PCR (Polymerase Chain Reaction) plate, nucleic acid extraction box and polymerase chain reaction device comprising structures of PCR plate and nucleic acid extraction box

Abstract

The PCR plate according to the present invention comprises: a main body part provided with one or more reaction holes; an insertion part which extends from the main body part, is inserted into the nucleic acid extraction cartridge, and is provided with an injection port through which a nucleic acid solution is injected; a flow path part which enables the nucleic acid solution to flow from the injection port to the reaction hole; and a blocking part which is mounted on the main body part and prevents the nucleic acid solution from flowing back from the reaction hole to the flow path part side.

Description

Technical Field

[0001] The present invention relates to a PCR plate, a nucleic acid extraction box and a polymerase chain reaction device comprising these structures, and in particular to a PCR plate, a nucleic acid extraction box and a polymerase chain reaction device comprising these structures capable of real-time detection of nucleic acid extraction, amplification reaction and amplification products. Background Art

[0002] Point of care (POC) diagnostic technology, which can accurately and quickly diagnose patients' diseases anytime and anywhere, is a very important evidence-based technology in the field of precision medicine and has attracted much attention. Symptom-based on-site diagnostic technology is a technology that uses disease symptoms such as cough, diarrhea, high fever, and genital abnormalities as a standard to quickly detect all infectious pathogens that cause disease symptoms at one time and confirm the causative pathogens, and then prescribe the best antibiotics and therapeutic agents. As a core new technology for future precision medicine, many related studies are currently being further developed. The advantage of this on-site diagnostic technology is that, just like the existing pregnancy test strips used to confirm whether a pregnancy is pregnant, and the blood glucose meter that can confirm blood sugar levels, even non-experts can make a quick and accurate diagnosis on site. At present, molecular diagnostic technology has attracted much attention as a core technology for future medicine. At present, multiple detection methods that can detect multiple pathogens at the same time have been developed. Using these technologies, we can accurately grasp the cause of infectious diseases and prescribe the best prescription, treat the disease as soon as possible, and significantly shorten the patient's recovery time, thereby improving medical quality and saving medical costs.

[0003] However, current molecular diagnostic systems take more than three hours to confirm results and require skilled experts to operate. Therefore, in order to perform POC molecular diagnostics required on-site, it is necessary to develop an automated small device that can perform the complex nucleic acid extraction process and real-time gene amplification detection in a fully automated manner and should ensure that non-professionals can operate it easily.

[0004] Representative molecular diagnostic methods include methods of diagnosis using polymerase chain reaction (PCR, Polymerase Chain Reaction, hereinafter referred to as "PCR"). Since it was invented by Kelly Mullis in 1985, polymerase chain reaction (PCR) has been widely used in molecular biology and molecular diagnosis because it can quickly and easily amplify specific DNA. Using PCR / RT-PCR, it is possible to confirm whether specific DNA / RNA exists in biological samples, so it is often used to diagnose infections of pathogenic microorganisms such as viruses. With the development of this PCR / RT-PCR technology into real-time quantitative PCR (Realtime PCR, Quantitative PCR) technology, the results can be known at the same time as the PCR ends, which not only simplifies the detection process and greatly shortens the detection time, but also can accurately determine the number of pathogens, so it is used as a standard diagnostic method for monitoring the treatment effects of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), etc. In addition, it can also detect gene expression patterns or gene mutations associated with specific diseases, so it is used as the most important disease diagnosis technology.

[0005] In order to perform this PCR, a nucleic acid extraction step is required, that is, to remove substances that hinder the PCR reaction from the biological sample and extract pure nucleic acid. The nucleic acid extraction process consists of multiple steps, and it requires proficiency in biological sample and nucleic acid extraction operations. When operated manually, there may be contamination problems due to operator errors, so in most cases, automated nucleic acid extraction equipment is used for molecular diagnosis.

[0006] PCR reactions and detection of reaction products require real-time quantitative PCR devices, so molecular diagnosis was previously mainly performed in large hospitals or professional clinical testing institutions.

[0007] Recently, through research and development, the entire process of nucleic acid extraction, PCR reaction, and reaction product detection has been automated, and a variety of automated systems and equipment that utilize these systems have been developed that allow PCR to be easily used even without professional skills.

[0008] However, existing devices have the problems of being too expensive or taking a long time to process, and having difficulty in performing multiple tests at the same time.

[0009] The basic principle of PCR is introduced below. The DNA double helix structure is heated at 95℃ to separate into single strands. Then the reaction solution is cooled to the annealing temperature, so that the complementary primers are selectively paired at both ends of the site to be amplified in the PCR reaction solution. The DNA polymerase sequentially connects the complementary four nucleoside triphosphates A, G, T, and C on each single strand to form a double helix structure. This reaction is an experimental process of repeatedly heating and cooling the PCR reaction solution 30 to 45 times (n) to make a specific DNA double helix structure undergo 2 n Geometric amplification. After the RT-PCR reaction synthesizes cDNA through reverse transcription, it can be amplified by PCR, thus expanding it into an RNA detection method.

[0010] The following is an introduction to the principle of real-time quantitative PCR, which was newly developed to formally apply PCR to molecular diagnosis. In order to quantitatively analyze the DNA amplified by the PCR reaction, a substance that produces fluorescence in proportion to the amount of DNA is added to the PCR reaction solution, and the fluorescence of each cycle is measured. The cycle where the critical fluorescence value is detected is found, thereby quantitatively measuring the initial target nucleic acid concentration.

[0011] Since the invention of PCR, a large number of pathogens and disease-related gene base sequences have been discovered through genome projects in the process of developing various application technologies, and molecular diagnostic technologies that amplify these disease-related DNA / RNA base sequences for qualitative and quantitative diagnosis have developed rapidly. Since the existing PCR temperature cycle takes about two hours, methods that can perform PCR more quickly and accurately are constantly being developed to enable on-site diagnosis (Lab Chip, 2016, 16, 3866-3884).

[0012] In order to perform a PCR reaction in a short time, the temperature of the reaction solution must be changed quickly. In addition, in order to amplify only the desired target through an accurate PCR reaction, the primers must be designed to bind to the desired target in a targeted manner, and the annealing temperature should be accurately adjusted during the PCR temperature cycling reaction.

[0013] To this end, a micro-PCR reaction vessel has been developed that has a smaller heat capacity and better heat transfer than the 0.2ml and 0.5ml reactors commonly used in previous laboratories. Since this microreactor uses less reaction solution and has a large surface area, it has a fast heat transfer rate and can achieve rapid heating and cooling. Add 10ul of PCR solution to a thin reaction tank of 17X15mm in size and 40-80um thick on a silicon wafer and cover it with a glass plate to maintain a large surface area (>100mm 2 / 10ul). However, using the existing Peltier heat block, it could not be shown that one cycle time can be shortened to about 3 minutes (Clin. Chem. 40 / 9, 1815-1818 (1994)).

[0014] In order to quickly perform thermal cycling on the PCR reactor, an early PCR reaction device was developed by repeatedly immersing the PCR reactor in a high-temperature water tank and a low-temperature water tank. (Turbo Thermalcycler.Bioneer Corp.Daejeon). This PCR device that allows the reactor to cycle in different temperature zones adopts a spatial movement method. Its advantage is that the PCR reaction can be quickly and accurately performed by immersing the reactor in a constant temperature water tank that accurately maintains the temperature in advance. However, since multiple constant temperature water tanks are required, the equipment is large and difficult to maintain. At the same time, the time difference temperature cycle PCR equipment that uses Peltier elements in a fixed block to change the temperature over time currently dominates.

[0015] The PCR method using microfluidics has also been developed into a spatial movement temperature cycle method and a time difference temperature cycle method. The spatial movement temperature cycle method can be roughly divided into an open method that flows continuously in a FIFO (First-In-First-Out) manner, and a closed method that moves repeatedly in different temperature intervals. As an open method, in 1994, Nakano et al. developed a method in which a capillary is wrapped around a cylindrical block with different temperature compartments and the PCR solution is continuously flowed into it. (Biosci.Biotech.Biochem., 58(2), 349-352, 1994). In 1998, Kopp et al. confirmed that a microfluidic PCR device that repeatedly flows through high and low temperature intervals in a microfluidic form allows 10ul of solution to undergo PCR through 20 cycles with a cycle of 4.5s (Science 2801046-1048, 1998).

[0016] The background technology of the present invention is disclosed in Korean Patent Publication No. 10-2016-0067872 (published on June 14, 2016, invention title: an analysis unit for performing polymerase chain reaction, an analysis device, an operation method of the analysis unit, and a manufacturing method of the analysis unit).

[0017] Previously, a high-speed polymerase chain reaction analysis plate was disclosed in Korean registered patent No. 10-2105558. However, if the disclosed analysis plate is used, amplification products between reaction wells may be mixed during the polymerase chain reaction process, resulting in reduced experimental accuracy. In order to solve these problems, an improved PCR plate that can quickly quantitatively and qualitatively analyze multiple targets is needed. Summary of the invention

[0018] [Technical problem to be solved by the invention]

[0019] The present invention is developed to solve the above-mentioned problem. One purpose of the present invention is to provide a PCR plate, a nucleic acid extraction box and a polymerase chain reaction device comprising these structures. The device can extract nucleic acids from biological samples, perform PCR reactions and scan excitation light of various bands and corresponding fluorescence to perform target nucleic acid detection in a fully automatic manner. Multiple targets can be detected in one operation. The device is easy to use and can obtain accurate results in a short time.

[0020] Another object of the present invention is to provide a PCR plate, a nucleic acid extraction box and a polymerase chain reaction device comprising these structures, which can quickly and repeatedly apply the accurate temperature required in the process of combining the temperature adjustment process required for the PCR process with the temperature required for the thermal denaturation process to the reaction target, ensuring that PCR can be performed quickly and accurately, thereby maximizing the reliability of the reaction.

