of the sample chamber 101 with the waste liquid channel 109. Each reagent chamber 102 is provided with a corresponding liquid flow channel 108. Therefore, the extraction rotary piston 2 is provided with multiple reagent communication grooves 21. Each reagent communication groove 21 and the waste liquid communication groove 22 extend along the axial direction of the extraction rotary piston 2. The multiple reagent communication grooves 21 and the waste liquid communication groove 22 are arranged side by side along the circumference of the extraction rotary piston 2. The starting ends of each reagent connecting groove 21 and the above-mentioned waste liquid connecting groove 22 can be aligned and connected with the liquid flow channel 108 of the sample chamber 101 to achieve communication with the sample chamber 101, so that the starting ends of each reagent connecting groove 21 and the above-mentioned waste liquid connecting groove 22 are all located at the same circumferential position of the extraction rotary piston 2, that is, their axial distance is zero. The terminal end of each reagent connecting groove 21 can be aligned and connected with the liquid flow channel 108 of the corresponding reagent chamber 102 to achieve communication with the reagent chamber 102, and the terminal end of the waste liquid connecting groove 22 can be aligned and connected with the waste liquid channel 109 to achieve communication with the waste liquid chamber 103. Therefore, the terminal ends of each reagent connecting groove 21 and the terminal ends of the waste liquid connecting groove 22 are respectively located at different circumferential positions of the extraction rotary piston 2, that is, the axial distance between any two terminal ends is greater than zero. At least part of the reagent connecting groove 21 and the above-mentioned waste liquid connecting groove 22 respectively have one or more curved segments 200, and the curved segments 200 correspond to the liquid flow channel 108 of the non-target reagent chamber 102 that needs to pass through and are located below the liquid flow channel 108 to avoid the liquid flow channel 108, thereby avoiding contact with the liquid flow channel 108. The parts of the reagent connecting groove 21 and the above-mentioned waste liquid connecting groove 22 except the curved segments 200 extend along straight lines and are parallel to each other. The "non-target reagent connecting groove" in this article is explained as follows: for example, for the reagent connecting groove 21 connecting the fifth reagent chamber for storing the eluent and the sample chamber 101, the first to fourth reagent chambers located between the two are non-target reagent chambers; for the waste liquid connecting groove 22, all reagent chambers are non-target reagent chambers; and so on.

[0048] Combine Figure 1 and Figure 2As shown, the vertical microfluidic chip also includes a positive and negative pressure pump for providing positive and negative pressure to the sample chamber 101, and the sample chamber 101 and the positive and negative pressure pump are interconnected. In the nucleic acid extraction and purification zone, only the sample chamber 101 is connected to the positive and negative pressure pump. A positive and negative pressure pump can provide the power for liquid circulation during nucleic acid extraction, purification, and transfer of the nucleic acid extract to the first amplification zone. The upper end of the body 1 (preferably the upper end surface of the body 1) is provided with an interface 13 for connecting to a positive and negative pressure pump. The interface 13 is connected to the sample chamber 101 through a microchannel provided in the body 1. When the positive and negative pressure pump provides positive pressure to the sample chamber 101, the liquid in the sample chamber 101 can be pushed into the waste liquid chamber 103 or the chamber of the first amplification zone; when the positive and negative pressure pump provides negative pressure to the sample chamber 101, the reagent in the reagent chamber 102 can be sucked into the sample chamber 101.

[0049] Combine Figures 1 to 3 As shown, the vertical microfluidic chip also includes a mixing piston 11 that can move or rotate to mix the materials in the sample chamber 101. The mixing piston 11 is movably or rotatably disposed in the sample chamber 101, or the mixing piston 11 is movably or rotatably disposed in the body 1 and communicates with the sample chamber 101. By reciprocating the mixing piston 11, the reagents and the like in the sample chamber 101 are disturbed, promoting mixing of the materials.

