Nucleic acid extraction and amplification device

Abstract

The present invention relates to a device and method for extracting a nucleic acid and amplifying the nucleic acid by repeating a PCR process twice and, more specifically, to a device and method for amplifying a nucleic acid by repeating a PCR process twice without contamination of a sample, the device including a cylinder (170) containing a second PCR reaction reagent (180) in a compartment of a nucleic acid extraction cartridge (100).

Description

Nucleic acid extraction and amplification device

[0001] The present invention relates to a device and method for extracting nucleic acids and amplifying them by repeating the PCR process twice, and more specifically, to a device and method capable of amplifying them by repeating the PCR process twice without contamination of a sample by including a cylinder (170) having a secondary PCR reaction reagent (180) provided inside a compartment of a nucleic acid extraction cartridge (100).

[0002] The process of extracting nucleic acids from various biological samples and amplifying them is widely used in diagnostic medicine, molecular biology, and other fields. Among these, point-of-care (POC) diagnostic technology, which enables accurate and rapid diagnosis of a patient's disease regardless of time or location, is attracting attention as a crucial technology for evidence-based precision medicine. Symptom-based point-of-care diagnostics, which rapidly screens for all infectious pathogens responsible for disease symptoms such as cough, diarrhea, high fever, and reproductive abnormalities, identify the causative pathogen, and prescribe optimal antibiotics and treatments, is a core new technology for future precision medicine. Much research is underway to further develop this technology.

[0003] Meanwhile, among the application techniques of Real-time PCR, double polymerase chain reaction (nested Real-time PCR) is a technique that can increase the specificity and sensitivity of PCR amplification, and is usually used when amplifying DNA or RNA with low purity. This method performs continuous PCR using two types of primers. The first PCR is performed using the primer of the target gene, and the second PCR is performed using the second primer attached to the sequence located within the first PCR product.

[0004] In addition, RT (Reverse transcription) Real-time PCR is a method that isolates RNA from a sample, synthesizes cDNA from the isolated RNA through reverse transcription, and then performs Real-time PCR again using the synthesized cDNA as a template, and shows very excellent sensitivity through two PCRs.

[0005] As described above, when going through two PCR steps, it takes a lot of time and money, and furthermore, the PCR reaction is stopped between the first and second PCR, and the tube is opened to mix the second reaction reagents, and then the PCR process is performed again. Therefore, the test procedure is not only cumbersome, but there is a fatal disadvantage that amplified genes, etc. can be contaminated during the process of opening and closing the tube. Therefore, when going through two PCR steps, there is a demand for an economical and highly sensitive diagnostic method that reduces the possibility of sample contamination.

[0006] To solve the problem of frequent false positive results due to cross-contamination caused by the need for complex nucleic acid extraction and amplification processes for POC molecular diagnosis required in the field and the frequent movement of samples, we aim to provide a nucleic acid extraction and amplification device that can prevent sample contamination by performing nucleic acid extraction and two PCR processes in one step.

[0007] In order to achieve the above purpose, the present invention is a nucleic acid extraction and amplification device including a nucleic acid extraction cartridge (100) including a compartment (140) equipped with an elution buffer and a compartment (150) equipped with a cylinder inside, and a nucleic acid amplification reaction unit (300), wherein the compartment (150) equipped with a cylinder includes a passage (160) through which a PCR reaction reagent (180) can move to the lower part of the compartment by the elevation of a cylinder (170) equipped with a PCR reaction reagent (180) inside, and the eluted nucleic acid and the PCR reaction reagent (180) can be moved to the nucleic acid amplification reaction unit (300) by the pressure change due to the elevation of the cylinder (170), and the nucleic acid amplification reaction unit (300) is connected to the outlet (190) of the compartment (140) equipped with an elution buffer and the inlet (195) of the compartment (150) equipped with a cylinder (170), respectively. A nucleic acid extraction and amplification device having a PCR zone (310) is provided.

[0008] In addition, the present invention may additionally include a compartment (110, 120, 130) equipped with a buffer used for nucleic acid extraction.

[0009] Additionally, in the present invention, the buffer may be a lysis buffer and a washing buffer.