[0021] [Technical solution]

[0022] The PC board of the present invention comprises: a main body portion, which is equipped with one or more reaction holes; an insertion portion, which is extended from the main body portion, inserted into a nucleic acid extraction box, and is equipped with an injection port for injecting a nucleic acid solution; a flow path portion, which can allow the nucleic acid solution to flow from the injection port to the reaction holes; and a blocking portion, which is installed on the main body portion to block the nucleic acid solution from flowing back from the reaction holes to the flow path portion.

[0023] In the present invention, the main body portion includes: a main frame portion, which is equipped with one or more reaction holes; a blocking accommodating portion, a recess of which is formed on the main frame portion and accommodates the blocking portion; a connecting flow path portion, which connects the blocking accommodating portion with the reaction hole; a storage portion, a recess of which is formed on the main body portion and is connected to the flow path portion, for supplying and accommodating the nucleic acid solution; a flow path guide portion, which is connected to the storage portion and is used to guide the nucleic acid solution to flow to one side of the blocking portion.

[0024] In the present invention, the connecting flow path portion may include a bottleneck portion that is narrowed from the blocking portion toward the reaction well side.

[0025] In the present invention, the accommodating portion blocking accommodating portion is divided into a plurality of accommodating portion blocking accommodating portions by a partition portion formed along the length direction of the storage portion, and the blocking portion can be installed on each of the divided accommodating portion blocking accommodating portions.

[0026] In the present invention, the blocking portion is composed of an elastically deformable material.

[0027] In the present invention, the main body portion includes: a main frame portion, equipped with one or more reaction holes; a circular accommodating portion blocking the accommodating portion, which is concavely formed in the main frame portion and arranged corresponding to each reaction hole, and is used to accommodate the circular blocking portion; and a connecting flow path portion, which is used to connect the accommodating portion blocking the accommodating portion and the reaction hole.

[0028] In the present invention, the blocking portion may be made of a material including a material capable of generating elastic deformation.

[0029] In the present invention, the thin sheet portion can be installed on the reaction hole by hot melting.

[0030] The nucleic acid extraction box of the present invention comprises: a box cover portion, which is equipped with a receiving portion equipped with multiple partition structures for holding solutions required for DNA extraction; a box body portion, which is combined with the box cover portion through an insertion structure and is equipped with a reaction receiving portion equipped with a solution introduced from the receiving portion to react with a sample or to perform cleaning.

[0031] In the present invention, the box cover portion includes a rubber portion which contacts one side of the accommodating portion of the box body portion and is composed of an elastically deformable material.

[0032] In the present invention, the nucleic acid extraction box may be equipped with a leak-proof rubber for preventing the nucleic acid solution from leaking, and the leak-proof rubber comprises an elastic material.

[0033] The nucleic acid extraction box assembly of the present invention includes: a nucleic acid extraction box; a PCR plate, which receives the nucleic acid solution from the nucleic acid extraction box and stores it in a reaction well containing a PCR mixed dry product. The nucleic acid extraction box includes: a box cover, which is equipped with a container equipped with multiple partition structures for holding the solution required for DNA extraction; a box body, which is combined with the box cover through an insertion structure, and is equipped with a reaction container equipped with a reaction container for reacting the solution introduced from the container with the sample for cleaning. The PCR plate includes: a main body, which is equipped with more than one reaction well; an insertion part, which extends from the main body, is inserted into the nucleic acid extraction box, and is equipped with an injection port for injecting the nucleic acid solution; a flow path part, which can make the nucleic acid solution flow from the injection port to the reaction well; a blocking part, which is installed on the main body to block the nucleic acid solution from flowing back from the reaction well to the flow path part.

[0034] In the present invention, the box cover portion includes a rubber portion which contacts one side of the accommodating portion of the box body portion and is composed of an elastically deformable material.

[0035] In the present invention, the nucleic acid extraction box may be equipped with a leak-proof rubber for preventing the nucleic acid solution from leaking, and the leak-proof rubber comprises an elastic material.

[0036] The polymerase chain reaction device of the present invention comprises: a nucleic acid extraction box; a PCR plate, which receives the nucleic acid solution from the nucleic acid extraction box and stores it in a reaction well containing a PCR mixed dry product. The nucleic acid extraction box comprises: a box cover, which is equipped with a container with multiple partition structures for holding the solution required for DNA extraction; and a box body, which is combined with the box cover by an insertion structure and is equipped with a reaction container for reacting with a sample or cleaning the solution introduced from the container. The PCR plate comprises a main body, which is equipped with at least one reaction well; an insertion part, which extends from the main body and is inserted into the nucleic acid extraction box, and is equipped with an injection port for injecting the nucleic acid solution; a flow path part, which can make the nucleic acid solution flow from the injection port to the reaction well; and a blocking part, which is installed on the main body to block the nucleic acid solution from flowing back from the reaction well to the flow path part.

[0037] In the present invention, the box cover portion includes a rubber portion which contacts one side of the accommodating portion of the box body portion and is composed of an elastically deformable material.

[0038] In the present invention, a leak-proof rubber for preventing leakage of the nucleic acid solution may be installed on the nucleic acid extraction box, and the leak-proof rubber comprises an elastic material.

[0039] [Effects of the invention]

[0040] According to the present invention, by extracting nucleic acid from a biological sample, performing a PCR reaction and scanning excitation light of various wavelengths and corresponding fluorescence, the reaction products can be automatically detected in real time, and multiple tests can be completed in one operation. It is easy to use and accurate results can be obtained in a short time.

[0041] In addition, the PCR mixed dried product can be prevented from backflowing through the reaction wells on the PCR plate, thereby preventing the PCR mixed dried product from being mixed into adjacent reaction wells and causing contamination.

[0042] In addition, according to the present invention, the accurate temperature required in the temperature adjustment process required for the PCR process combined with the thermal denaturation step can be applied to the reaction target at one time, quickly and in real time, ensuring that PCR can be performed accurately, thereby maximizing the reliability of the reaction.

[0043] That is, if the existing temperature control method of moving the reaction liquid to increase the temperature is adopted, it is not conducive to the PCR reaction because the temperature cannot be increased uniformly. In addition, the temperature of the entire reactant cannot be uniformized by moving the reaction liquid to increase the temperature in sequence, so other reactions are likely to occur. The present invention can solve the above problems. By keeping the temperature range set on the heating block in a constant temperature state and directly pressurizing the entire reaction liquid to increase the temperature, the temperature required for the PCR reaction can be very effectively increased.

[0044] In addition, in order to minimize the time delay in changing the temperature from high to low, the block-shaped heating block structure is arranged side by side and spaced apart. When the PCR plate is pressurized, the heating blocks with different temperatures are pressurized in real time by changing the position of the heating blocks, which can significantly solve the problem caused by the time delay required in the temperature change process.

[0045] In addition, the PCR plate is implemented in an insertable structure that is inserted into the nucleic acid extraction box, so the nucleic acid extraction box can be universal, and the PCR plates used in various test kits can be stored in a small space, and the appropriate PCR plate can be inserted and used as needed during the test. A reaction well equipped in the PCR plate can analyze up to 6 fluorescence values, and the reaction wells of the PCR plate can be increased to 8 as needed. Therefore, all pathogens that may be contained in the patient's biological samples related to the symptoms can be amplified and detected, providing symptom-based multiple molecular diagnostic tests.

[0046] In addition, according to the present invention, the constant temperature plate is divided into regions with a first temperature and a second temperature gradient. When the heating block is pressurized by the driving module, the region with a set temperature (first temperature or second temperature) corresponding to the temperature of the heating block is moved accordingly, and the upper and lower surfaces of the PCR plate are contact-pressurized at the same time, thereby achieving double efficiency compared to a constant temperature plate method that maintains a single temperature.

[0047] In addition, the present invention adopts a movable constant temperature plate structure, which is driven by a sliding belt to ensure the reliability of the product structure and movement. In addition, the heating time of the target inner plate is synchronized with the upper and lower surfaces, which can shorten the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a structural diagram of components constituting the main components of the polymerase chain reaction device according to an embodiment of the present invention; Figures 2 to 7 Schematic diagram for explaining the structure of the temperature control module in the embodiment of the present invention.

[0049] Figures 8 to 13 is a schematic diagram of an embodiment of a PCR plate applied to an embodiment of the present invention;

[0050] Figures 14 to 19 is a schematic diagram of another embodiment of a PCR plate applied to an embodiment of the present invention;

[0051] Figure 20 to Figure 23 is a conceptual diagram for explaining the structure and operation of the constant temperature plate and translation drive module applied to an embodiment of the present invention;

[0052] Fig.24 is a three-dimensional conceptual diagram of a nucleic acid extraction box according to an embodiment of the present invention, showing the structure of the PCR plate insertion and combination;

[0053] Fig.25 yes Fig.24 Exploded stereogram of

[0054] Fig.26 Yes Display Fig.24 A schematic diagram of the internal structure of the box cover portion in the structure shown;

[0055] Fig. 27 It is displayed in perspective Fig.24 Schematic diagram of the binding state of the structure shown;

[0056] Fig.28 is an assembly stereogram of another embodiment of the nucleic acid extraction box of the present invention;

[0057] Fig.29 is a cross-sectional view of another embodiment of the nucleic acid extraction box of the present invention

[0058] Figure 30 to Figure 32 is a schematic diagram of the working state of the lower part of the box structure of an embodiment of the present invention;

[0059] Fig.33 is a schematic diagram of the overall structure and layout of the polymerase chain reaction device according to the embodiment of the present invention;

[0060] Fig.34 yes Fig.33 An enlarged view of the combined layout of the main parts of the embodiment of the present invention;

[0061] Fig.35 Shows Fig.34 A conceptual vertical section of the section showing the layout of the concave structure;

[0062] Fig.36 yes Fig.35 a side perspective cutaway view of the structure shown;

[0063] Fig.37 is a three-dimensional conceptual diagram of a nucleic acid extraction box according to another embodiment of the present invention;

[0064] Fig.38 yes Fig.37 a cross-sectional view of the structure shown;

[0065] Fig.39 yes Fig.38 Schematic diagram of using a punch to punch holes in leak-proof rubber;

[0066] Fig.40 yes Fig.39 Schematic diagram of using a pressing fixture to pressurize the leak-proof rubber;

[0067] Fig.41 It is a schematic diagram of the leakproof rubber preventing the residual liquid from leaking;

[0068] Fig.42 is a schematic diagram of another embodiment of a PCR plate;

[0069] Fig.43 yes Fig.42 a cross-sectional view of the structure shown;

[0070] Fig.44 It is a schematic diagram of mounting the sheet on a PCR plate;

[0071] Fig.45 The test process for confirming whether the solutions between reaction wells are mixed during PCR is shown;

[0072] Fig.46 The mixed flow test process of the reaction wells of the PCR plate is shown. DETAILED DESCRIPTION

[0073] Hereinafter, an embodiment of the PCR plate, the nucleic acid extraction box and the polymerase chain reaction device including these structures of the present invention will be described with reference to the accompanying drawings. In the process, for the sake of clarity and convenience of the description, the thickness of the lines or the size of the components in the accompanying drawings may be exaggerated.

