HIGH-SPEED POLYMERASE CHAIN REACTION ANALYSIS PLATE

Abstract

The present invention relates to the structure of an analysis plate applied to a high-speed polymerase chain reaction (PCR), and to a PCR analysis plate used for implementing an analysis of a real-time PCR, a real-time nested PCR and a post-PCR lateral flow hybridization reaction. The present invention is provided with: a check valve for enabling the maintaining of positive pressure when an elastic film expands into a convex form by having a solution pushed therein by the positive pressure; a lateral flow analysis module for analyzing a post-PCR follow-up PCR or lateral flow; and a shut-off valve enabling the controlling of the movement of the solution after each reaction ends. A high-speed PCR analysis plate may be provided whereby, by pressing, by means of a temperature-controllable heating block, the elastic film, which is in a convex form by the solution, of a PCR unit, a PCR solution may undergo rapid temperature circulation with minimum heat resistance, and a PCR dried material and a nucleic acid solution may be homogenized and mixed.

Claims

1. A high-speed polymerase chain reaction (PCR) analysis plate comprising: a base substrate in which an inlet is formed; a reaction unit formed as a closed structure on the base substrate by fusing a sealing film having elasticity to the base substrate into a shape of a closed line including the inlet; and a shut-off valve provided between the reaction unit and a flow channel unit connected to the inlet of the reaction unit.

2. A high-speed polymerase chain reaction (PCR) analysis plate comprising: a base substrate in which an inlet is formed; a reaction unit formed as a closed structure on the base substrate by fusing a sealing film having elasticity to the base substrate into a shape of a closed line including an inlet; and a valve unit including a check valve configured to prevent a backflow of a solution injected into the reaction unit and a flow channel unit connected to the inlet of the reaction unit.

3. The high-speed PCR analysis plate of claim 1, wherein the reaction unit the base substrate further includes: a first PCR unit and a second PCR unit spaced apart therefrom, which are formed as closed structures in which a sealing film having elasticity is fused into a shape of a closed line, wherein the first PCR unit and the second PCR unit communicate with each other through a second flow channel unit formed in a lower portion of the base substrate; and a shut-off valve configured to intermittently control a flow of a fluid in the second flow channel unit between the first PCR unit and the second PCR unit.

4. The high-speed PCR analysis plate of claim 3, wherein the second PCR unit is formed by fusing a sealing film having elasticity into a shape of closed lines forming a plurality of second PCR units having second inlets and further includes the second flow channel unit which starts at an outlet of the first PCR unit and is branched off at a branching point into a plurality of flow channels connected in parallel to the inlets of the plurality of second PCR units.

5. The high-speed PCR analysis plate of claim 12, further comprising: an outlet formed at one end of the reaction unit of the base substrate; a lateral flow analysis unit communicating with the outlet through a second flow channel unit connected to the outlet, wherein the lateral flow analysis unit includes a lateral flow analysis module with a fixed nucleic acid probe and a second inlet and is formed by fusing a sealing film with elasticity into a shape of a closed line; and a shut-off valve configured to intermittently control a flow of a solution in the second flow channel unit connecting the outlet of the reaction unit and the inlet of the lateral flow analysis unit.

6. The high-speed PCR analysis plate of claim 3, further comprising: a lateral flow analysis unit disposed to be spaced apart from the second PCR including a third inlet, and a lateral flow analysis module with a fixed nucleic acid probe, and formed by fusing a sealing film having elasticity into a shape of a closed line, wherein the second PCR unit includes a second outlet; a third flow channel unit connecting the second outlet of the second PCR unit and the third inlet of the lateral flow analysis unit; and a second shut-off valve configured to intermittently control a flow of a solution in the third flow channel unit.

7. The high-speed PCR analysis plate of claim 1, wherein: the check valve is formed of an elastic material and opens by pressure of injecting a solution into a flow channel; a surface of the check valve coming into contact with an inner side of a sealing film is implemented in a shape of a cone, a two-tier cylinder in which a top diameter is no more than of a bottom diameter, or a cylinder having projections formed on an upper surface and allows a solution to permeate; and a lower surface of the check valve is implemented as a smooth mirror surface and comes into close contact with a surface for a solution injection hole in the base substrate.

8. The high-speed PCR analysis plate of claim 1, wherein: the shut-off valve is formed by fusing the sealing film having elasticity into a shape of a close line including an inlet and an outlet of the shut-off valve and opens in such a way that the sealing film is stretched due to pressure of a solution entering through the inlet and the solution exits through the outlet; and when an upper portion of the sealing film is pressed with a valve compression unit and the sealing film having elasticity comes into close contact with the base substrate, the inlet and the outlet are closed as all the solution in an inner space of the shut-off valve is drained.

9. The high-speed PCR analysis plate of claim 1, wherein the closed line formed by fusing the sealing film having elasticity to the base substrate has a width of 0.5 mm to 2 mm.

10. The high-speed PCR analysis plate of claim 1, wherein the closed line formed by fusing the sealing film having elasticity to the base substrate is formed to be projected to a height of 2 mm or less on the base substrate S.

11. The high-speed PCR analysis plate of claim 5, wherein the lateral flow analysis module has a structure of lateral flow paper with a fixed nucleic acid probe, includes a loading pad attached to a front end and an absorption pad attached to a rear end, and includes one or more nucleic acid probes fixed to a plurality of linear arrays arranged perpendicularly to a flow of a solution.