[0010] In addition, the nucleic acid extraction and amplification device of the present invention may additionally include a load unit (200), and the load unit (200) is composed of a load support unit (240) in which a magnetic load (210) and a pumping load (230) are combined, and the magnetic load (210) is covered with a strip (220), and the pumping load (230) may be fastened to a cylinder (170) provided inside a compartment of a nucleic acid extraction cartridge (100).

[0011] In addition, the present invention is such that when the cylinder (170) rises, the cylinder bottom surface is positioned between the diameters of the inlet port of the flow path (160), so that the PCR reaction reagent (180) provided inside the cylinder (170) can move to the lower part of the compartment through the flow path (160) and be mixed with the first PCR product (320).

[0012] The present invention provides a method for extracting and amplifying nucleic acids, including a step of: attaching a pumping rod (230) to a cylinder (170) of a nucleic acid extraction cartridge (100) and then raising the cylinder (170), thereby causing an aqueous solution of nucleic acids eluted from a chamber (140) equipped with an elution buffer to be filled into a PCR zone (310) of a nucleic acid amplification reaction unit (300) through an outlet (190) and then performing PCR;

[0013] In addition, the present invention may include a step of further raising a cylinder (170) connected to a pumping rod (230) so that a first PCR product (320) is moved to a compartment (150) equipped with a cylinder (170) through an inlet / outlet (195) by negative pressure generated while simultaneously mixing a second PCR reaction reagent (180) equipped inside the cylinder with the first PCR product (320); a step of lowering the cylinder (170) so that a mixture of the first PCR product (320) and the second PCR reaction reagent (180) is moved to a PCR zone (310) of a nucleic acid amplification reaction unit (300) so that a second PCR is performed.

[0014] Additionally, in the present invention, the second PCR reaction reagent (180) provided inside the cylinder (170) can be mixed with the first PCR product (320) through the passage (160) that is opened by the rise of the cylinder (170).

[0015] In addition, in the present invention, the nucleic acid-eluted aqueous solution comprises the steps of: introducing a sample into a compartment (110) equipped with a lysis buffer to separate the nucleic acid, then introducing a magnetic bead (400) to attach the nucleic acid to the magnetic bead (400), and introducing a magnetic rod (210) covered with a strip (220) to capture the magnetic bead (400) to which the nucleic acid is attached; moving the magnetic rod (210) to a compartment (120, 130) equipped with a washing buffer and washing the magnetic bead (400); moving the magnetic rod (210) to a compartment (140) equipped with an elution buffer and eluting the nucleic acid from the magnetic bead (400) into an aqueous solution; It can be obtained through a step of moving the magnetic rod (210) to a compartment (130) containing a washing buffer and separating the magnetic bead (400).

[0016] According to the present invention, DNA contamination can be prevented by performing nucleic acid extraction and two PCR processes in one step in one system, and an economical nucleic acid extraction and amplification device and method can be provided.

[0017] Figures 1 and 2 illustrate schematic diagrams of a nucleic acid extraction and amplification device according to the present invention.

[0018] Figures 3 to 5 illustrate nucleic acid extraction and amplification steps using the present invention.

[0019] The terminology used in the present invention is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms “comprising,” “having,” “including,” or “having” indicate the presence of a feature, number, step, operation, component, sub-part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, sub-parts, or combinations thereof.

[0020] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0021]

[0022] In one embodiment of the present invention, the present invention is a nucleic acid extraction and amplification device including a nucleic acid extraction cartridge (100) including a compartment (140) equipped with an elution buffer and a compartment (150) equipped with a cylinder inside, and a nucleic acid amplification reaction unit (300), wherein the compartment (150) equipped with the cylinder includes a passage (160) through which a PCR reaction reagent (180) can move to the lower part of the compartment by the elevation of a cylinder (170) equipped with a PCR reaction reagent (180) inside, and the eluted nucleic acid and the PCR reaction reagent (180) can be moved to the nucleic acid amplification reaction unit (300) by the pressure change due to the elevation of the cylinder (170), and the nucleic acid amplification reaction unit (300) is connected to an outlet (190) of the compartment (140) equipped with an elution buffer and an inlet (195) of the compartment (150) equipped with a cylinder (170), respectively. A nucleic acid extraction and amplification device having a PCR zone (310) is provided.

[0023] In another embodiment of the present invention, a compartment (110, 120, 130) equipped with a buffer used for nucleic acid extraction may be additionally included.

[0024] In another embodiment of the present invention, the buffer may be a lysis buffer and a washing buffer.