[0074] In addition, the terms described below are defined in consideration of the functions of the present invention, and may vary according to the intention or practice of the user or operator. Therefore, the definitions of these terms should be made based on the overall content of this specification.

[0075] Figure 1 is a diagram of the main components of the polymerase chain reaction device according to the embodiment of the present invention; Figures 2 to 7 is a schematic diagram for explaining the structure of a temperature control module in an embodiment of the present invention; Figures 8 to 13 is a schematic diagram of an embodiment of a PCR plate applied to an embodiment of the present invention; Figures 14 to 19 is a schematic diagram of another embodiment of a PCR plate applied to an embodiment of the present invention; Figure 20 to Figure 23 is a conceptual diagram for explaining the structure and operation of the constant temperature plate and translation drive module applied to an embodiment of the present invention; Fig.24 is a three-dimensional conceptual diagram of a nucleic acid extraction box according to an embodiment of the present invention, showing the structure of the PCR plate insertion and combination; Fig.25 yes Fig.24 An exploded perspective view of the structure shown; Fig.26 yes Fig.24 A schematic diagram of the internal structure of the box cover portion in the structure shown; Fig. 27 It is described in perspective Fig.24 Schematic diagram of the binding state of the structure; Fig.28 is an assembly stereogram of another embodiment of the nucleic acid extraction box of the embodiment of the present invention; Fig.29 is a cross-sectional view of another embodiment of the nucleic acid extraction box of the embodiment of the present invention; Figure 30 to Figure 32 is a schematic diagram of the working state of the lower part of the box structure of an embodiment of the present invention; Fig.33 is a schematic diagram of the overall structure and layout of the polymerase chain reaction device according to the embodiment of the present invention; Fig.34 yes Fig.33 An enlarged view of the combined layout of the main parts of the embodiment of the present invention; Fig.35 yes Fig.34 Conceptual image of a partial vertical section showing the layout of the concave structure; Fig.36 yes Fig.35 a side perspective cutaway view of the structure shown; Fig.37 is a three-dimensional conceptual diagram of a nucleic acid extraction box according to another embodiment of the present invention; Fig.38 yes Fig.37 a cross-sectional view of the structure shown; Fig.39 yes Fig.38 Schematic diagram of using a punch to punch holes in leak-proof rubber; Fig.40 yes Fig.39 Schematic diagram of using a pressing fixture to pressurize the leak-proof rubber; Fig.41 It is a schematic diagram of the leakproof rubber preventing the residual liquid from leaking; Fig.42 is a schematic diagram of another embodiment of a PCR plate; Fig.43 yes Fig.42 a cross-sectional view of the structure shown; Fig.44 It is a schematic diagram of mounting the sheet on a PCR plate; Fig.45 The test process for confirming whether the solutions between reaction wells are mixed during PCR is shown; Fig.46 The mixed flow test process of the reaction wells of the PCR plate is shown.

[0076] Reference Figure 1 It can be seen that the polymerase chain reaction device described in the embodiment of the present invention can be equipped with a temperature control module, which is implemented by a heating block structure that contacts the PCR reaction plate and applies a specific temperature. The accurate temperature required in the annealing process of the temperature adjustment process required for the PCR process and the temperature required for the thermal denaturation process can be quickly, real-time, and applied to the reaction target at one time without time difference, ensuring that accurate PCR can be performed, thereby maximizing the reliability of the reaction.

[0077] The temperature control module of the present invention pressurizes the PCR reaction plate in real time by changing the positions of the heating blocks respectively set to the first temperature and the second temperature, so as to minimize the time delay in the process of changing the temperature from the first temperature to the relatively lower second temperature, and vice versa, from the second temperature to the relatively higher first temperature, thereby significantly improving the problem caused by the time delay required in the temperature change process.

[0078] In addition, the present invention can further realize a constant temperature plate structure arranged at the lower part of the PCR plate and operated by a sliding horizontal moving structure. In this case, by maintaining the temperature of the PCR plate at the first temperature or the second temperature, the time required for applying the temperature change can be reduced to the maximum extent, thereby maximizing the reaction speed.

[0079] The polymerase chain reaction device of the embodiment of the present invention comprises: a PCR plate (200) for receiving a nucleic acid solution from a nucleic acid extraction box (100) and receiving the nucleic acid solution in a reaction hole (W) containing a PCR mixed dried product; and a temperature control module (300) comprising a pair of heating blocks (310, 320) arranged on one side of the PCR plate (200), adjacent to the reaction hole (W) and applying different temperatures, capable of horizontal operation and vertical movement. The PCR plate (200) comprises: a main body, which is equipped with at least one reaction hole (W); an insertion part (220) extending from the main body (210), inserted into the nucleic acid extraction box (100), and equipped with an injection port (h1) for injecting the nucleic acid solution; and a flow path part (230) capable of allowing the nucleic acid solution to flow from the injection port (h1) to the reaction hole (W); and a blocking part (240) installed on the main body (210) to block the nucleic acid solution from flowing back from the reaction hole (W) to the flow path part (230).

[0080] The present invention provides convenience through the above structure, and even non-professionals can freely perform nucleic acid extraction by inserting the required sample through the nucleic acid extraction box (100). At the same time, the temperature can be quickly and accurately controlled by using the heating block (310, 320) structure, and the heating block can directly apply the temperature cycle target temperature required for amplification of the PCR plate (200) to the reaction solution in the PCR plate (200) by extrusion with the film.

[0081] In addition, the present invention can provide a polymerase chain reaction (hereinafter referred to as "PCR") device implemented as a system, which can detect reactants by real-time scanning of excitation light and corresponding fluorescence of various bands below the PCR plate (200) through a scanning module.

[0082] Figures 2 to 7 It is a schematic diagram for explaining the structure of the temperature control module (300) of the present invention.

[0083] Figure 2 and Figure 3 A perspective view schematically shows a temperature control module of the present invention.

[0084] Reference Figure 2 and Figure 3 It can be seen that the temperature control module (300) has the function of performing constant temperature control on the PCR plate (200), and the PCR plate (200) is used to extract nucleic acid from a biological sample and mix it with a polymerase, and to accommodate and store a PCR (polymerase chain reaction) premix or nucleic acid solution injected from the nucleic acid extraction box (100).

[0085] The temperature control module (300) is equipped with a first heating block (310), which includes a first pressurizing surface (G1) corresponding to the surface of the reaction hole (W) realized on the PCR plate (200), and the temperature is maintained at a set temperature within the range of the temperature required for thermal denaturation (hereinafter referred to as the "first temperature") through a heating unit. At the same time, it is equipped with a second heating block (320), which is spaced apart from the first heating block (310) and arranged at a position corresponding to the first heating block (310), and is provided with a second pressurizing surface (G2) corresponding to the surface of the reaction hole (W), and the temperature is maintained at a set temperature within the range of the temperature required for annealing (hereinafter referred to as the "second temperature") through a heating unit. In particular, the structures of the first heating block (310) and the second heating block (320) can be operated horizontally and moved up and down.

[0086] In one embodiment, the first heating block (310) and the second heating block (320) have a three-dimensional structure and can be equipped with a flat pressure surface on the lower surface, and the first heating block (310) and the second heating block (320) can be spaced apart from each other to ensure that they have temperatures in different temperature ranges.

[0087] like Figure 2 and Figure 3 As shown, the first heating block (310) and the second heating block (320) are arranged opposite to each other in a structure facing each other, and the upper surface can perform the pressurizing function of the flat structure as a whole, and the upper part thereof can adopt a rectangular solid structure. The implementation of the rectangular solid structure is an embodiment, and any three-dimensional shape is included in the gist of the present invention as long as it has a pressurizing surface of a flat structure for pressurization.

[0088] In addition, the first heating block (310) and the second heating block (320) are arranged on the side, and the adjacent surfaces are realized in a structure spaced apart from each other, and can maintain different set temperatures respectively.

[0089] For example, the first temperature of the first heating block (310) can be set within the range of 94-96°C, which is suitable for the thermal denaturation step of separating double-stranded DNA (including DNA extracted from biological samples). In an embodiment of the present invention, it can be maintained at 95°C.

[0090] In addition, the second temperature of the second heating block (320) can be set within the range of 50-65°C, which is the temperature required for the primer annealing step of the primer binding to the separated model DNA. In one embodiment of the present invention, it can be maintained at 55°C.

[0091] The first heating block (310) and the second heating block (320) are not formed by accommodating water or heat transfer fluid inside, but are formed by a metal material body having a large heat capacity and high heat transfer efficiency, and can always maintain a set temperature through an internal heating unit. To this end, the heating unit should be controlled by temperature regulation so that a temperature sensor can be installed inside to maintain a constant temperature.