[0050] Magnetic beads (not shown in the figure) for adsorbing nucleic acids are provided in the sample chamber 101. Figure 2 and Figure 3 As shown, a magnetic body 12 is provided near the sample chamber 101 of the body 1 and is embedded in the magnet hole of the body 1. The magnetic body 12 is preferably an electromagnet. When the electromagnet is energized, the magnetic beads can attract nucleic acids; when the electromagnet is de-energized, the magnetic beads release the nucleic acids. In another embodiment, the magnetic body 12 can also be a removable magnet.

[0051] like Figure 3As shown, the chamber of the first amplification zone (i.e., the chamber located between the extraction rotary piston 2 and the amplification rotary piston 3) includes a first amplification chamber 104, and the first amplification chamber 104 is used to perform a first round of PCR amplification on the extracted nucleic acid. Amplification reagents are pre-installed in the first amplification chamber 104. A first liquid inlet channel 110 and a first liquid outlet channel 111 connected to the first amplification chamber 104 are provided on the main body 1. The first amplification chamber 104 is located below the extraction rotary piston 2, and the extraction rotary piston 2 is also provided with a nucleic acid transfer groove 23 that can connect the liquid flow channel 108 of the sample chamber 101 and the first liquid inlet channel 110. The nucleic acid transfer groove 23 is provided on the outer circumferential surface of the extraction rotary piston 2, and the central angle between the starting end and the ending end of the nucleic acid transfer groove 23 is greater than zero, preferably greater than 90 degrees. When the extraction rotary piston 2 rotates to a certain position, the starting end of the nucleic acid transfer groove 23 is aligned and connected with the liquid flow channel 108 of the sample chamber 101 above, and the ending end is aligned and connected with the first liquid inlet channel 110 below. The positive and negative pressure pumps provide positive pressure to the sample chamber 101 to transfer the nucleic acid extract from the sample chamber 101 to the first amplification chamber 104.

[0052] The chamber of the first amplification zone also includes a buffer chamber 105 for receiving the amplification product of the first amplification chamber 104. The first amplification chamber 104 can be connected to the second amplification chamber in the second amplification zone through the buffer chamber 105. The buffer chamber 105 is located between the extraction rotary piston 2 and the amplification rotary piston 3. A second liquid inlet channel 112 and a second liquid outlet channel 113 connected to the buffer chamber 105 are provided on the body 1. The extraction rotary piston 2 is also provided with a pipetting connecting groove 24 that can connect the first liquid outlet channel 111 and the second liquid inlet channel 112. The pipetting connecting groove 24 extends in a straight line along the axial direction of the extraction rotary piston 2 and is located as a whole on the right side of the terminal end of the nucleic acid transfer groove 23. When the pipetting connecting groove 24 connects the first liquid outlet channel 111 and the second liquid inlet channel 112, the nucleic acid transfer groove 23 connects the liquid flow channel 108 of the sample chamber 101 with the first liquid inlet channel 110.

[0053] Combine Figures 4a to 5 As shown, the extraction rotary piston 2 includes a first body 201 and a first sealing layer 202 coated on the first body 201. The reagent connecting groove 21, the waste liquid connecting groove 22, the nucleic acid transfer groove 23, and the pipetting connecting groove 24 are provided on the first sealing layer 202; alternatively, the reagent connecting groove 21, the waste liquid connecting groove 22, the nucleic acid transfer groove 23, and the pipetting connecting groove 24 are provided on the first body 201 and extend through the first sealing layer 202. The reagent connecting groove 21, the waste liquid connecting groove 22, the nucleic acid transfer groove 23, and the pipetting connecting groove 24 are respectively surrounded by the first sealing layer 202. The first sealing layer 202 is made of a flexible material, such as rubber.

[0054] The buffer chamber 105 is connected to a quantitative chamber 106 provided in the body 1 via an air flow channel. The vertical microfluidic chip further includes a quantitative piston 14 slidably disposed in the quantitative chamber 106. By moving the quantitative piston 14, the amplification product in the first amplification chamber 104 can be drawn into the buffer chamber 105, and the amplification product in the buffer chamber 105 can be quantitatively distributed to the second amplification zone.