12. The high-speed PCR analysis plate of claim 1, wherein: one or more pairs of primers or a primer/probe, which is dried material, is applied to a portion adjacent to the inlet of the reaction unit; when a target nucleic acid solution containing polymerases is injected, as air inside the flow channel unit and the reaction unit is compressed, the target nucleic acid solution is mixed with the dried material, and the sealing film having elasticity is stretched and forms a convex shape; and when an operation of pressing the convex surface of the sealing film having elasticity with a pressing block is repeatedly performed, the injected solution moves in the reaction unit, and a PCR reactant solution becomes homogeneous.

13. The high-speed PCR analysis plate of claim 1, wherein: one or more primers or a PCR mixture including a primer/probe, which is dried material, is applied to a portion adjacent to the inlet of the reaction unit; when a target nucleic acid solution is injected, as air inside the flow channel unit and the reaction unit is compressed, the solution is mixed with the dried material, and the sealing film having elasticity is stretched and forms a convex shape; and when an operation of pressing the convex surface of the sealing film having elasticity with a pressing block is repeatedly performed, the injected solution moves, and a PCR reactant solution becomes homogeneous.

14. The high-speed PCR analysis plate of claim 3, wherein, in the second reaction unit, a PCR mixture for nested PCR including a primer/probe or a primer and a fluorescent dye for DNA detection, which is dried material, is applied.

15. The high-speed PCR analysis plate of claim 5, wherein the second reaction unit includes a labeled primer having the ability to complementarily bind to a 5 position of a single strand of amplified DNA and a mixture for a DNA polymerase reaction, which are dried materials, and probes of a hybridization analysis module have nucleic acid sequences complementary to single-helix DNA synthesized by the labeled primer and are fixed in a solid state.

16. The high-speed PCR analysis plate of claim 12, wherein the labeled nested primer is labeled with a fluorescent material, a chemiluminescent material, or gold nanoparticles.

17. The high-speed PCR analysis plate of claim 2, wherein the reaction unit on the base substrate further includes: a first PCR unit and a second PCR unit spaced apart therefrom, which are formed as closed structures in which a sealing film having elasticity is fused into a shape of a closed line, wherein the first PCR unit and the second PCR unit communicate with each other through a second flow channel unit formed in a lower portion of the base substrate; and a shut-off valve configured to intermittently control a flow of a fluid in the second flow channel unit between the first PCR unit and the second PCR unit.

18. The high-speed PCR analysis plate of claim 2, further comprising: an outlet formed at one end of the reaction unit of the base substrate; a lateral flow analysis unit communicating with the outlet through a second flow channel unit connected to the outlet, wherein the lateral flow analysis unit includes a lateral flow analysis module with a fixed nucleic acid probe and a second inlet and is formed by fusing a sealing film with elasticity into a shape of a closed line; and a shut-off valve configured to intermittently control a flow of a solution in the second flow channel unit connecting the outlet of the reaction unit and the inlet of the lateral flow analysis unit.

19. The high-speed PCR analysis plate of claim 2, wherein: the check valve is formed of an elastic material and opens by pressure of injecting a solution into a flow channel; a surface of the check valve coming into contact with an inner side of a sealing film is implemented in a shape of a cone, a two-tier cylinder in which a top diameter is no more than of a bottom diameter, or a cylinder having projections formed on an upper surface and allows a solution to permeate; and a lower surface of the check valve is implemented as a smooth mirror surface and comes into close contact with a surface for a solution injection hole in the base substrate.

20. The high-speed PCR analysis plate of claim 2, wherein the closed line formed by fusing the sealing film having elasticity to the base substrate has a width of 0.5 mm to 2 mm.

21. The high-speed PCR analysis plate of claim 2, wherein: one or more pairs of primers or a primer/probe, which is dried material, is applied to a portion adjacent to the inlet of the reaction unit; when a target nucleic acid solution containing polymerases is injected, as air inside the flow channel unit and the reaction unit is compressed, the target nucleic acid solution is mixed with the dried material, and the sealing film having elasticity is stretched and forms a convex shape; and when an operation of pressing the convex surface of the sealing film having elasticity with a pressing block is repeatedly performed, the injected solution moves in the reaction unit, and a PCR reactant solution becomes homogeneous.

22. The high-speed PCR analysis plate of claim 2, wherein: one or more primers or a PCR mixture including a primer/probe, which is dried material, is applied to a portion adjacent to the inlet of the reaction unit; when a target nucleic acid solution is injected, as air inside the flow channel unit and the reaction unit is compressed, the solution is mixed with the dried material, and the sealing film having elasticity is stretched and forms a convex shape; and when an operation of pressing the convex surface of the sealing film having elasticity with a pressing block is repeatedly performed, the injected solution moves, and a PCR reactant solution becomes homogeneous.

Description

DESCRIPTION OF DRAWINGS

[0039] FIG. 1 is a set of conceptual diagrams illustrating the operation mechanism of a PCR analysis plate according to one embodiment of the present invention including a shut-off valve and a fused elastic film.

[0040] FIG. 2 is a set of conceptual diagrams illustrating the operation mechanism of a PCR analysis plate according to another embodiment of the present invention including a check valve and a fused elastic film.

[0041] FIG. 3 is a perspective assembly view of the PCR analysis plate of FIG. 2 having a check valve and two PCR units, which shows an elastic film to be fused, the check valve, and a sealing agent for forming a flow channel.