[0092] That is, when the PCR premix or nucleic acid solution is injected into the PCR plate (200), when the first temperature needs to be applied, the first heating block (310) is translated to be adjacent to the surface of the PCR plate (200). That is, the first pressurizing surface (G) has a flat plate structure, and can heat the entire surface of the PCR plate (200) at the same temperature and the same pressure at the same time, so that a uniform temperature can be transferred to the entire sample.

[0093] Furthermore, when it is necessary to apply the second temperature required for the binding step, the second heating block (320) is translated and positioned above the PCR plate (200), so that the entire surface of the PCR plate (200) can be heated at the same time with the same pressure.

[0094] That is, it does not require additional time to prepare for the set temperature reaction, and can heat the entire surface of the PCR plate (200) at the same pressure and the same temperature through simple horizontal operation, so compared with the existing method of controlling the set temperature, a fast and accurate PCR reaction can be performed.

[0095] In addition, since the first heating block (310) and the second heating block (320) are set to different temperatures, considering that the second heating block (320) can always be heated by the first heating block (310) using radiant heat and conductive heat, a cooling fan unit (340) that can achieve a cooling effect can be provided in the interval space between the two structures.

[0096] Importantly, the second heating block (320) can relatively maintain a second temperature, such as an annealing temperature of 55°C, and can be equipped with a divergent cooling mode on the top, which can minimize the thermal interference of the first heating block (310) and easily dissipate the overflowed heat through the cooling fan unit. For example, in the present invention, the second heating block (320) can also include a temperature adjustment mode portion (321) implemented on the side of the second pressurizing surface (G2). The temperature adjustment mode portion (321) adopts a structure in which multiple protruding modes are configured on the upper part, which can expand the contact surface area with the air, improve the heat dissipation efficiency, and is conducive to maintaining a constant low temperature.

[0097] Different from the method of moving the reaction sample for PCR reaction or moving it to other heating areas by setting a time, the above-mentioned present invention adopts a heating block structure. When the reaction sample is fixed, the uniform temperature can be increased from the upper part as a whole at the same time, and the precise transmission of the first temperature and the second temperature can be achieved.

[0098] In addition, the first heating block (310) or the second heating block (320) in the present invention can be linked with a driving module (330) that performs a horizontal operation or an up-and-down movement operation. The driving module (330) includes a guide member (331, 332) that passes through the first heating block (310) and the second heating block (320), and the first heating block (310) and the second heating block (320) can move up and down along the guide member (331, 332).

[0099] That is, the first heating block (310) and the second heating block (320) in the present invention are arranged at intervals from each other, and cross each other as the driving module (330) operates to achieve up and down movement. In addition, it also includes: elastic members (S1, S2) arranged below the guide members (331, 332), which can provide appropriate elasticity to play a buffering role when the heating blocks (310, 320) pressurize the PCR plate (200) (see Figure 5 ).

[0100] In addition, the polymerase chain reaction device of the embodiment of the present invention further includes: a constant temperature plate (350) linked to the temperature control module (300).

[0101] like Figure 2 and Figure 3 As shown, the constant temperature plate (350) is arranged below the heating block (310, 320) structure constituting the temperature control module (300). After the PCR plate (200) enters, the first heating block (310) or the second heating block (320) of the temperature control module (300) pressurizes the PCR plate (200) through horizontal operation and up and down movement operation, so that the PCR plate (200) can have the same temperature as the first heating block (310) or the second heating block (320).

[0102] To this end, the constant temperature plate (350) further includes: a translation drive module (400), which translates toward the lower part of the translation PCR plate (200).

[0103] To this end, the constant temperature plate (350) further includes: a translation driving module (400), and the translation driving module (400) translates toward the lower part of the PCR plate (200).

[0104] like Figure 2 and Figure 3 As shown, the translation driving module (400) includes: a moving rod (420) and a driving motor unit (410) connected to one end of the constant temperature plate (350), and a conversion plate (430) that converts the rotational force of the driving motor unit (410) into the translational force of the moving rod (420).

[0105] This translation drive module (400) can make the constant temperature plate (350) translate along the lower direction of the temperature control module (300). In particular, the constant temperature plate (350) according to the embodiment of the present invention can be realized by dividing into a first area heated to a first temperature and a second area separated from the first area and heated to a second temperature.

[0106] In the embodiment of the present invention, the temperature control module (300) and the constant temperature plate (350) can be integrated by using the guide components (331, 332) as a medium.

[0107] That is, if driven by the dynamic driving module (400), the constant temperature plate (350) and the temperature control module (300) including the heating blocks (310, 320) move together.

[0108] At this time, the constant temperature plate (350) includes a first area heated to a first temperature and a second area spaced apart from the first area and heated to a second temperature. When configured, the first pressurizing surface (G1) of the first heating block (310) corresponds to the upper part of the first area. At the same time, at the same temperature, the PCR plate (200) is placed between the upper and lower constant temperature plates (350) and the heating blocks (310, 320) to achieve pressurization.

[0109] That is, in an implementation example of the present invention, the constant temperature plate (350) is divided into areas having a first temperature and a second temperature gradient. When the heating block is pressurized by the translation drive module (400), the sliding structure is translated to correspond to the set temperature (first temperature or second temperature) area corresponding to the temperature of the heating block (310, 320). By simultaneously performing contact pressure on the upper and lower surfaces of the PCR plate (200), the efficiency is increased by 2 times compared to the method of maintaining a single temperature of the constant temperature plate (350).

[0110] Figure 4 Observed from the rear Figure 3 Cross-sectional view of the medium temperature control module. Figure 5 This is a cross-sectional view of the temperature control module viewed from the front.

[0111] Therefore, the temperature control module (300) of the present invention is equipped with a driving module (330) for realizing horizontal operation and vertical movement of the first heating block (310) and the second heating block (330), and realizes the automatic operation of these heating modules.

[0112] Reference Figures 2 to 5 It can be seen that the driving module (330) performs an action to move the first heating block (310) and the second heating block (320), and at the same time, the translation driving module (400) translates the first heating block (310) and the second heating block (320), so that the contact part with the surface of the reaction hole (W) on the PCR plate (200) can be changed to the first pressure surface (G1) or the second pressure surface (G2).

[0113] The first heating block (310) and the second heating block (320) are arranged side by side in a mutually isolated state, and are respectively provided with through guide grooves (312, 322; see Figure 2 ) structure, the first heating block (310) and the second heating block (320) are mounted on a guide member that passes through the guide grooves (312, 322). Thus, the first heating block (310) and the second heating block (320) move up and down along with the guide members (331, 332) and can pressurize the PCR plate (200) from the top.

[0114] Of course, in this case, a constant temperature plate (350) is arranged at the lower part of the first heating block (310) and the second heating block (320), corresponding to the first temperature area with the heating blocks and the area with the same temperature as the second temperature, respectively, and the PCR plate can be pressurized from the upper and lower parts.

[0115] As described above, the temperature control module (300) of the present invention can directly apply the first temperature and the second temperature to the entire PCR plate surface for the entire PCR object in the PCR plate (200), thereby ensuring good results in terms of application speed and reaction efficiency.

[0116] In addition, the heating block accessory of the temperature control module (300) must be located at the upper part which can be translated at any time, and is a structure that will only move downward when pressed against the PCR plate (200).

[0117] In order to realize such a structure, a first elastic member (S1) can be included, which is a first elastic auxiliary material (S1) with restoring force. The first elastic member is configured to be inserted into the structure inside the driving frame and always rises upward when not pressed. The present invention also has a second elastic auxiliary material (335), which can transmit stress when contacting the PCR plate (200) to achieve pressurization, thereby preventing the applied pressure from being too large ( Figure 5 ). As shown in the figure, the second elastic auxiliary material (335) is realized by a leaf spring structure, and when pressure is applied to the first and second heating blocks (310, 320) below, a certain buffer force is generated to ensure that excessive pressure is not applied to the surface of the PCR plate (200).

[0118] In addition, in the present invention, the PCR plate (200) has a plate-like structure with reaction holes. At this time, the PCR plate (200) is at a constant temperature. For example, in order to maintain it within the second temperature range (e.g., 55° C.), a constant temperature plate (350) structure is added below.

[0119] This is because, when the temperature control module (300) of the present invention is in close contact with a first heating block at a first temperature (e.g., 95° C.) and the temperature is increased, or when it is in close contact with a second heating block at a second temperature (e.g., 55° C.), the PCR plate (200) must quickly reach the target temperature in order to improve the internal amplification efficiency.

[0120] Therefore, in the example of the present invention, it also includes: a constant temperature plate (350), which is arranged at the lower part of the PCR plate (200) to keep the temperature of the PCR plate (200) at the second temperature.

[0121] It is worth mentioning that although the method of applying a certain temperature by fixedly installing the constant temperature plate (350) structure is well achieved, as mentioned above, the constant temperature plate (350) itself can be divided into areas where the first temperature and the second temperature are applied, so that the constant temperature plate can be implemented as a translational structure.

[0122] Figure 6 Yes Figure 2 A schematic diagram showing that the constant temperature plate (350) in the structure shown is translated toward the lower part of the temperature control module and enters therein through the translation drive module (400), Figure 7 yes Figure 6 Schematic diagram of the constant temperature plate (350) being moved outward to change the temperature zone during operation.

[0123] Right now, Figure 6 In the structure shown, after the first temperature is applied to the first heating block (310), the first area of ​​the constant temperature plate (350) will move together with the first heating block toward the lower part of the PCR plate, so that the first heating block (310) corresponds to the upper surface of the PCR plate (200), and then Figure 7 As shown, when the second area and the second heating block are translated toward the lower part of the translational PCR plate, the second heating block (320) will be translated to correspond to the upper surface of the PCR plate (200).

[0124] The translational movement of the constant temperature plate (350) is achieved by sliding, and can contact and move with a sliding belt that contacts the side of the constant temperature plate (350).