[0055] like Figure 3 As shown, the chamber of the second amplification zone (i.e., the chamber located below the amplification rotary piston 3) includes multiple second amplification chambers 107, which are used to perform a second round of PCR amplification on the products of the first round of amplification. Multiple second amplification chambers 107 are arranged side by side from left to right along the axis of the amplification rotary piston 3. The body 1 is provided with a liquid flow channel 108 that communicates with each second amplification chamber 107.

[0056] The connection and disconnection between the second liquid outlet channel 113 of the buffer chamber 105 and the liquid flow channel 108 of each second amplification chamber 107 are realized by the connecting groove provided on the amplification rotary piston 3. Figure 6a and Figure 6b As shown, the amplification rotary piston 3 is provided with an amplification connecting groove 31 capable of connecting the first liquid outlet channel 111 with the liquid flow channel 108 of the second amplification chamber 107. Specifically, the first liquid outlet channel 111 is connected to the amplification connecting groove 31 via the second liquid outlet channel 113 of the buffer chamber 105. The amplification connecting groove 31 is capable of connecting the second liquid outlet channel 113 and the liquid flow channel 108 of the second amplification chamber 107. The amplification rotary piston 3 is capable of selectively connecting the second liquid outlet channel 113 with the liquid flow channel 108 of one of the second amplification chambers 107, while disconnecting the other liquid flow channels 108; as the amplification rotary piston 3 rotates a certain angle, the amplification rotary piston 3 connects the second liquid outlet channel 113 with the liquid flow channel 108 of another second amplification chamber 107, while disconnecting the other liquid flow channels 108.

[0057] The layout of the amplification connecting groove 31 on the amplification rotary piston 3 is shown in FIG. Figure 6a and Figure 6b As shown. Figure 6a and Figure 6bAs shown, the amplification connecting groove 31 is provided on the outer circumferential surface of the amplification rotary piston 3, and each second amplification chamber 107 corresponds to an amplification connecting groove 31. The amplification connecting groove has a starting end and an ending end, and the central angle between the starting end and the ending end is greater than zero; specifically, the central angle is 180 degrees. A plurality of amplification connecting grooves 31 are arranged side by side along the circumferential direction of the amplification rotary piston 3. Each amplification connecting groove 31 includes three straight line segments connected in sequence, adjacent straight line segments are perpendicular to each other and there is an arc transition between the two to form a corner segment 300. The starting ends of the amplification connecting grooves 31 are arranged at intervals along the circumference of the amplification rotary piston 3 and are located at the same circumferential position of the amplification rotary piston 3. The lengths of the amplification connecting grooves 31 are different from each other, and the ending ends of any two adjacent amplification connecting grooves 31 are spaced apart by a distance in the axial direction and circumferential direction of the amplification rotary piston 3.

[0058] Reference Figures 6a to 7 As shown, the amplification rotary piston 3 includes a second body 301 and a second sealing layer 302 covering the second body 301. An amplification communication channel 31 is formed in the second sealing layer 302; alternatively, the amplification communication channel 31 is formed in the second body 301 and extends through the second sealing layer 302. The amplification communication channel 31 is surrounded on all sides by the second sealing layer 302. The second sealing layer 302 is made of a flexible material, such as rubber.

[0059] The extraction rotary piston 2 and the amplification rotary piston 3 can be driven to rotate by a power source, which can be a motor. Figure 2 As shown, the extraction rotary piston 2 has a driving end for engaging with a power source, and the driving end has a connecting hole 203 in the form of a polygonal hole or a special-shaped hole for inserting and connecting the output shaft of the motor. The amplification rotary piston 3 has a driving end for engaging with a power source, and the driving end has a connecting hole 303 in the form of a polygonal hole or a special-shaped hole for inserting and connecting the output shaft of the motor.