[0042] FIG. 4 is top and bottom plan views of the PCR analysis plate of FIG. 3, which shows the elastic film to be fused, the check valve, and the sealing agent for forming the flow channel.

[0043] FIG. 5 is a cross-sectional view of the PCR analysis plate of FIG. 4 taken along the line A-A.

[0044] FIG. 6 shows a plan-view photograph (FIG. 6A) and an oblique-view photograph (FIG. 6B) showing the actual appearance of the PCR analysis plate of FIGS. 3 and 4 and shows that the elastic film forms a convex shape in which positive pressure is maintained as a reaction solution is injected into a receiving unit.

[0045] FIG. 7 is top and bottom plan views of a PCR analysis plate having a check valve and eight PCR units and a cross-sectional view of the PCR analysis plate taken along the line A-A.

[0046] FIG. 8 is a set of conceptual diagrams illustrating the operation mechanism of a PCR analysis plate for nested PCR according to the present invention having a check valve, a first PCR unit, and a second PCR unit and enabling nested PCR.

[0047] FIG. 9 is a plan view of the PCR analysis plate of the present invention having a check valve, a first PCR unit, and a second PCR unit and enabling nested PCR.

[0048] FIG. 10 is a perspective assembly view of the PCR analysis plate for nested PCR of FIG. 8 having a check valve, a first PCR unit, a second PCR unit, a shut-off valve, and a purification unit, which shows an elastic film to be fused, the check valve, the purification unit, the shut-off valve, and a sealing film for forming a flow channel.

[0049] FIG. 11 is a lower assembly view of the PCR analysis plate of FIG. 10.

[0050] FIG. 12 is a diagram illustrating a PCR analysis plate according to still another embodiment of the present invention.

[0051] FIG. 13 is a set of conceptual diagrams sequentially illustrating the operation mechanism of a PCR analysis plate of the present invention enabling post-PCR lateral flow analysis.

[0052] FIG. 14 is a plan view of the PCR analysis plate of FIG. 13.

[0053] FIG. 15 is a set of conceptual diagrams sequentially illustrating the operation mechanism of a PCR analysis plate of the present invention enabling, after a PCR reaction, an asymmetric PCR reaction or an extension reaction using a nested primer to be performed, followed by lateral flow analysis.

[0054] FIG. 16 is a plan view of the PCR analysis plate of FIG. 15.

[0055] FIGS. 17A, 17B, and 17C are, respectively, a top plan view, a bottom plan view, and a cross-sectional view of a PCR analysis plate according to yet another embodiment of the present invention having two PCR units.

[0056] FIGS. 18A, 18B, and 18C are, respectively, a top plan view, a bottom plan view, and a cross-sectional view of a PCR analysis plate according to yet another embodiment of the present invention having eight PCR units.

MODE OF THE INVENTION

[0057] Advantages and features of the present invention and a method of achieving the same will become apparent with reference to the exemplary embodiments described below in detail as well as the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied into other forms. Rather, the exemplary embodiments introduced herein are provided so that the disclosed contents may be thorough and complete, and that the spirit of the present invention may be sufficiently conveyed to those skilled in the art.

[0058] The terms used herein have been used only for the purpose of describing particular embodiments and are not intended to limit the present invention. In the present specification, singular expressions include plural expressions unless the context clearly indicates otherwise. It will be further understood that the terms comprises, comprising, includes, including, has, and/or having, when used herein, specify the presence of stated features, integers, steps, operations, elements, components and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups thereof.

[0059] FIG. 1 is a set of conceptual diagrams illustrating the main parts of a high-speed PCR analysis plate (hereinafter referred to as PCR plate) according to one embodiment of the present invention. FIG. 2 is a set of conceptual diagrams illustrating a case where a check valve rather than a shut-off valve as in FIG. 1 is provided. FIG. 3 is a perspective assembly view illustrating one example of the PCR plate of FIG. 2 having a check valve and two PCR units, which shows an elastic film to be fused, a check valve, and a sealing agent for forming a flow channel. FIG. 4 is top and bottom plan views of the PCR plate of FIG. 3, and FIG. 5 is a cross-sectional view of the PCR plate of FIG. 4 taken along the line A-A.

[0060] Referring to FIGS. 1 to 4, a PCR plate of the present invention may have a structure including a base substrate S, a reaction unit W which is provided on the base substrate, receives a nucleic acid solution, and includes at least one accommodation region where a PCR mixture including a primer, a primer/probe, or a primer and a probe, which is in the form of dried material, is accommodated, and an elastic film F which seals the inside of the reaction unit W.

[0061] Specifically, as in the structure shown in FIG. 3, the PCR plate of the present invention is implemented as a plate-shaped structure including one surface portion and the other surface portion opposite to the one surface portion.

[0062] Here, in the one surface portion, there is provided a reaction unit W which implements accommodation regions W1, W2 for accommodating a PCR mixture including a primer, a primer/probe, or a primer and a probe, which is in the form of dried material and, by receiving a nucleic acid solution injected by a nucleic acid extraction cartridge or an external injection device, realizing PCR. The reaction unit W may be implemented as a structure in which an elastic film F is fused to the base substrate S, and according to one exemplary embodiment of the present invention, the reaction unit W may also be implemented as an isolated space as shown in FIG. 4 using a projecting partition wall GA having a height of 2 to 4 mm, preferably 2 to 3 mm, and more preferably 2 mm or less. When the partition wall is implemented with a height of more than 4 mm, there is a disadvantage in that when a high-temperature heating block and a low-temperature heating block come into contact with the elastic film at a later time point, temperature (heat) is not precisely transferred to reactants at the bottom. It is most preferred that the partition wall is implemented with a height of 2 mm or less, and even when the reaction unit is implemented through fused regions, it is preferred that the fused regions are implemented with a height (thickness) of 2 mm or less.