[0125] In addition, references 6 and Figure 7 , which is a conceptual diagram of the bottom surface of the temperature control module (300) according to the present invention. As shown in the figure, the constant temperature plate (350) is configured between the PCR plate (200) and the scanning module (not shown) below: Figure 1 Between the symbol 500), in order to transmit the laser irradiated into the scanning module to the PCR plate (200) and detect the laser, the penetrating part (H) that guides the light of the scanning module (500) is a through structure and is configured in large numbers.

[0126] Therefore, in the present invention, the integrated system equipped with the scanner can realize nucleic acid extraction, PCR process and detection process in one system. In order to be applicable to the diagnosis of various diseases, a separate PCR plate structure can be adopted.

[0127] refer to Figure 8 , Fig.13 It is a schematic diagram of an implementation example of a PCR plate (200) suitable for use in the present invention.

[0128] refer to Figure 8 , Figure 2 and Figure 3 Conceptual diagram, the PCR plate (200) in the present invention includes a main body (210), an inserting part (220), a flow part (230) and a blocking part.

[0129] The main body (210) has one or more reaction holes (W1...Wn). The reaction holes (W) are on the plate surface, and are used to obtain PCR (polymerase chain reaction) premix or nucleic acid solution (hereinafter referred to as "nucleic acid solution") from the nucleic acid extraction box (100), or to receive a PCR mixture dry product including primers, primer / probes or primer probes.

[0130] The insertion part (220) is extended from the main body (210) and inserted into the nucleic acid extraction box (100). The reagent kit has an injection port (h1) for injecting nucleic acid. The insertion part (220) is extended from one end of the main body (210) and is combined with the structure inserted in the nucleic acid extraction box (100).

[0131] In particular, the structure of the injection port (h1) for injecting the nucleic acid solution into the insertion part (220) is provided so as to introduce the nucleic acid solution from the nucleic acid extraction box (100). In addition, the PCR plate (200) is connected to the injection port (h1) and connected to the flow part (230) on the surface of the main body (210), thereby connecting a plurality of reaction wells (W).

[0132] The reaction hole (W) in the present invention is Figure 8 In the structure shown, a structure with 8 reaction holes (W1~W8) is realized. Not only can a structure with more than one reaction hole be realized, but the structure of the reaction hole can also be realized as a recessed structure by processing the surface of the main body (210).

[0133] It is worth mentioning that in the implementation examples of the present invention, Figure 8 As shown, a structure in which a constant reaction hole (W) area is divided in the surface area of ​​the main body (210), and the primers dried in the reaction hole (W) and the nucleic acid solution injected into the nucleic acid extraction box (100) can be dispersed and mixed in the reaction hole (W).

[0134] The circulation part (230) allows the nucleic acid solution to flow from the injection port (h1) to the reaction hole (W). The blocking part (240) is installed on the main body (210) to prevent the nucleic acid solution from flowing back from the reaction hole (W) into the circulation part (230). The blocking part (240) includes an elastically deformable material. The elastically deformable material of the blocking part (240) is composed of rubber, silicone, etc.

[0135] The main body (210) includes a main frame (211), a blocking receiving portion (213), a connecting and circulating portion (215), a storage portion (217), and a flow path guiding portion (219). The main frame (211) has one or more reaction holes (W1...Wn) (W).

[0136] The blocking receiving portion (213) is recessed in the main frame portion (211), and the receiving blocking portion (240) is formed with a recess corresponding to the size of the blocking portion (240) in order to accommodate the blocking receiving portion (213).

[0137] The connecting and circulating part (215) is connected to the blocking and receiving part (213) and the respective reaction wells (W). The nucleic acid solution is supplied to the reaction wells (W) through the connecting and circulating part (215).

[0138] The storage part (217) is recessed in the main body (210), communicated with the circulation part (230), and contains the nucleic acid solution. The storage part (217) contains the supplied nucleic acid solution and stores it to supply the respective reaction wells (W).

[0139] The flow path guide part (219) is connected to the storage part (217) and guides the nucleic acid solution to flow into the blocking part (240).

[0140] The connecting and circulating part (215) has a relatively narrow bottleneck part (215a) formed from the blocking part (240) to the reaction hole (240). The bottleneck part (215a) suddenly forms a narrow structure in the connecting and circulating part (215), and forms a structure in which the part accommodating the blocking part (240) is slightly protruded in the blocking accommodating part (213).

[0141] refer to Fig.10 , downward pressure is applied above the blocking portion (240), and the blocking accommodating portion (213) is inserted, and the width of the bottleneck portion (215a) connected to the circulation portion (215) is narrow.

[0142] refer to Fig.11 When the bottleneck (215a) is sealed and pressurized with a transparent film, the blocking portion (240) is pressurized from four sides of the flow portion (215) to block the flow of the nucleic acid solution. Therefore, due to the weak pressure difference between adjacent reaction holes (W), the flow of the nucleic acid solution is blocked.

[0143] refer to Fig.12 If the nucleic acid solution continues to be injected into the blocking portion (240), the blocking portion (240) is pushed from the storage portion (217) through the flow path guide portion (219), so that the nucleic acid solution flows into each reaction hole (W).

[0144] refer to Fig.13 After the nucleic acid solution flows into each reaction hole (W), when the temperature change of each reaction hole (W) causes the pressure to rise, pressure is applied from the reaction hole (W) to the blocking part (240), and the blocking part (240) can tightly push the flow path guiding part (219), thereby preventing the nucleic acid solution from flowing back from the reaction hole (W) to the flow part (230).

[0145] In addition, the PCR plate (200) is composed of a sealing cover auxiliary material (uncoated) including a plurality of reaction holes (W), and the cover auxiliary material can be made of a thin film material with a transparent material having light transmittance.

[0146] The cover auxiliary material is tightly attached to the surface of the reaction hole (W), so that a cavity is generated inside the reaction hole (W). Thereafter, the PCR (polymerase chain reaction) premix injected into the nucleic acid extraction box (100) and the nucleic acid solution expel the air layer in the cavity and are injected into the reaction hole (W).

[0147] In particular, in the present invention, if Figure 8As shown, when the flow portion (230) connected to the reaction hole (W) in the main body (210) is connected to the injection port (h1), it extends through the main body (210) to the end of the main body (210) and is respectively connected to multiple reaction hole ends in the opposite direction of the insertion portion (220).

[0148] That is, Figure 2 As shown, when the PCR premix and nucleic acid solution are injected into the nucleic acid extraction box (100) through the injection port (h1) below the insertion part, the flow starts from the circulation part (230), flows horizontally (x1), branches to the left and right from the end of the main body (210), and can be connected to the inlet on each reaction hole (W). The reason for forming such flow is that there is a small amount of air layer in the reaction hole (W) area sealed by the cover auxiliary material, and the injected PCR premix and nucleic acid solution are Figure 8 The structure is the same, with the center line (Cx) of the main body as the reference, starting from the upper area, the air layer is pushed upward to the area (Cb) below the main body (210).

[0149] Therefore, relatively speaking, the PCR reaction mixture is placed in the upper area (Ca) of the reaction hole (W), so that the area where the heating block (310, 320) of the present invention performs pressurization and the area where the scanner module performs detection are performed in the upper area (Ca), so that the detection accuracy, the efficiency of the PCR reaction, and the efficiency of temperature control can be improved.

[0150] In addition, the PCR plate (200) of the present invention can be made of a synthetic resin material with high light transmittance. According to the function of the scanner module, it is made of a material with high light transmittance, thereby improving the detection efficiency.

[0151] This material can be applied to a variety of synthetic resin materials such as transparent PP, PE, PPA, PMMA, PC, etc., but is not limited thereto, and can be applied to any material that can ensure a certain degree of light transmittance.

[0152] However, the PCR plate (200) can maintain a constant temperature by applying a pyrogen in the lower thermostatic plate (350). The temperature control module of the PCR (polymerase chain reaction) premix or nucleic acid solution filled in the reaction well (W), the dried primer and the probe at least one of which contains a PCR reactant. In order to ensure its temperature maintenance efficiency, the thickness of the main body (210) should be within the range of 1.0 mm to 3.0 mm. When the thickness is less than 1.0 mm, the high temperature heat set at the first temperature is easily transferred to the lower part of the main body (210), causing thermal interference with the thermostatic plate (350), making it difficult to control the temperature. When the thickness exceeds 3.0 mm, it is easy to control the temperature of the substance contained in the reaction well (W), and the temperature of the thermostatic plate (350) below is difficult to control and maintain a constant temperature.

[0153] That is, in the present invention, Figure 4 and Figure 5 The first heating block (310) and the second heating block (320) are translated, and the first heating surface (G1) or the second heating surface (G2) in contact with the surface of the reaction hole (W) is pressurized by the structure in contact with the upper surface of the covering partition wall type and the covering partition wall type cover auxiliary material, so that the set temperature is set to the first temperature or the second temperature to control the temperature of the reactant.

[0154] In addition, when the PCR plate (200) is subjected to temperature cycling, Figure 2 and Figure 3 In the temperature control module (300) of the structure shown, the first heating block (310) is translated so that it is opposite to the upper surface of the PCR plate (200), and after the lower surface of the PCR plate (200) is translated together with the first heating block (310) in the first area of ​​the constant temperature plate (350), the first heating block (310) moves downward to perform contact pressure. In order to lower the temperature to the second temperature, the upper surface of the PCR plate (200) is translated so that it is opposite to the second heating block (320). On the lower surface of the PCR plate (200), after the second area of ​​the constant temperature plate (350) is translated together with the second heating block (320), the second heating block (320) moves downward and performs contact pressure. This driving mechanism can achieve simultaneous heating and cooling of the upper and lower surfaces of the PCR plate (200). In the present invention, the constant temperature plate (350) and the first and second heating blocks (310, 320) are an integrated translation structure, and pressure on the upper and lower parts of the PCR plate (200) can be achieved at the same temperature at the same time.