[0060] This embodiment also provides a method for nucleic acid amplification, which uses the vertical microfluidic chip described above. The method comprises the following steps:

[0061] S1. Add the nucleic acid sample into the sample chamber 101;

[0062] S2. Rotate the extraction rotary piston 2 to connect the reagent chamber 102 and the sample chamber 101 through the reagent connecting groove 21, so that the reagent is transferred to the sample chamber 101 and mixed to react;

[0063] S3, rotating the extraction rotary piston 2 so that the waste liquid connecting groove 22 on it connects the sample chamber 101 with the waste liquid chamber 103 provided on the body 1, and transferring the waste liquid after the reaction to the waste liquid chamber 103;

[0064] Repeat steps S2 and S3, sequentially adding lysate, protease, rinse solution, and eluent into the sample chamber 101 for reaction, and draining the waste liquid into the waste liquid chamber 103 after each reaction;

[0065] S4. Rotate the extraction rotary piston 2 to connect the sample chamber 101 and the first amplification chamber 104 through the nucleic acid connecting groove, and transfer the nucleic acid extract in the sample chamber 101 to the first amplification chamber 104.

[0066] S5. Rotate the extraction rotary piston 2 to close the microchannel between the sample chamber 101 and the first amplification chamber 104, and perform the first round of amplification reaction. Specifically, the extraction rotary piston 2 closes the microchannel between the first amplification chamber 104 and the buffer chamber 105.

[0067] S6. Rotate the extraction rotary piston 2 so that the pipetting connecting groove 24 on the extraction rotary piston 2 connects the first amplification chamber 104 with a buffer chamber 105 provided on the main body 1, and transfer the amplification product to the buffer chamber 105;

[0068] S7, rotating the amplification rotary piston 3, so that the amplification connecting groove 31 is connected to the second amplification chamber 107, and the first round of amplification products are transferred to the second amplification chamber 107. Specifically, the amplification connecting groove 31 connects the buffer chamber 105 with the second amplification chamber 107.

[0069] Step S7 is specifically implemented as follows:

[0070] Step S7-1: Rotate the amplification rotary piston 3 to connect the first amplification connecting groove 31 with the corresponding first second amplification chamber 107, and move the quantitative piston 14 in the quantitative chamber 106 connected to the buffer chamber 105 to quantitatively push the amplification product in the buffer chamber 105 into the first second amplification chamber 107;

[0071] Step S7-2: Continue rotating the amplification rotary piston 3 to connect the second amplification connecting groove 31 with the corresponding second amplification chamber 107, and move the quantitative piston 14 in the quantitative chamber 106 connected to the buffer chamber 105 to quantitatively push the amplification product in the buffer chamber 105 into the second amplification chamber 107.

[0072] Repeat the above steps S7 - 1 and S7 - 2 to push the amplification products into each second amplification chamber 107 in sequence.

[0073] The use process of the PCR nucleic acid extraction and amplification microfluidic chip of this embodiment is described as follows.

[0074] 1. Nucleic acid extraction and purification steps

[0075] 1. Open the cover 15 , add the sample into the sample chamber 101 through the hole above the sample chamber 101 , and then close the cover 15 .

[0076] 2. Rotate the extraction rotary piston 2 to a certain angle to connect the first reagent chamber 102 with the sample chamber 101. Then, generate negative pressure through the positive and negative pressure pumps to transfer the lysate in the first reagent chamber 102 to the sample chamber 101. Then, drive the mixing piston 11 to mix the lysate and nucleic acid sample and react.

[0077] 3. The magnetic body 12 begins to generate a magnetic field to adsorb the magnetic beads. The extraction rotary piston 2 then continues to rotate a certain angle to connect the sample chamber 101 and the waste liquid chamber 103. The positive and negative pressure pumps generate positive pressure to discharge the waste liquid in the sample chamber 101 that is not adsorbed on the magnetic beads into the waste liquid chamber 103.

[0078] 4. Rotate the extraction rotary piston 2 to a certain angle to connect the second reagent chamber 102 with the sample chamber 101. Then, the positive and negative pressure pumps generate negative pressure to transfer the protease in the second reagent chamber 102 to the sample chamber 101. At the same time, the magnetic body 12 stops generating a magnetic field to stop the adsorption of the magnetic beads, and then the mixing piston 11 is driven to perform a mixing reaction.

[0079] 5. The magnetic body 12 begins to generate a magnetic field to adsorb the magnetic beads. The extraction rotary piston 2 then continues to rotate a certain angle to connect the sample chamber 101 and the waste liquid chamber 103. The positive and negative pressure pumps generate positive pressure to discharge the waste liquid in the sample chamber 101 that is not adsorbed on the magnetic beads into the waste liquid chamber 103.