[0063] In addition, in the following embodiments of the present invention, the concept of fusing is defined as a state in which a film material is in close contact with a base substrate without any lifting, and although the definition generally refers to the fusing achieved by ultrasonic waves, heating, pressing, or the like, it is considered that the concept also includes modifications of close contact implemented using a predetermined adhesive material or attachment.

[0064] In addition, in implementing the reaction unit W, although FIG. 3 illustrates the implementation of two accommodation regions W1, W2 and FIG. 7 illustrates the implementation of eight accommodation regions, the present invention is not limited thereto, and of course, it is also possible to implement the reaction unit W as a structure having more than one or two accommodation regions.

[0065] In addition, the PCR plate of the present invention may be formed by fusing an elastic film F having elasticity onto the reaction unit W into the shape of a closed line and may be implemented as a structure which seals the inside of the accommodation regions. Hereinafter, in the present invention, the expression having the shape of a closed line defines a processed structure in the form of a fused linear region in which when predetermined outer edge portions of an elastic film are fused to a base substrate, since the fused regions are fused to the base substrate perfectly, the structure is implemented as a structure in which the fused regions and the inside space are sealed.

[0066] The elastic film F is disposed on the base substrate S such that the elastic film F seals all of the accommodation regions W1, W2, a flow channel unit J, and a valve unit to be described below.

[0067] As illustrated in FIG. 3, the partition wall GA implements the accommodation regions W1, W2 and the flow channel unit J connected to the accommodation regions W1, W2. The flow channel unit J is capable of guiding the nucleic acids injected through an injection hole hl formed at one end of the base substrate opposite the accommodation regions W1, W2 to the accommodation regions W1, W2 via a check valve depression K.

[0068] In this case, a PCR mixture including a primer, a primer/probe, or a primer and a probe, which is in the form of dried material for realizing PCR and is accommodated in the accommodation regions W1, W2, reacts with the injected nucleic acids, causing a PCR reaction.

[0069] In order to realize high-speed PCR, a process of alternately bringing high- and low-temperature heating blocks HB into close contact with upper portions of the accommodation regions W1, W2 is required so that the temperature conditions capable of inducing PCR can be applied. In this case, heat transfer is affected by how close the heating blocks HB come into contact with the elastic film F.

[0070] According to one embodiment of the present invention, since the elastic film forming the accommodation regions W1, W2 of the PCR plate 200 is stretched to form a convex shape by the injection of a solution, when the heating blocks come into close contact with the convex part of the elastic film surface, the elastic film forms uniform close contact with the heating blocks HB, and a rapid PCR reaction is enabled.

[0071] In addition, when the elastic film F having a convex shape due to the injected solution is pressed, since the nucleic acid solution injected from the outside is moved and homogeneously mixed with a PCR mixture including a primer, a primer/probe, or a primer and a probe, which is in the form of dried material, accommodated in an accommodation region, a PCR reaction is uniformly implemented.

[0072] However, when pressure is applied on the upper surface of the accommodation regions W1, W2 by the heating blocks HB as shown in FIG. 2, a phenomenon in which reactants undergoing a mixed reaction again flow reversely through the flow channel unit J occurs. Accordingly, in order to prevent the backflow, the valve units are provided in the present invention, and a shut-off valve D (FIG. 1) or a check valve V (FIG. 2) for preventing the backflow of a solution pushed out by the heating blocks is disposed on the base substrate.

[0073] In one embodiment of the present invention, the shut-off valve is configured as a part that performs an intermittent function of blocking or applying the flow of a fluid on a base substrate, and is defined, in the structure of FIG. 10, as the concept of a region D implemented by a pair of holes hd1, hd2 realizing the introduction of a fluid into the base substrate S and the elastic film member F provided on top. In addition, the check valve V is an independent structure implemented between the elastic film and the base substrate and is defined as a structure that controls the flow of a fluid.

[0074] In addition, according to one exemplary embodiment of the present invention, the PCR plate includes all structures having one or more of a shut-off valve D disposed between a flow channel unit J connected to an inlet of a reaction unit W and the reaction unit W and a valve unit including a check valve V for preventing the backflow of a solution injected into the reaction unit W and the flow channel unit J connected to the inlet of the reaction unit W.

[0075] That is, in one embodiment of the present invention, the above-described check valve V is implemented in the valve unit portion of FIG. 3 and seated so as to be spaced from the inside of the check valve depression K communicating with one end of the flow channel unit J communicating with the reaction unit W.

[0076] The check valve V may be operated to close a fluid inlet hole Ha for introducing fluid into the valve unit by floating.

[0077] To describe functions of the check valve V of the present invention in detail, as shown in FIGS. 2 to 4, in the PCR plate of the present invention, the injection path for injecting nucleic acids into the reaction unit W is applied through the injection hole hl provided at one end of the plate, and the applied nucleic acids pass through the check valve depression K and the flow channel unit again and move toward the rear end of the PCR plate to the reaction unit W.