[0155] Due to this operation, the upper and lower surfaces of the PCR plate (200) are contacted and pressed at the same time, which can increase the efficiency by 2 times compared with the method of maintaining a single temperature constant temperature plate (350). Therefore, when the heating time of the target plate is extended, it can be carried out at the same time. The advantage is that the inspection time can be reduced by 1 / 2.

[0156] Figures 14 to 19 This is another embodiment of the PCR plate (200) applicable to the present invention.

[0157] refer to Fig.14 , Figure 2 and Figure 3 In the conceptual diagram, another exemplary PCR plate (200) of the present invention includes a main body (210), an inserting part (220), a circulation part (230), and a blocking part (240). The description of the main body (210), the inserting part (220), the circulation part (230), and the blocking part (240) of the PCR plate (200) is replaced by the above description.

[0158] In another example of the PCR plate (200) of the present invention, the blocking accommodating portion (213) is divided into a plurality of partitions (214) formed in the length direction of the storage portion (217). The blocking portion (240) is installed in each of the partitioned blocking accommodating portions (213) in the partition (214).

[0159] refer to Fig.16 A plurality of blocking parts (240) are respectively inserted and installed in each blocking accommodating part (213) from top to bottom. The elastically deformable blocking part (240) is inserted into the blocking accommodating part (213). The elastically deformable blocking part (240) seals the blocking accommodating part (213).

[0160] refer to Fig.17 After the blocking portion (240) is sealed, the flow caused by the pressure difference of each blocking accommodating portion (213) is cut off by the spacer portion (214).

[0161] refer to Fig.18 After the nucleic acid solution is injected, it passes through the storage part (217) through the connecting flow part (215), pushes the spacer part (240), and flows into each reaction hole (W).

[0162] refer to Fig.19 After the nucleic acid solution flows in, the temperature of each reaction hole (W) changes, causing the pressure to rise, and the blocking part (240) is pressurized in the reaction hole (W). The blocking part (240) can be connected to the flow part (215) by tightly pressurizing to cut off the backflow of the nucleic acid solution.

[0163] Figure 20 to Figure 23 It is a drawing that details the structure and operation of this constant temperature plate and translation drive module.

[0164] Fig. 20 Shows Figure 2 and Figure 3 The structure in which the constant temperature plate (350) is installed, Fig. 22 The structure with only the thermostatic plate separated is shown.

[0165] refer to Fig. 20 and Fig.21 In the example of the present invention, the constant temperature plate (350) is arranged at Figure 2 and Figure 3 Under the temperature control module (300) heating block (310, 320) structure shown, after the PCR plate (200) is configured, when the PCR plate (200) is pressurized by the horizontal movement and up and down movement of the first heating block (310) or the second heating block (320) of the upper temperature control module (300), the temperature can be the same as that of the first heating block (310) or the second heating block (320).

[0166] like Fig. 20As shown, the constant temperature plate (350) is provided with a partition (Ss) for dividing the first area (a1) for maintaining the first temperature and the second area (a2) for maintaining the second temperature, and the first area (a1) and the second area (a2) are connected with reference to two sides (a3, a4) of the partition (Ss).

[0167] A connector structure (Ca, Cb) is installed at one end of the constant temperature plate (350) and is capable of transmitting a signal for applying power or controlling the power supply.

[0168] In particular, the translational movement of the constant temperature plate (350) is achieved by sliding, and the plate moves by contacting a sliding belt that contacts the side of the constant temperature plate (350), thereby simplifying the structure and improving mobility.

[0169] At this time, the second area (a2) may also be provided with a light-transmitting portion (H) which can transmit the detection light of the scanning module (500) to scan the concentration of the amplification reactants.

[0170] Temperature sensors (Sa, Sb) are arranged in the first area (a1) and the second area (a2) to measure the temperatures of the corresponding areas, thereby performing control and management.

[0171] Fig.23 Shows Fig. 22 The lower surface is provided with a connector portion (Cc, Cd) for accommodating control signals and power supply, and a temperature sensor (Sa, Sb) is placed therein to achieve constant temperature maintenance of the first temperature and the second temperature.

[0172] Although the temperature maintenance of the first area or the second area of ​​the constant temperature plate (350) can be achieved by installing various heating means, hot wires or heating resistors inside the plate, in a specific implementation example of the present invention, the electrode and temperature sensor circuit are implemented on an epoxy printed circuit, heating paint is sprayed between the electrodes, and then the metal plates of the first area and the second area are bonded to their respective temperature sensors and heating paint to achieve this effect.

[0173] In the present invention, the constant temperature plate (350) further comprises a translation driving module (400) that translates below the PCR plate (200).

[0174] like Figure 2 and Figure 3 As shown, the translation drive module (400) is composed of a moving rod (420) and a driving motor unit (410) connected to one end of the constant temperature plate (350), and a conversion plate (430) that converts the rotational force of the driving motor unit (410) into the translational force of the moving rod (420).

[0175] like Fig.21As shown, the PCR plate (200) is inserted into the nucleic acid extraction box (100), and the nucleic acid solution extracted from the nucleic acid extraction box (100) is injected into the injection port (h1) in combination with the structure of the connecting passage. Subsequently, the extracted nucleic acid solution is injected into one or more reaction wells containing a dried PCR mixture containing at least one side of the primer and the probe, and moved to the accommodating PCR plate (200).

[0176] Subsequently, in order to make the reaction well portion of the PCR plate (200) form the first temperature or the second temperature, the first heating block (310) or the second heating block (320) of the temperature control module (300) of the present invention will be lowered.

[0177] At this time, the translation driving module (400) translates together with the constant temperature plate (350) and the temperature control module (300), and the PCR plate (200) is inserted between the constant temperature plate (350) and the temperature control module (300).

[0178] When the heating block (310, 320) translated by the translation driving module (400) applies pressure to the PCR plate (200), the constant temperature plate (350) having a set temperature (first temperature or second temperature) corresponding to the temperature of the heating block (310, 320) naturally corresponds to the first area or the second area.

[0179] That is, when the first area (a1) of the constant temperature plate (350) moves horizontally toward the bottom of the PCR plate (200), the first heating block ( Figure 2 , 310) are simultaneously translated to be opposite to the upper surface of the PCR plate (200), and then the heating block (310, 320) is lowered to contact the upper surface of the PCR plate (200).

[0180] In addition, when the second area (a2) moves horizontally below the PCR plate (200), the second heating block (eh, 320) moves horizontally at the same time to face the upper surface of the PCR plate (200), and then the heating block (310, 320) descends to contact the upper surface of the PCR plate (200).

[0181] In summary, when the PCR plate (200) is subjected to temperature cycling, in order to raise the temperature to the first temperature, the first heating block (310) is translated so that it is opposite to the upper surface of the PCR plate (200), and after the lower surface of the PCR plate (200) is translated in the first area of ​​the constant temperature plate (350), the first heating block (310) moves downward to achieve contact pressure.

[0182] Furthermore, in order to cool down to the second temperature again, the upper surface of the PCR plate (200) is translated to be opposite to the second heating block (320), and on the lower surface of the PCR plate (200), the second area of ​​the constant temperature plate (350) is translated to the second heating block (320), and then the second heating block (320) is moved downward to perform contact pressure, and the upper surface of the PCR plate (200) is driven to be heated and cooled simultaneously.

[0183] Similarly, by simultaneously contacting and pressing the upper and lower surfaces of the PCR plate (200), the efficiency is increased by 2 times compared to the method of maintaining a single temperature of the constant temperature plate (350).

[0184] Below, we will refer to Figures 24 to 32 It is explained that the PCR plate (200) of the present invention contains a PCR (polymerase chain reaction) premix for nucleic acid extract and a nucleic acid solution nucleic acid extraction box (100).

[0185] Fig.24 It is a three-dimensional conceptual diagram of the nucleic acid extraction box of the present invention, showing the structure of the PCR plate insertion. Fig.25 yes Fig.24 Separated stereogram of Fig.26 yes Fig.25 Schematic diagram of the internal structure of the box cover part (R1) in the structure.

[0186] refer to Figure 24 to Figure 26 According to the present invention, the nucleic acid extraction box (100) is connected to a box cover part (R1) having a plurality of spacer structure accommodating parts (22, 23, 24, 25, 26) for containing a solution required for DNA extraction, and the box cover part (R1) is connected through an insertion structure, and can be composed of a box body part (R2), including a reaction accommodating part (11) for reacting with a sample or cleaning a solution introduced from the accommodating parts (22, 23, 24, 25, 26).

[0187] In this case, a piston (18) is included, through which the PCR premix or nucleic acid solution purified from the reaction container (11) is injected into the injection port (h1) of the PCR plate (200) connected to the structure inserted into the box body (R2).

[0188] The box cover (R1) of the present invention contacts one side of each reaction containing part (11) of the box body (R2), and includes a rubber part (30) containing an elastically deformable material. The rubber part (30) is composed of an elastically deformable material, rubber, and silica gel.

[0189] In the present invention, when the rubber part (30) is assembled on the box cover part (R1) and the box body part (R2), a seal can be formed between the box cover part (R1) and the box body part (R2), and it rises along the wall surface of the reaction containing part (11) of the box body part (R2), thereby preventing the nucleic acid solutions from mixing with each other.

[0190] Nucleic acid extraction kit reference Figures 25 to 32 The operation of the present invention is explained as follows. Fig. 27 yes Fig.24 A transparent stereogram showing the internal structure after combination.

[0191] The bottom surface of the nucleic acid extraction box body (R2) of the present invention is provided with a rotary valve (19) with a passage (19-1) formed inside. The rotary valve can be rotated to connect the spaces of the various receiving parts (11, 13, 14, 15, 16, 17) of the box body (R2) with the passage of the rotary valve (19). After a specific receiving part is connected to the passage, the piston (18) is activated to extract the solution contained in the receiving part and transfer the substance to other receiving parts or a PCR plate (200).