[0080] 6. Rotate the extraction rotary piston 2 to a certain angle to connect the third reagent chamber 102 with the sample chamber 101. Then, negative pressure is generated by the positive and negative pressure pumps to transfer the rinse liquid in the third reagent chamber 102 to the sample chamber 101. At the same time, the magnetic body 12 stops generating a magnetic field to stop the adsorption of the magnetic beads. Then, the mixing piston 11 is driven to perform a mixing reaction and perform the first rinse of the nucleic acid.

[0081] 7. The magnetic body 12 begins to generate a magnetic field to adsorb the magnetic beads. The extraction rotary piston 2 then continues to rotate a certain angle to connect the sample chamber 101 and the waste liquid chamber 103. The positive and negative pressure pumps generate positive pressure to discharge the waste liquid in the sample chamber 101 that is not adsorbed on the magnetic beads into the waste liquid chamber 103.

[0082] 8. Rotate the extraction rotary piston 2 to a certain angle to connect the fourth reagent chamber 102 with the sample chamber 101. Then, generate negative pressure through the positive and negative pressure pumps to transfer the rinse solution in the fourth reagent chamber 102 to the sample chamber 101. At the same time, the magnetic body 12 stops generating a magnetic field to stop the adsorption of the magnetic beads. Then, drive the mixing piston 11 to perform a mixing reaction and perform a second rinse on the nucleic acid.

[0083] 9. The magnetic body 12 begins to generate a magnetic field to adsorb the magnetic beads. The extraction rotary piston 2 then continues to rotate a certain angle to connect the sample chamber 101 and the waste liquid chamber 103. The positive pressure generated by the positive and negative pressure pumps is used to discharge the waste liquid in the sample chamber 101 that is not adsorbed on the magnetic beads into the waste liquid chamber 103.

[0084] 10. Rotate the extraction rotary piston 2 to a certain angle to connect the fifth reagent chamber 102 with the sample chamber 101. Then, generate negative pressure through the positive and negative pressure pumps to transfer the eluate in the fifth reagent chamber 102 to the sample chamber 101. At the same time, the magnetic body 12 stops generating a magnetic field to stop the adsorption of the magnetic beads. Then, drive the mixing piston 11 to perform a mixing reaction, and elute the extracted nucleic acid to form a nucleic acid extract.

[0085] 2. First round PCR amplification steps

[0086] 1. Rotate the extraction rotary piston 2 to connect the first amplification chamber 104 with the sample chamber 101 , and then use the positive and negative pressure pumps to generate positive pressure to discharge the nucleic acid extract in the sample chamber 101 into the first amplification chamber 104 .

[0087] 2. Turn the extraction rotary piston 2 to close the microchannel and perform the first round of PCR amplification reaction.

[0088] 3. Rotate the extraction rotary piston 2 to connect the first amplification chamber 104 with the buffer chamber 105, and then the quantitative piston 14 transfers the liquid into the buffer chamber 105 for mixing.

[0089] 3. Second round PCR amplification steps

[0090] 1. Rotate the amplification rotary piston 3 to connect the buffer chamber 105 with the first second amplification chamber 107 (chamber L1), and then transfer the liquid into the chamber L1 through the quantitative piston 14.

[0091] 2. Turn the amplification rotary piston 3 to transfer the liquid into cavities L2-L12 in sequence.

[0092] 3. Perform the second round of PCR amplification.

[0093] This embodiment can realize PCR nested amplification, integrating nucleic acid extraction, amplification and detection in one; it can simultaneously realize PCR real-time fluorescence detection of 1-48 gene loci; it adopts a vertical structure to effectively eliminate the influence of bubbles, ensure accurate liquid intake, and realize precise liquid quantification; it has a compact and simple structure and is easy to mass produce. The liquid path of the microfluidic chip is switched by rotating the piston, which is easy to operate and controllable, and easy to automate. It achieves full sealing without aerosol contamination, and does not require additional air circuits. It only needs to control a positive and negative pressure pump and a quantitative piston to provide the power for the flow of liquid during the entire operation. It is easy to operate and has a simple structure.