[0078] The backflow phenomenon refers to a process in which reactants inside the accommodation region flow in the reverse direction of the injection path and are discharged to the outside. In the present invention, the backflow phenomenon may occur when an injected nucleic acid solution flows through the flow channel, causing the air in the flow channel to be compressed and the elastic film to be stretched to form a convex shape and thereby form positive pressure, when a convex elastic film part of the accommodation region is pressed and a dried material in the accommodation region is mixed with the solution, or when additional positive pressure is formed inside the accommodation region by the heating blocks alternately applying high temperature and low temperature to a convex elastic film part of the accommodation region for PCR, in which case, reactants inside the accommodation region flow in the reverse direction of the injection path and are discharged to the outside.

[0079] FIG. 5 is a cross-sectional view of the PCR plate of FIG. 4 taken along the line A-A, and FIGS. 1 and 2 are conceptual diagrams illustrating operation mechanisms of PCR plates according to embodiments of the present invention.

[0080] Hereinafter, the operation mechanism of a PCR plate having a structure having a check valve as implemented in one embodiment of the present invention will be described with reference to FIGS. 3 to 5.

[0081] As shown in FIG. 5, in the PCR plate according to one embodiment of the present invention, a fluid inlet hole Ha through which a nucleic acid fluid introduced from the outside is injected may be disposed below a check valve depression K where a check valve V is placed, and a path-forming block Va structure for ensuring a fluid movement path may be implemented below the fluid inlet hole Ha.

[0082] In the PCR plate, a fluid containing nucleic acids introduced from the outside is injected into the injection hole hl through the bottom of the plate, and the injected fluid moves to the check valve depression K through an inflow path.

[0083] Hereinafter, the above-described operation will be described in detail with reference to FIG. 2.

[0084] FIG. 2 is a set of conceptual diagrams illustrating a process in which a fluid containing nucleic acids moves to a reaction unit W in a PCR plate structure.

[0085] Referring to FIG. 2, when a fluid containing a nucleic acid extract is introduced from the outside through the injection hole hl, the fluid moves along a path (in the direction indicated by the arrows) from the bottom of the fluid inlet hole Ha toward the check valve depression K, thereby causing the check valve V to slightly float (FIG. 2A).

[0086] Subsequently, as the elastic film slightly stretches due to the pressure caused by the injection of the fluid containing a nucleic acid extract, the check valve V floats, and the fluid containing a nucleic acid extract moves easily to the reaction unit W, and after moving, the fluid reacts with a PCR mixture including a primer, a primer/probe, or a primer and a probe, which is in the form of dried material and has already been provided in the reaction unit W, that is, an accommodation region, and thereby PCR is performed (FIG. 2B).

[0087] In this case, the pressure on the inside of the elastic film sealing the upper portion of the accommodation region is increased due to the injection of the fluid, causing the surface to convexly bulge out as shown in FIG. 2B. The surface of the elastic film under the influence of positive pressure as such may effectively bring the solution in the reaction unit of the PCR plate into close contact with the heating blocks HB as will be described below, and this may be advantageous for securing precise temperature conditions.

[0088] Subsequently, when equipment which is capable of applying a temperature required for a thermal denaturation process and an exact temperature required for an annealing process and performing heating and pressing applies pressure on the inside of the reaction unit (accommodation region) periodically while controlling temperature as required for a PCR process to induce PCR, positive pressure (PP) is formed inside the accommodation region, and reactants are homogeneously mixed.

[0089] At the same time, the positive pressure causes a backflow phenomenon in which the mixed reactants are pushed out of the accommodation region reversely.

[0090] When such a backflow phenomenon occurs, the check valve V is pressed downward as shown in FIG. 2B, in which case, the check valve V naturally blocks the fluid inlet hole Ha. When the check valve V blocks the fluid inlet hole Ha as such, reactants inside the accommodation region cannot flow reversely to the outside and continue to completely react in the accommodation region.

[0091] Therefore, in one embodiment of the present invention, the check valve V is implemented as a three-dimensional structure having an upper surface having irregularities and a lower surface enabling close contact.

[0092] That is, the lower surface of the check valve blocks the inlet by coming into close contact with the inlet, and the irregularities on the upper surface face the inner surface of the elastic film, in which case the check valve is pressed, and when a solution is applied to an upper portion of the check valve excluding the irregularities and thus positive pressure is applied, the check valve strongly seals the area around the inlet.

[0093] In other words, a structure is implemented so that the check valve V in the check valve depression K floats and opens due to the inflow of the fluid introduced into the reaction unit W, and at the same time, the lower surface of the check valve V is capable of closing the fluid injection hole, and in the upper surface of the check valve V, only the center part comes into close contact with the elastic film, and the remainder comes into contact with the solution.

[0094] In this case, although the check valve may be implemented in various structures, materials, and shapes, the structure, material, or shape of the check valve may be variously changed, and for example, the check valve may have the shape of a cone, a two-tier cylinder in which a top diameter is no more than of a bottom diameter, or a cylinder having projections formed on an upper surface thereof. However, it is preferred that the entire lower surface V2 of the check valve is formed of an elastic material such as silicone rubber so that the check valve can efficiently close the entire fluid inlet hole Ha.