[0192] like Fig.25 and Fig.26 As shown, the lid (R1) is internally formed with various solution receiving parts (22, 23, 24, 25, 26) for containing the solution required for DNA extraction, and the bottom surface of the receiving part is sealed with a film or the like, and the design is easy to be penetrated by the penetration needle of the main body receiving part. In addition, five holes (21-1, 21-2, 27, 28, 29) are formed.

[0193] The first container (22) of the box cover (R1) contains a mixed buffer, the second container (23) contains a first washing buffer, the third container (24) contains a second washing buffer, the fourth container (25) contains a third washing buffer, and the fifth container (26) contains an elution buffer.

[0194] The PCR plate (200) is covered with a plastic film (polyethylene, polypropylene, PET, etc.), and the reaction wells contain a mixture of at least one PCR dry material including dry PCR primers and probes. Figure 8 The structure is the same as described in .

[0195] The application of the nucleic acid extraction kit in the present invention can be carried out in the following order.

[0196] 1. Input biological samples

[0197] The box body (R2), the box cover (R1) and the PCR plate (200) are installed on the automated equipment described later in a combined state, and the biological sample (blood) is put into Fig.25 In the first hole (21-1) shown.

[0198] 2. Nucleic acids flow out of the cells and bind to the droplets.

[0199] like Fig.30 As shown, through the rotation of the rotary valve (19) configured at the bottom of the box body (R2) and the action of the piston (18), the binding buffer in the first container (22) is poured into the reaction container (11), so that the biological sample is mixed with the beads (magnetic beads with a silica gel coating) on ​​the magnetic plate (MT).

[0200] The magnetic plate (MT) used in the present invention is installed at the end of the through-pipe extended from the reaction containing part (11) of the box body (R2). The nucleic acid extracted from the cells containing the biological sample is dissolved on the magnetic plate and performs the function of binding with the dispersed magnetic bead surface.

[0201] At this time, the magnetic beads can be suspended in binding buffer and used instead of the magnetic plate.

[0202] Subsequently, a sensor (ultrasonic processing head) is placed in the sealed second hole (21-2) on the box body (R2) and ultrasonic waves are applied, and the ultrasonic waves are transmitted through the plastic to mix the biological sample, the plate and the binding buffer to homogenize the reaction solution. At this time, the biological tissue contained in the biological sample will also be broken, and nucleic acids will flow out, and the flowing nucleic acids will be bound to the surface of the beads.

[0203] A magnetic rod is placed in the third hole (27) of the cartridge body (R2), the beads are fixed to the wall of the reaction container, and the remaining solution is transferred to the first container by the rotation of the rotary valve and the movement of the piston.

[0204] 3.1 cleaning

[0205] pass Fig. 27 By rotating the rotary valve of the box body (R2) and moving the piston, the first washing buffer in the second container (23) is poured into the reaction container (11) and mixed with the beads bound with the nucleic acid.

[0206] Afterwards, remove Fig.25 The magnetic bar in the third hole (27) applies ultrasonic waves to the sensor in the second hole (21-2) to perform a first cleaning. After the first cleaning, the substances non-specifically bound to the beads except nucleic acid are cleaned.

[0207] The magnetic beads put into the third hole (27) are fixed on the wall surface of the reaction container, and the primary washing liquid is transferred to the second container (23) through the rotation of the rotary valve and the movement of the piston.

[0208] 4.2 cleaning

[0209] pass Fig. 27By rotating the rotary valve of the box body (R2) and moving the piston, the second washing buffer in the third container (24) is poured into the reaction container (11) and mixed with the beads bound to the nucleic acid.

[0210] Afterwards, remove Fig.25 The magnetic rod in the third hole (27) is placed in the sensor of the second hole (21-2), and ultrasonic waves are applied to the sensor to perform two washes. After two washes, the substances non-specifically bound to the beads are washed away except nucleic acids.

[0211] The magnetic bar inserted into the third hole (27) is fixed to the wall of the reaction container, and the secondary washing liquid is transferred to the third container (24) through the rotation of the rotary valve and the movement of the piston.

[0212] 5.3 times cleaning

[0213] Fig. 27 In the box body (R2) shown in the figure, through the rotation of the rotary valve and the movement of the piston, the third washing buffer on the fourth container (25) is put into the reaction container (11) and mixed with the beads bound to the nucleic acid.

[0214] Then, remove Fig.25 The magnetic rod on the third hole (27) is placed, and ultrasonic waves are applied to the sensor in the second hole (21-2) to perform three washes. After three washes, the substances non-specifically bound to the beads are washed away except nucleic acids.

[0215] The magnetic bar inserted into the third hole (27) is fixed to the wall of the reaction container, and the three washing solutions are transferred to the fourth container (25) through the rotation of the rotary valve and the movement of the piston.

[0216] 6. Nucleic acid elution

[0217] pass Fig. 27 By rotating the rotary valve of the box body (R2) and moving the piston, the elution buffer in the fifth container (26) is poured into the reaction container (11) and mixed with the beads bound to the nucleic acid.

[0218] Then, remove Fig.25 The magnetic rod on the third hole (27) is placed in the second hole (21-2), and ultrasonic waves are applied to dissolve the nucleic acid bound to the bead surface in the elution buffer.

[0219] 7. Generate PCR Master Mix

[0220] A magnetic bar is placed in the third hole (27) to fix the beads on the wall of the reaction container. The piston is selected by rotating the rotary valve, and the elution buffer containing the dissolved nucleic acid is placed in the sixth container (17) to mix with the PCR material [a mixture of polymerase, dNTP, etc.] contained in the sixth container to generate a PCR premix. The "PCR premix" used in the present invention is defined as consisting of the above substances.

[0221] 8. Transfer of PCR Plates

[0222] pass Fig. 27 The rotation of the rotary valve of the box body (R2) and the movement of the piston inject the PCR premix and nucleic acid solution generated in the sixth container into the PCR plate (200) and mix them with at least one of the primers and probes on the PCR plate (200). Fig.32 As shown, this injection process relies on the piston (18) to move along the rotary valve flow path (Y) under pressure and be injected into the injection port (h1).

[0223] Thereafter, a heating rod is placed in the fourth hole (29), and the cover plate membrane at the entrance of the PCR reaction plate is pressurized and heated, thereby sealing the PCR reaction plate.

[0224] 9. PCR reaction

[0225] Finally, the PCR plate (200) contains at least nucleic acid extracted from a biological sample, polymerase, dNTP, one of primers and probes, and other buffers.

[0226] Therefore, on the PCR plate (200), pressurized heating is performed by the temperature control module of the present invention, thereby performing a PCR reaction.

[0227] Figure 33 to Figure 36 The overall structure and component layout of the equipment constituting the polymerase chain reaction system of the present invention are shown in FIG.

[0228] The polymerase chain reaction device according to the embodiment of the present invention is equipped with a nucleic acid extraction box assembly including a nucleic acid extraction box (100) and a PCR plate (200).

[0229] like Fig.35 As shown, after the nucleic acid extraction box (100) is installed inside the polymerase chain reaction system of the embodiment of the present invention, the PCR plate (200) can be installed on the side. The reaction hole portion corresponding to the main body of the PCR plate (200) is exposed to the outside, and a temperature control module (300) is arranged above it.

[0230] Fig.34 , Fig.33 It is an enlarged view of the main parts of the present invention. Fig.35 yes Fig.34 Conceptual image of a partial vertical section showing the structure of the depression. Fig.35 yes Fig.34 Conceptual side elevation cutaway view of the structure shown.

[0231] like Figure 34 to Figure 36 As shown, according to the present invention, a PCR premix containing nucleic acid extracted from the inside of a nucleic acid extraction box (100) and a nucleic acid solution are injected into a PCR plate (200) with a reaction hole (W). A first heating block (310) is arranged on the upper part of the PCR plate (200) to form a first heating surface (G1) corresponding to the surface of the reaction hole (W), and the temperature required for thermal denaturation can be maintained by a heating unit. At the same time, a second heating block (320) is arranged, which adopts a structural translation that changes the heating area by translation. Therefore, the PCR premix and nucleic acid solution injected into the reaction hole (W) are directly heated by the heating block to meet the first temperature (95°C) required for thermal denaturation or the second temperature (55°C) required for annealing.

[0232] At the same time, if Fig.35 and Fig.36 As shown, a constant temperature plate (350) is arranged at the lower part of the PCR plate (200) to keep the temperature of the PCR plate (200) at a constant level.

[0233] A scanning module (500) is arranged at the lower part of the constant temperature plate (350), and the light (L) irradiated by the irradiation part (E1) reaches the PCR plate (200) via the light-transmitting part (H) of the constant temperature plate (350), thereby completing the fluorescence detection.

[0234] In the implementation example of the present invention, as described above, when the temperature control module (300) is in close contact with the first heating block (310) of the first temperature (e.g., 95° C.) and the temperature is increased, or when the temperature control module (300) is in close contact with the second heating block (320) of the second temperature (e.g., 55° C.), if the PCR plate (200) is maintained within a certain temperature range, it is easier to control the temperature of the internal amplification reactants. Therefore, maintaining the constant temperature plate (350) at the second temperature will become an important factor in improving the reliability of the reaction. When the temperature of the PCR plate (200) is increased to 95° C., the first constant temperature zone is closely attached to the first temperature block, and the heat conduction speed is increased, so that the temperature of the PCR plate can be quickly increased to 95° C. within 2-3 seconds.

[0235] When performing RT / PCR to detect RNA targets, a PCR premix or PCR plate containing RT-PCR reaction dried products is used. After adjusting the low temperature block to the RT reaction temperature, close to the PCR reaction plate, the RT reaction time is maintained, and the PCR reaction can be performed after the reverse transcription reaction.

[0236] Through the PCR reaction, the presence or absence of amplified nucleic acid or the concentration of amplified nucleic acid can be determined, and this information can be used for diagnosis. At this time, conventional nucleic acid detection methods can be used to confirm the presence or absence of amplified nucleic acid or its concentration.