[0025] In another embodiment of the present invention, two compartments equipped with a dissolution buffer may be provided.

[0026] In the present invention, the dissolution buffer facilitates the extraction of nucleic acids from a sample and their binding to a magnetic material, and may have various compositions depending on the type of nucleic acid to be extracted.

[0027] In another embodiment of the present invention, one compartment equipped with a washing buffer may be provided.

[0028] In another embodiment of the present invention, three compartments equipped with washing buffers may be provided.

[0029] In the present invention, the washing buffer may be 100% ethanol or 70% ethanol.

[0030] In another embodiment according to the present invention, the nucleic acid extraction and amplification device may additionally include a load unit (200), and the load unit (200) is composed of a load support unit (240) in which a magnetic rod (210) and a pumping rod (230) are combined, the magnetic rod (210) is covered with a strip (220), and the pumping rod (230) may be fastened to a cylinder (170) provided inside a compartment of a nucleic acid extraction cartridge (100).

[0031] In the present invention, the load support member (240) may have a bar or a plate shape with a flat surface, but is not limited thereto.

[0032] In the present invention, the empty hole in front of the first compartment (110) can be provided without a partition as a space for positioning the magnetic rod (210) for fastening the integral pumping rod (230).

[0033] In another embodiment according to the present invention, when the cylinder (170) rises, the bottom surface of the cylinder is positioned between the diameters of the inlet port of the flow path (160), so that the PCR reaction reagent (180) provided inside the cylinder (170) can move to the lower part of the compartment through the flow path (160) and be mixed with the first PCR product (320).

[0034] In addition, in an embodiment of the present invention, a method for extracting and amplifying nucleic acids is provided, including a step of performing PCR by filling an aqueous solution in which nucleic acids are eluted from a chamber (140) equipped with an elution buffer through an outlet (190) into a PCR zone (310) of a nucleic acid amplification reaction unit (300) by a negative pressure generated by connecting a pumping rod (230) to a cylinder (170) of a nucleic acid extraction cartridge (100) and then raising the cylinder (170).

[0035] Magnetic beads (400) capable of binding to nucleic acids and eluting the bound nucleic acids can be divided into a liquid transfer method and a bead transfer method depending on the type of process for eluting the nucleic acids bound to the magnetic beads. The bead transfer method used in the present invention typically utilizes cartridges of reaction containers each pre-filled with a dissolution solution, a washing solution, and an elution solution, and is also implemented by sequentially transferring magnetic beads from one container to a container containing another reaction solution using a magnetic rod. This bead transfer method eliminates the need for a process of transferring magnetic beads and a process of dispensing and removing a reaction solution by using a container pre-filled with a reagent, so that the reaction time can be shortened.

[0036] In another embodiment of the present invention, the present invention may include a step of further raising a cylinder (170) connected to a pumping rod (230) to thereby move a first PCR product (320) through an inlet (195) to a compartment (150) equipped with a cylinder (170) by negative pressure, and at the same time, mixing a second PCR reaction reagent (180) equipped inside the cylinder with the first PCR product (320); and a step of lowering the cylinder (170) to move a mixture of the first PCR product (320) and the second PCR reaction reagent (180) to a PCR zone (310) of a nucleic acid amplification reaction unit (300) to thereby perform a second PCR.

[0037] In another embodiment of the present invention, the second PCR reaction reagent (180) provided inside the cylinder (170) can be mixed with the first PCR product (320) through the passage (160) opened by the rise of the cylinder (170).

[0038] In another embodiment of the present invention, a nucleic acid-eluted aqueous solution comprises the steps of: introducing a sample into a compartment (110) equipped with a lysis buffer to separate nucleic acids, then introducing magnetic beads (400) to attach nucleic acids to the magnetic beads (400), and introducing a magnetic rod (210) covered with a strip (220) to capture the magnetic beads (400) to which the nucleic acids are attached; moving the magnetic rod (210) to a compartment (120, 130) equipped with a washing buffer and washing the magnetic beads (400); moving the magnetic rod (210) to a compartment (140) equipped with an elution buffer and eluting the nucleic acids from the magnetic beads (400) into an aqueous solution; It can be obtained through a step of moving the magnetic rod (210) to a compartment (130) containing a washing buffer and separating the magnetic bead (400).