[0094] The above embodiment is intended only to illustrate the technical concepts and features of the present invention and is a preferred embodiment. Its purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. It is not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the principles of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A vertical microfluidic chip for nucleic acid amplification, comprising a body with a chamber, characterized in that: The chamber comprises: a first amplification chamber for performing a first round of amplification on the nucleic acid extract; and a second amplification chamber, which is used to perform a second round of amplification on the product of the first round of amplification; The body is provided with a first liquid outlet channel communicating with the first amplification chamber and a liquid flow channel communicating with the second amplification chamber; The vertical microfluidic chip also includes: an amplification rotary piston rotatably disposed in the body about a horizontally extending rotation axis, wherein the first amplification chamber is located above the amplification rotary piston, and the second amplification chamber is located below the amplification rotary piston; and an amplification connecting groove is provided on the amplification rotary piston for connecting the first liquid outlet channel with the liquid flow channel of the second amplification chamber; Among them, there are multiple second amplification chambers, and multiple second amplification chambers are arranged in parallel from left to right along the axial direction of the amplification rotary piston; the outer circumferential surface of the amplification rotary piston is provided with multiple amplification connecting grooves; each second amplification chamber is provided with a liquid flow channel, and each second amplification chamber corresponds to an amplification connecting groove, and multiple amplification connecting grooves are arranged in parallel along the circumferential direction of the amplification rotary piston; each amplification connecting groove has a starting end and an ending end respectively, and the starting end of each amplification connecting groove is arranged at the same circumferential position of the amplification rotary piston at intervals, and each amplification connecting groove includes three straight line segments connected in sequence, adjacent straight line segments are perpendicular to each other and there is an arc transition between the two to form a corner segment, the lengths of each amplification connecting groove are different, and the ending ends of any two adjacent amplification connecting grooves are spaced a distance apart in the axial direction and circumferential direction of the amplification rotary piston.

2. The vertical microfluidic chip according to claim 1, characterized in that: A central angle between the starting end and the ending end of the amplification connecting groove is greater than zero.

3. The vertical microfluidic chip according to claim 1, characterized in that: The amplification rotary piston includes an amplification rotary piston body and a sealing layer coated on the amplification rotary piston body. The amplification connecting groove is opened on the sealing layer or opened on the amplification rotary piston body and passes through the sealing layer.

4. The vertical microfluidic chip according to claim 1, characterized in that: The vertical microfluidic chip also includes an extraction rotary piston arranged above the first amplification chamber. The extraction rotary piston can be rotatably arranged in the body around a horizontally extending rotation axis. The body is provided with a first liquid inlet channel connected to the first amplification chamber, and the extraction rotary piston is provided with a nucleic acid transfer groove that can be connected to the first liquid inlet channel.

5. The vertical microfluidic chip according to claim 4, characterized in that: An amplification reagent is provided in the first amplification chamber, and the chamber further includes a buffer chamber for receiving the amplification product of the first amplification chamber. The buffer chamber is located between the extraction rotary piston and the amplification rotary piston. The first liquid outlet channel can be connected to the amplification connecting groove through the buffer chamber. A second liquid inlet channel and a second liquid outlet channel connected to the buffer chamber are provided on the main body. The extraction rotary piston is also provided with a pipetting connecting groove capable of connecting the first liquid outlet channel and the second liquid inlet channel. The amplification connecting groove can connect the second liquid outlet channel and the liquid flow channel of the second amplification chamber.

6. The vertical microfluidic chip according to claim 1, characterized in that: The chamber also includes a buffer chamber for receiving the amplification product of the first amplification chamber. The buffer chamber is located above the amplification rotary piston. The first liquid outlet channel can be connected to the amplification connecting groove through the buffer chamber. The buffer chamber is connected to a quantitative chamber opened in the main body through an air flow channel. The vertical microfluidic chip also includes a quantitative piston slidably arranged in the quantitative chamber.

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