[0237] For example, DNA intercalating dyes can be used, using the DNA bead intercalation fluorescent dye SYBR green method, using probes with multiple fluorophores and dampeners,

[0238] Reference Figures 37 to 44 It can be seen that in other embodiments of the present invention, the main body (210) of the PCR plate (200) includes a main frame (211), a blocking accommodating portion (213), and a connecting flow path portion (215). The main frame (211) has more than one reaction hole (W1...Wn) (W).

[0239] The blocking accommodating portion (213) is recessed in the main frame portion (211) and corresponding to each reaction hole (W), forming a circular shape to accommodate the circular blocking portion (240). The size of the blocking accommodating portion (23) corresponds to the blocking portion (240) to accommodate the blocking portion (240).

[0240] The connecting flow path part (215) is connected to the blocking accommodating part (213) and each reaction well. The nucleic acid solution can be supplied to each reaction well (W) through the connecting flow path part (215).

[0241] The blocking part (240) contains an elastically deformable material. The blocking part (240) is an elastically deformable material, and is composed of rubber, silicone, etc. When the nucleic acid solution is injected into each reaction hole (W), the blocking part (240) configured on each blocking accommodating part (213) is made of rubber, etc., has a small deadweight, and generates a small resistance to each blocking accommodating part (213).

[0242] Therefore, the nucleic acid solution is injected through one flow path portion (230), and an equal amount of the nucleic acid solution is uniformly injected into each reaction well (W).

[0243] Reference Fig.43 and Fig.44 It can be seen that a hole is formed below each blocking accommodating portion (213), and the hole and the blocking portion (240) are in contact with each other by their own weight. When the pressure of each reaction hole (W) is higher than the pressure of the connecting flow path portion (215), the blocking portion (240) and the hole of the blocking accommodating portion (240) are tightly fitted, realizing the function of a check valve to prevent the nucleic acid solution from flowing back.

[0244] Fig.45Among the reaction wells (W), the test was performed in the outermost reaction wells W1 and W8 among the 8 reaction wells (W1 to W8). During the PCR reaction, in order to confirm whether the nucleic acid solution was mixed between the reaction wells (W), the reaction wells were dried and then PCR was performed.

[0245] The reaction wells (W) were checked at 5, 15, and 25 minutes of PCR, and as time went on, it was found that the nucleic acid solution was mixed with the reaction wells (W). After 15 minutes, it was confirmed that all reaction wells (W1 to W8) were mixed.

[0246] exist Fig.46 In the reaction wells (W), the test was performed at the outermost reaction wells W1 and W8. After the outermost reaction wells were dried, PCR was performed to confirm that the solutions between the reaction wells were not mixed due to the effect of the check valve.

[0247] Reference Figures 37 to 41 It can be seen that holes (21-1, 21-2, 27, 28, 29) are formed on the nucleic acid extraction box (100). A rubber (40) for preventing liquid leakage is installed on each hole (21-1, 21-2, 27, 28, 29) of the nucleic acid extraction box (100). The rubber (40) for preventing liquid leakage is made of a material such as elastic rubber.

[0248] The liquid-leakage-proof rubber (40) can prevent the nucleic acid solution remaining on the nucleic acid extraction box (100) from leaking out through each hole (21-1, 21-2, 27, 28, 29) of the nucleic acid extraction box (100).

[0249] Reference Fig.39 It can be seen that the leak-proof rubber (40) is punctured by the half-moon-shaped punching hole (500), forming a hole through which air can flow.

[0250] Reference Fig.40 It can be seen that when the nucleic acid solution flows in, the pressing clamp of the leak-proof rubber (40) is pressed down and the solution is smoothly sucked in through the holes of the leak-proof rubber (40).

[0251] Reference Fig.41 It can be seen that when the nucleic acid extraction box (100) is discarded, the anti-leakage rubber (40) can seal the nucleic acid extraction box (100) to prevent the nucleic acid solution inside the nucleic acid extraction box from flowing out.

[0252] Reference Fig.44 It can be seen that the thin sheet portion is heat-fused to the reaction hole (W). The thin sheet portion (250) is heat-fused to each reaction hole (W) to maximize the volume of each reaction hole (W), and heat is quickly transferred in the reaction hole (W), so that the reaction of the nucleic acid solution can be quickly carried out.

[0253] The present invention has been described above with reference to an embodiment shown in the accompanying drawings of the present invention. However, this is only an exemplary introduction. Through the above description, a technician with general knowledge in the field of the technology can implement various modifications and equivalent other embodiments. Therefore, the true technical protection scope of the present invention should be determined according to the following claims.

Claims

1. A PCR plate, characterized in that: include: A main body, which is equipped with one or more reaction holes; An insertion portion, which is extended from the main body, inserted into the nucleic acid extraction box, and is equipped with an injection port for injecting nucleic acid solution; a flow path portion that enables the nucleic acid solution to flow from the injection port to the reaction well; A female cut-off portion is mounted on the main body portion to prevent the nucleic acid solution from flowing back from the reaction hole to the flow path portion.

2. The PCR plate according to claim 1, characterized in that: The main body comprises: A main frame portion, which is equipped with one or more reaction holes; A blocking accommodating portion, which is formed concavely on the main frame portion and accommodates the blocking portion; A connecting flow path portion, which connects the blocking accommodating portion and the reaction hole; a storage portion, which is formed concavely on the main frame portion, communicated with the flow path portion, and contains the nucleic acid solution; The flow path guide part is communicated with the storage part and guides the nucleic acid solution to flow toward the blocking part.

3. The PCR plate according to claim 2, characterized in that: The connecting flow path has a bottleneck portion whose width gradually narrows from the blocking portion toward the reaction well.

4. The PCR plate according to claim 2, characterized in that: The blocking accommodating portion is divided into a plurality of parts on a partition wall portion formed along the length direction of the storage portion. A blocking portion is installed on each of the blocking accommodating portions.

5. The PCR plate according to claim 1, characterized in that: The blocking portion is made of a material including a material capable of generating elastic deformation.

6. The PCR plate according to claim 1, characterized in that: The main body comprises: A main frame portion, which is equipped with one or more reaction holes; A blocking accommodating portion is formed by being recessed on the main frame portion and is respectively arranged corresponding to the reaction holes to form a circular structure to accommodate the circular blocking portion; A connecting flow path portion connects the blocking accommodating portion and the reaction hole.

7. The PCR plate according to claim 6, characterized in that: The blocking portion is made of a material including a material capable of generating elastic deformation.

8. The PCR plate according to claim 6, characterized in that: A thin sheet portion is disposed in the reaction well by hot melting.

9. A nucleic acid extraction kit, characterized in that: include: A box cover portion, which is equipped with a receiving portion with multiple partition structures for containing solutions required for DNA extraction; The box body is combined with the box cover through an insertion structure and is equipped with a reaction receiving part, so that the solution introduced from the receiving part reacts with the sample or performs washing.

10. The nucleic acid extraction kit according to claim 9, characterized in that: The box cover portion includes a rubber portion which is joined to a receiving side surface of the box body portion and contains a material capable of generating elastic deformation.

11. The nucleic acid extraction kit according to claim 9, characterized in that: The nucleic acid extraction box is provided with a leak-proof rubber for preventing the nucleic acid solution from leaking, and the leak-proof rubber comprises a material capable of generating elastic deformation.

12. A nucleic acid extraction kit assembly, characterized in that: include: Nucleic acid extraction kit; A PCR plate, which receives the nucleic acid solution from the nucleic acid extraction box and stores the nucleic acid solution on a reaction R plate containing a PCR mixed dried product; The nucleic acid extraction kit comprises: A box cover portion, which is equipped with a plurality of partition structure accommodating portions for containing solutions required for DNA extraction; a box body part, which is combined with the box cover part through an insertion structure and is equipped with a reaction receiving part for reacting the solution introduced from the receiving part with the sample or for washing; The PCR plate comprises: A main body, which is equipped with one or more reaction holes; An insertion portion, which is extended from the main body, inserted into the nucleic acid extraction box, and is equipped with an injection port for injecting the nucleic acid solution; a flow path portion that enables the nucleic acid solution to flow from the injection port to the reaction well; The blocking part is mounted on the main body part and blocks the nucleic acid solution from flowing back from the reaction hole to the flow path part.

13. The nucleic acid extraction box assembly according to claim 12, characterized in that: The box cover part includes a rubber part, which is connected to a side surface of the accommodating part of the box body part and contains a material that can generate elastic deformation.

14. The nucleic acid extraction box assembly according to claim 12, characterized in that: The nucleic acid extraction box is provided with a leak-proof rubber for preventing the nucleic acid solution from leaking, and the leak-proof rubber comprises a material capable of generating elastic deformation.

15. A polymerase chain reaction device, characterized in that: include: Nucleic acid extraction kit; A PCR plate, which receives the nucleic acid solution from the nucleic acid extraction box and stores the nucleic acid solution on a reaction R plate containing a PCR mixed dried product; ， The nucleic acid extraction kit comprises: A box cover portion, which is equipped with a plurality of partition structure accommodating portions for containing solutions required for DNA extraction; a box body part, which is combined with the box cover part through an insertion structure and is equipped with a reaction receiving part for reacting the solution introduced from the receiving part with the sample or for washing; The PCR plate comprises: A main body, which is equipped with one or more reaction holes; An insertion portion, which is extended from the main body, inserted into the nucleic acid extraction box, and is equipped with an injection port for injecting the nucleic acid solution; a flow path portion that enables the nucleic acid solution to flow from the injection port to the reaction well; The blocking part is installed on the main body and blocks the nucleic acid solution from flowing back from the reaction hole to the flow path part.

16. The polymerase chain reaction device according to claim 15, characterized in that: The box cover part includes a rubber part which is joined to a side surface of the box body receiving part and contains a material capable of generating elastic deformation.

17. The polymerase chain reaction device according to claim 15, characterized in that: The nucleic acid extraction box is provided with a leak-proof rubber, which comprises a material capable of producing elastic deformation to prevent leakage of the nucleic acid solution.

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