[0095] FIG. 6 shows actual photographs of a PCR plate implemented according to FIGS. 2 and 5, and FIG. 6A is an image of the PCR plate before injecting a fluid containing nucleic acids, and FIG. 6B is an image of the PCR plate after injecting a nucleic acid solution, which shows that the elastic film in the accommodation region has a convex shape due to the positive pressure applied by the injection of the reaction solution. When high- and low-temperature heating blocks maintaining constant temperatures alternately press the surface of the elastic film having an upwardly convex shape as shown in FIG. 6B, since the convex surface of the elastic film of the accommodation region is pressed, heat is transferred, and as the reaction solution is moved, the PCR solution is uniformly mixed. The backflow caused by the additional pressure applied due to the contact may be controlled through the check valve.

[0096] The PCR plate is usable for the analysis of real-time PCR amplification. In order to perform a real-time PCR reaction, a fluorescence value from the PCR unit is measured each cycle, a cycle in which fluorescence is increased above a threshold fluorescence value is identified, and thereby the concentration of a target is measured.

[0097] In order to accomplish the above-described objectives, according to one exemplary embodiment of the present invention, the base substrate may be made of a transparent synthetic resin. One example of the transparent synthetic resin is a polymer based on polyethylene (PE), polypropylene (PP), polyester (PET), polycarbonate (PC), or polymethacrylate (PMMA). In addition, it is required that the transparent synthetic resin is a polymer not containing a fluorescent material so that various fluorescent materials used for real-time quantitative PCR can be measured.

[0098] Hereinabove, the operation principle of the simplest example of the PCR plate which is the basis of the present invention has been described in detail.

[0099] The above-described embodiment is an example of performing a single PCR reaction, and in the present invention, there are additional embodiments for achieving high performance while using the above-described basic principle.

[0100] The following is one exemplary embodiment and is an application example relating to the performance of nested PCR which is known as the most efficient method for amplifying trace amounts of targets in a sample containing nucleic acid materials having complex nucleic acid sequences.

[0101] FIG. 8 is a set of conceptual diagrams illustrating a PCR plate of the present invention enabling nested PCR and illustrating the operation mechanism of the PCR plate having a check valve, a first PCR unit, and a second PCR unit. FIG. 9 is a plan view of the PCR plate of the present invention of FIG. 8 having a check valve, a first PCR unit, and a second PCR unit and enabling nested PCR.

[0102] FIG. 10 is an exploded perspective view illustrating the assembly of the PCR plate of FIGS. 8 and 9.

[0103] First, referring to FIGS. 9 and 10, the PCR plate according to the present embodiment of the present invention is implemented to include a pair of reaction units, which are spaced apart from each other, on a base substrate S. That is, a first PCR unit W1 and a second PCR unit W2 are provided. In particular, the first PCR unit W1 and the second PCR unit W2 are implemented as spaces formed by fusing an elastic sealing film, and between the first PCR unit W1 and the second PCR unit W2, a shut-off valve D for controlling the flow of a fluid in a second flow channel unit J2 connecting an outlet of the first PCR unit W1 and an inlet of the second PCR unit W2 is disposed.

[0104] That is, the present embodiment of the present invention is different from the embodiment illustrated in FIGS. 2 to 4 including the above-described check valve in that the structure according to the present embodiment has the structure of FIG. 3 but further includes a second PCR unit, a flow channel J2 connected to the first PCR unit W1, and a shut-off valve D for controlling the flow of a solution midway through the flow channel.

[0105] In order to improve the efficiency of secondary nested PCR, a purification unit FT containing an absorbent for removing inhibitory materials inhibiting the secondary nested PCR, such as primers and pyrophosphate generated in the primary PCR, may be added to a flow channel before the second PCR unit W2. The second PCR unit W2 is manufactured in the same manner as the first PCR unit W1. The second PCR unit W2 is manufactured by fusing an elastic film to the base substrate into the shape of a closed line including an inlet, wherein, before sealing by fusing the elastic film, additional primers, probes, deoxynucleoside triphosphates (dNTPs), and a polymerase required for the nested PCR are added to the base substrate in the form of dried materials.

[0106] FIG. 10 is an overall assembly view of a nested PCR plate. FIG. 11 is a lower assembly view of the nested PCR plate of FIG. 10.

[0107] In order to manufacture the nested PCR plate of the present invention, a flow channel on the lower surface of the base substrate S and a purification unit FT containing an absorbent are formed by turning over the base substrate S so that the lower surface thereof faces upward and placing the absorbent in the purification unit FT and sealing with a film F3, and therefore, the flow channel and the purification unit are formed at one time.

[0108] Subsequently, after turning over the base substrate S so that the upper surface thereof faces upward, a check valve V is installed, a first PCR dried material and a second PCR dried material are placed in their respective positions in the first PCR unit W1 and the second PCR unit W2, and the elastic film F is fused, and thereby a nested PCR plate of the present invention is obtained.

[0109] The absorbent is a porous particle allowing penetration of molecules depending on the sizes of molecules and allows the penetration of small molecules such as primers or pyrophosphate while blocking the penetration of amplified DNA with a large molecular size, which is a product of PCR. As the absorbent, a combination of Sephadex (G-25 or G-50), porous ceramics, and the like may be used.

[0110] The operation principle of the primary PCR of the present invention may be described as follows based on the structure of FIG. 10 with reference to the cross-sectional conceptual diagrams of FIG. 8.

[0111] As shown in FIG. 8A, it is required that the shut-off valve is kept closed at all times until the primary PCR is completed. A nucleic acid extract or the like injected through the injection hole hl passes through the check valve V, and the primary PCR is performed in the first PCR unit W1. While the primary PCR is performed, the check valve V is in a closed state as shown in FIG. 8B and prevents the backflow of reactants. The primary PCR is performed by alternately pressing the bulged part of the elastic film of the first PCR unit W1 with heating blocks HB1.