[0039]

[0040] Hereinafter, the present invention will be described in more detail through specific examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0041]

[0042] Example. Nucleic acid extraction and amplification process.

[0043] Example 1. Nucleic acid extraction step

[0044] To perform PCR, a nucleic acid extraction step is required to remove substances that inhibit the PCR reaction from a biological sample and extract pure nucleic acids.

[0045] Accordingly, the present invention goes through a step of isolating nucleic acids by introducing a sample into a first compartment (110) of a nucleic acid extraction cartridge (100) equipped with a lysis buffer capable of isolating nucleic acids from a biological sample.

[0046] Here, the biological sample means all types of samples from which DNA can be extracted, such as the subject's blood, saliva, tissue, etc., and the lysis buffer is a solution that uses a chemical method to lyse cells and elute contaminants such as proteins, RNA, and other substances out of the cells, including DNA, and can be used without limitation in type as long as it is used for general DNA extraction, and preferably includes a solution of urea and Tris-HCl alone or in combination, and in addition to the lysis buffer, a protein hydrolytic enzyme such as proteinase K or RNase can be additionally added.

[0047] After this, a magnetic bead (400) is introduced into the first compartment (110) to attach the nucleic acid separated from the sample to the magnetic bead (400), and a magnetic rod (210) covered with a strip (220) is introduced into the first compartment (110) to capture the magnetic bead (400) to which the nucleic acid is attached.

[0048]

[0049] Example 2. Washing step

[0050] When the magnetic rod (210) on which the magnetic beads (400) with attached nucleic acids are captured is moved to the second compartment (120) equipped with a washing buffer (100% ethanol), and the magnetic rod (210) is removed from the strip (220), the magnetic beads (400) are separated from the strip (220) and washed by the washing buffer.

[0051] After this, the magnetic rod (210) covered with a strip (220) is reinserted into the second compartment (120) to capture the magnetic beads (400) with attached nucleic acids, and then moved to the third compartment (130) equipped with a washing buffer (70% ethanol), and the washing step is repeated, and the magnetic rod (210) covered with a strip (220) is reinserted into the third compartment (130) to capture the magnetic beads (400) with attached nucleic acids.

[0052]

[0053] Example 3. Elution step

[0054] The magnetic rod (210) on which the magnetic beads (400) to which the nucleic acids are attached are captured is moved to the fourth compartment (140) equipped with an elution buffer, the magnetic rod (210) is separated from the strip (220), the strip (220) is removed, and the nucleic acids are eluted from the magnetic beads (400) in an aqueous solution. After about 5 minutes, the magnetic rod covered with the strip is inserted to capture the magnetic beads from which the nucleic acids have been eluted, and the magnetic rod is moved to the third compartment, and the magnetic rod is removed from the strip to separate the magnetic beads.

[0055]

[0056] Example 4. First and second PCR steps

[0057] After the pumping rod (230) is connected to the cylinder (170) provided in the fifth compartment (150), the cylinder (170) is raised, and the resulting negative pressure causes the aqueous solution in which the nucleic acid in the fourth compartment (140) is eluted to fill the PCR zone (310) of the nucleic acid amplification reaction unit (300) through the outlet (190), thereby performing the first PCR.

[0058] After this, the pumping rod (230) connected to the cylinder (170) located in the fifth compartment (150) is further raised, and the first PCR product (320) is moved to the fifth compartment (150) through the inlet / outlet (195) by the negative pressure generated, and at the same time, the second PCR reaction reagent (180) provided inside the cylinder (170) is mixed with the first PCR product (320) through the passage (160) opened by the raising of the cylinder (170), and thereafter, the cylinder (170) located in the fifth compartment (150) is lowered, and the mixture of the first PCR product (320) and the second PCR reaction reagent (180) is moved to the PCR zone (310) of the nucleic acid amplification reaction unit (300), and the second PCR is performed to amplify the nucleic acid.

[0059]

[0060] As described above, the best practice embodiments have been disclosed in the drawings and specifications. While specific terminology has been used herein, it is solely for the purpose of describing the present invention and is not intended to limit the scope of the invention as defined in the claims. Therefore, those skilled in the art will understand that various modifications and equivalent embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

[0061]

[0062] [Explanation of symbols]

[0063] 100 nucleic acid extraction cartridges

[0064] 105 compartment top plate

[0065] 110 First compartment

[0066] 120 Second compartment

[0067] 130 Third compartment

[0068] 140 4th compartment

[0069] 150 Fifth compartment

[0070] 160 euros

[0071] 165 air inlet

[0072] 170 cylinders

[0073] 172 cylinder top plate

[0074] 174 cylinder lower plate

[0075] 176 Cylinder joint top plate

[0076] 178 Cylinder joint lower plate

[0077] 180 Secondary PCR Reagents

[0078] 190 outlet

[0079] 195 Inlet / Outlet

[0080] 200 load section

[0081] 210 Magnetic Rod

[0082] 220 strips

[0083] 230 pumping rod

[0084] 240 Road Support Unit

[0085] 300 nucleic acid amplification reaction section

[0086] 310 PCR zone

[0087] 320 1st PCR product

[0088] 400 magnetic beads

Claims

1. A step in which a first PCR is performed after an aqueous solution containing nucleic acids eluted from a compartment equipped with an elution buffer is filled into the PCR zone of the nucleic acid amplification reaction section through an outlet by the negative pressure generated by attaching a pumping rod to the cylinder of a nucleic acid extraction cartridge and raising the cylinder; A step in which the first PCR product is moved into the compartment equipped with the cylinder through the inlet and outlet by the negative pressure generated by further raising the cylinder until the upper plate of the compartment is located between the upper and lower plates of the cylinder fastening portion, and at the same time, the second PCR reaction reagent equipped inside the cylinder is mixed with the first PCR product through the passage opened by the further raising of the cylinder; A method for extracting and amplifying nucleic acids, comprising: a step of lowering a cylinder to move a mixture of a first PCR product and a second PCR reaction reagent to a PCR zone of a nucleic acid amplification reaction section to perform a second PCR; A nucleic acid extraction and amplification method that can quickly and simply perform two PCR processes inside a nucleic acid extraction and amplification device by raising the pumping load twice and then lowering it once, and can prevent contamination due to external exposure of the sample.

2. In paragraph 1, the aqueous solution from which the nucleic acid is eluted is A step of introducing a sample into a chamber equipped with a lysis buffer to separate nucleic acids, then introducing magnetic beads to attach nucleic acids to the magnetic beads, and then introducing a magnetic rod covered with a strip to capture the magnetic beads to which nucleic acids are attached; A step of moving the magnetic rod to a chamber equipped with a washing buffer and washing the magnetic beads; A step of moving the magnetic load to a chamber equipped with an elution buffer and eluting nucleic acids from the magnetic beads into an aqueous solution; A method for extracting and amplifying nucleic acids, characterized in that it is obtained through the step of moving the magnetic rod to a compartment containing a washing buffer and separating magnetic beads.

3. A nucleic acid extraction and amplification device including a nucleic acid extraction cartridge and a nucleic acid amplification reaction unit, the nucleic acid extraction and amplification device including a compartment equipped with an elution buffer and a compartment equipped with a cylinder inside, The above nucleic acid amplification reaction section is connected to the outlet of the compartment equipped with the elution buffer and the inlet of the compartment equipped with the cylinder, and includes a PCR zone. A nucleic acid extraction and amplification device characterized in that a compartment having a cylinder inside is opened when the upper plate of the compartment is positioned between the upper plate and the lower plate of the cylinder fastening part due to the rise of the cylinder, and the PCR reaction reagent provided inside the cylinder moves to the lower part of the compartment through the passage and is then mixed with the first PCR product. A nucleic acid extraction and amplification device that can quickly and simply perform two PCR processes inside the device by raising the pumping load twice and then lowering it once, and can prevent contamination due to external exposure of the sample.

4. A nucleic acid extraction and amplification device characterized in that it further includes a compartment equipped with a buffer used for nucleic acid extraction in the third paragraph.

5. A nucleic acid extraction and amplification device characterized in that, in paragraph 4, the buffer is a lysis buffer and a washing buffer.

6. In paragraph 3, The nucleic acid extraction and amplification device may additionally include a loading unit, The above load section is composed of a load support section in which a magnetic load and a pumping load are combined, The above magnetic load is covered with a strip, A nucleic acid extraction and amplification device characterized in that the above pumping rod can be fastened to a cylinder provided inside a compartment of a nucleic acid extraction cartridge.

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