[0112] Subsequently, when the primary PCR of the introduced reactants is terminated, the piston PG pressing the shut-off valve D is raised as shown in FIG. 8C, allowing the solution contained in the first PCR unit W1 under positive pressure to move along the flow channel, pass through the shut-off valve D and then the purification unit FT, and reach the second PCR unit W2.

[0113] In this case, in order to move as much entire solution as possible, the solution is pressed with the front heating block HB1 while the rear heating block HB2 is in a lifted state so that the solution is moved completely, and then the shut-off valve D is pressed with the piston to block the flow channel.

[0114] Subsequently, the solution is alternately pressed with high- and low-temperature heating blocks HB2, which forms PCR cycles where the amplified products of primary PCR are subjected to a PCR reaction. In this case, in order to perform a real-time PCR reaction, a fluorescence value from the PCR unit is measured each cycle, a cycle in which fluorescence is increased above a threshold fluorescence value is identified, and thereby the concentration of a target is measured. Therefore, it is required that the PCR plate is manufactured using the above-described transparent polymer materials.

[0115] FIG. 12 is a diagram illustrating a PCR plate according to still another embodiment of the present invention.

[0116] The structure of FIG. 12 is one example of a PCR plate of the present invention which enables multiplex nested PCR for analyzing a large number of targets to be performed.

[0117] That is, in various embodiments of the present invention, structures enabling a primary PCR reaction and then a plurality of secondary real-time PCR reactions are implemented.

[0118] The structure of FIG. 12 is one example of implementing the gist of the present invention, and FIG. 12 is a plan view of an exemplary PCR plate enabling a primary multiplex PCR reaction and then secondary real-time PCR reactions in eight secondary reaction units to be performed.

[0119] That is, the structure of FIG. 12 has the same basic structure as the structure of FIGS. 9 and 10 in the way an injection hole, a check valve CB, a first PCR unit W1, and a shut-off valve D are arranged and the first PCR unit W1 is configured. However, the structure of FIG. 12 is different from the structure of FIGS. 9 and 10 in that the second PCR unit is implemented as multiple units rather than as a single unit. Therefore, the structure of FIG. 12 is the same as the structure of FIGS. 9 and 10 in terms of operation principle and different in that the flow channel provided after the purification unit FT is formed such that eight secondary PCR units W8 are connected in parallel. The structure of FIG. 12 operates according to the above-described process and has the advantage of being able to analyze a large number of targets through real-time PCR at the same time.

[0120] FIG. 13 is a set of diagrams illustrating the structure of a PCR plate according to yet another embodiment of the present invention enabling post-PCR lateral flow analysis. FIG. 14 is a plan view of the PCR plate of FIG. 13.

[0121] Referring to FIGS. 13 and 14, since the PCR plate according to the present embodiment of the present invention has a sealed system formed by fusing films F to upper and lower surfaces of a base substrate S made of a polymer, amplification can be completed in a short time, and therefore, the PCR plate is ideal for lateral flow analysis integrated with PCR carried out by, after PCR, passing PCR products through the paper in which nucleic acid probes are fixed to respective bands and analyzing the bands thus hybridized.

[0122] Specifically, in order to perform lateral flow analysis after PCR using amplified PCR products, it is necessary that single-helix DNA is finally produced. To this end, when preparing the above-described PCR dried material, a dried material containing an excessive amount of one primer is prepared. When an asymmetric PCR reaction is performed using this PCR material, a PCR product containing an excessive amount of only one strand is finally produced. (Wooddell, C I; Burgess, R (1996). Use of Asymmetric PCR to Generate Long Primers and Single-stranded DNA for Incorporating Cross-linking Analogs into Specific Sites in a DNA Probe (PDF). Genome Res. (6): 886-892).

[0123] The plate for post-PCR lateral flow analysis according to the present invention is manufactured to be the same as the PCR plate of FIGS. 9 and 10 in terms of having a structure including a check valve, a first PCR unit W1, and a shut-off valve D and in the way the plate is operated. However, the plate for post-PCR lateral flow analysis according to the present invention is manufactured by completing a lower flow channel of the base substrate with a sealing agent, installing the check valve above the lower flow channel and placing the above-described asymmetric PCR dried material and a lateral flow analysis module (LSM) in respective positions, and then fusing an elastic film.

[0124] Here, the lateral flow analysis module (LSM) used is a paper module in which a loading pad L1 is attached to a front end and an absorption pad L3 is attached to a rear end and which has bands L2 including a large number of probes fixed thereon in a middle portion of the paper.

[0125] In this case, the base substrate S is preferably implemented to include a depression so that the lateral flow analysis module can be seated therein. According to the present invention, it is possible to manufacture a PCR lateral flow analysis plate which enables, by a simple process, a PCR reaction to be performed in a confined space at high speed and lateral flow probe hybridization analysis to be performed.

[0126] As shown in FIG. 13, the working principle of the PCR of the present embodiment is basically the same as that of the plate having one PCR unit. However, it is required that the shut-off valve D is kept closed at all times until the PCR is completed. When PCR is terminated, the piston PG pressing the shut-off valve D is raised, causing the solution contained in the PCR unit under positive pressure to move along the flow channel, pass through the shut-off valve D, and enter the lateral flow analysis unit LS. In this case, in order to move as much entire solution as possible, the convex part of the reaction unit is pressed with the heating block HB and thus, the solution is moved. Subsequently, the solution is absorbed by the loading pad L1 and slowly moves along the paper in which probes are fixed in the form of bands L2 and finally reaches the absorption pad L3, and when the solution encounters probes complementary to the targets, since hybridization occurs, the movement of the solution is stopped, and the color of the fluorescent label or gold nanoparticles appears. Through this, various targets can be qualitatively detected. In this case, the temperature of the PCR plate should be kept constant so that hybridization can occur accurately. For this, the temperature of the metal plate on which the PCR plate is placed should be controlled to be constant, and by pressing the heating block HB down while adjusting the temperature of the heating block HB to the same temperature and pressing the shut-off valve with the piston, the temperature of the PCR plate is kept constant.

[0127] FIG. 15 is a set of conceptual diagrams sequentially illustrating the operation mechanism of a PCR plate of the present invention enabling, after a PCR reaction, a nested asymmetric PCR reaction or an extension reaction using a nested primer to be performed, followed by lateral flow analysis. FIG. 16 is a plan view of the PCR plate of FIG. 15.

[0128] This structure has the most complex form among the structures derived from the present invention and enables a primary PCR reaction, a secondary, nested asymmetric PCR reaction, and then lateral flow analysis to be performed. For this, a first shut-off valve D1 for transferring a solution carrying products of the primary PCR reaction to the secondary PCR reaction and a second shut-off valve D2 for moving a solution from the secondary PCR reaction to a lateral flow analyzer LS are installed.

[0129] In addition, a purification unit FT for removing materials inhibiting the secondary PCR reaction from the products of the primary PCR reaction is provided in the flow channel after the first shut-off valve.

[0130] As the secondary PCR reaction, a nested asymmetric PCR reaction, a nested primer extension reaction, or the like may be used. In this case, a primer for forming a single helix should be labeled with fluorescence or the like, and a dried material including the primer should be input in the second reaction unit.

[0131] In order to manufacture the PCR analysis plate of the present invention, a flow channel on the lower surface of the base substrate and a purification unit containing an absorbent are formed by turning over the base substrate so that the lower surface thereof faces upward and placing the absorbent in the purification unit and sealing with a film, and therefore, the flow channel and the purification unit are formed at one time. Subsequently, after turning over the base substrate so that the upper surface thereof faces upward, a check valve, a first PCR dried material, a second PCR dried material, and a lateral flow module are placed in their respective positions, and an elastic film is fused, and thereby the nested PCR lateral flow analysis plate of the present invention is obtained. The absorbent is the porous particle described above, and as the absorbent, a combination of Sephadex (G-25 or G-50), porous ceramics, and the like may be used.

[0132] Referring to FIGS. 15 and 16, the operation principle of the primary PCR reaction of the present invention is as follows:

[0133] (a) It is required that the first shut-off valve D1 and the second shut-off valve D2 are kept closed at all times until the primary PCR reaction is completed.

[0134] (b) The primary PCR reaction is performed in the reaction unit by the operation of the check valve V as described above in the embodiment of FIG. 9.

[0135] (c) Subsequently, when the primary PCR reaction is terminated, the piston PG1 pressing the first shut-off valve D1 is raised, allowing the solution contained in the first PCR unit under positive pressure to move along the flow channel, pass through the first shut-off valve D1 and then the purification unit FT, and reach the second PCR unit W2. In this case, in order to move as much entire solution as possible, the solution is pressed with the front heating block1 HB1 while the rear heating block HB2 is in a lifted stated so that the solution is moved completely, and then the first shut-off valve D1 is pressed with the piston PG1 to block the flow channel. Subsequently, the solution is alternately pressed with high- and low-temperature heating blocks HB2, which forms secondary PCR cycles, and thereby a secondary PCR reaction is performed. In this case, both the first shut-off valve D1 and the second shut-off valve D2 should be in a closed state.

[0136] (d) After the secondary PCR reaction is terminated, the second shut-off valve D2 opens to allow the secondary PCR solution to flow to a loading pad of the lateral flow analysis unit LS. When single-helix DNA strands generated in the secondary PCR reaction encounter target probe nucleic acids and stop moving, the targets can be detected by way of detecting labels in each band.

[0137] Although the present invention has been described based on specific embodiments thereof, it should be obvious to those of ordinary skill in the technical field to which the present invention pertains that the technical spirit of the present invention is not limited to the embodiments, that modifications or changes may be made within the scope described in the claims, and that such modifications or changes are encompassed within the scope of the following claims.

DESCRIPTION OF REFERENCE NUMERALS

[0138] S: base substrate [0139] W: reaction unit [0140] F: elastic film [0141] W1: first PCR unit [0142] W2: second PCR unit [0143] W3: third PCR unit [0144] J: flow channel unit [0145] J2: second flow channel [0146] J3: third flow channel [0147] V: check valve [0148] Va: path-forming block [0149] K: check valve depression [0150] Ha: inlet hole [0151] D, D1, D2: shut-off valve [0152] LS: lateral flow analysis unit [0153] HB, HB1, HB2: heating block [0154] FT: purification unit [0155] hl: injection hole [0156] hd1, hd2: hole [0157] L1: loading pad [0158] L2: band with fixed probe [0159] L3: absorption pad