Reaction tube for controlling sample quantitation, PCR device, and method for controlling sample quantitation

Abstract

According to one embodiment of the present invention, a reaction tube for controlling sample quantitation comprises: a reaction body portion; a reaction channel portion disposed inside the reaction body portion and allowing movement of a sample; a reaction space portion connected to the reaction channel portion and storing a portion of the sample; and a reaction reservoir portion connected to the reaction channel portion and storing a remaining portion of the sample.

Description

Reaction tube for sample quantitative control, PCR device, and sample quantitative control method

[0001] The present invention relates to a reaction tube for sample quantitative control, a PCR device equipped with a reaction tube, and a method for sample quantitative control of a PCR device. More specifically, the invention relates to a reaction tube for sample quantitative control, a PCR device equipped with a reaction tube, and a method for sample quantitative control of a PCR device comprising a reaction body portion, a reaction channel portion located inside the reaction body portion for sample movement, a reaction space portion connected to the reaction channel portion for storing a portion of the sample, and a reaction reservoir portion connected to the reaction channel for storing the remainder of the sample.

[0002] Polymerase Chain Reaction (PCR) is a molecular biological technique that replicates and amplifies desired portions of DNA. This technique allows for the selective amplification of only specific desired DNA fragments within a sample. Because it requires a short amplification time, offers high accuracy, and can measure even small amounts of DNA, it plays an important role in various fields such as molecular biology, medicine, forensic science, and biological classification.

[0003] Meanwhile, RT-PCR (Real-time PCR) has established itself as a universal technology for confirming infectious diseases. However, the technological level of RT-PCR devices as diagnostic equipment has stagnated due to the advancement and optimization of bio-related technologies, while latecomers continue to develop their own equipment. Most RT-PCR devices are operated by specialized testing laboratories, focusing their technological capabilities on processing large volumes of samples simultaneously.

[0004] However, a difficulty in utilizing RT-PCR devices for point-of-care diagnosis is that the extraction and fixation of target DNA or RNA from samples requires the use of separate automated equipment or manual work by skilled personnel, making it difficult for unskilled workers to easily perform in a medical setting. Furthermore, since most automated equipment is designed to process a large number of samples simultaneously, it is difficult to apply in cases where the number of samples to be processed at one time is small, such as in primary care institutions.

[0005] In addition, there is a need to develop a device that can perform the entire process from DNA extraction to real-time PCR in a single step without the intervention of a skilled operator. One such method involves using centrifugal force to transfer samples to PCR reaction tubes, which is being developed as a very stable and reliable method in terms of injection. However, accurately controlling the required amount in the reaction tube is very difficult. Consequently, variations in injection volume are inevitable due to the characteristics of the fluid; these changes affect efficiency and can lead to errors in PCR measurements. In particular, since the dilution effect of samples and reagents caused by over-injection significantly impacts the PCR reaction, a method for absolute quantification is crucial, and research and development are required for technologies to accurately control the sample content related to this.

[0006] The present invention was conceived to solve the above-mentioned problems and aims to provide a reaction tube for sample quantification control, a PCR apparatus equipped with a reaction tube, and a method for sample quantification control of a PCR apparatus, which controls the quantification of a sample to minimize the dilution effect between the sample and the reagent, thereby reducing errors caused by differences in the injection content of the sample in the PCR reaction.

[0007] The present invention aims to provide a reaction tube for sample quantitative control that reduces reaction error between a sample and a reagent by moving and separating an excess sample injected into a separate space formed within the reaction tube, a PCR device equipped with the reaction tube, and a method for sample quantitative control of a PCR device.

[0008] A reaction tube for sample quantitative control according to one embodiment of the present invention comprises: a reaction body portion; a reaction channel portion located inside the reaction body portion for sample movement; a reaction space portion connected to the reaction channel portion for storing a portion of the sample; and a reaction reservoir portion connected to the reaction channel for storing the remainder of the sample.

[0009] The above reaction space section is located below the above reaction reservoir section.

[0010] The reaction channel section comprises a reaction channel connection section into which a sample is injected from the outside to the inside of a reaction body section; a reaction channel introduction section connected to the reaction channel connection section and moving the sample with a cross-sectional area smaller than that of the reaction channel connection section; a reaction channel inclination section formed at an angle with respect to the direction of the reaction introduction section, with one end connected to the reaction channel introduction section and the other end connected to a reaction space section; and a reaction channel branching section, with one end branching from the reaction channel inclination section and the other end connected to the reaction reservoir section.

[0011] The above reaction channel inclination includes a first reaction channel inclination formed to allow the sample to move from the top to the bottom, a second reaction channel inclination connected to the first reaction channel inclination and formed to allow the sample to move from the bottom to the top, and a third reaction channel inclination connected to the second reaction channel inclination and formed to allow the sample to move from the top to the bottom.

[0012] The angle formed by the reaction channel slope and the reaction channel branch is 30 to 150 degrees.

[0013] A PCR device according to one embodiment of the present invention comprises one or more reaction tubes for sample quantification control.

[0014] A sample quantity control method using a reaction tube for sample quantity control according to an embodiment of the present invention comprises: a first step in which a sample is injected into a separation chamber and the separation chamber is rotated so that the sample passes through a separation channel and is injected into a reaction chamber; a second step in which a portion of the sample passes through a reaction channel and is injected into a reaction tube to completely fill the reaction tube, and then the remaining portion of the sample is injected into a reaction reservoir; and a third step in which mineral oil passes through the reaction channel and is injected into the reaction reservoir.

[0015] According to the reaction tube for sample quantitative control, the PCR device equipped with the reaction tube, and the method for sample quantitative control of the PCR device of the present invention, the sample introduced from the interface tube has the effect of increasing the accuracy of the PCR reaction by preventing volume deviation within the reaction tube.

[0016] FIG. 1(a) shows a cross-sectional schematic diagram of a PCR device equipped with a plurality of reaction tubes, and FIG. 1(a) shows a cross-sectional schematic diagram of a rotating PCR device equipped with a plurality of reaction tubes.

[0017] Figure 2(a) shows a schematic cross-sectional view of a reaction tube, and Figure 2(b) shows a schematic cross-sectional view of a reaction tube filled with a sample.

[0018] Figure 3(a) shows a photograph of a reaction tube filled with 20 µl of sample, Figure 3(b) shows a photograph of a reaction tube filled with 25 µl of sample, Figure 3(c) shows a photograph of a reaction tube filled with 32 µl of sample, and Figure 3(d) shows a photograph of a reaction tube filled with 38 µl of sample.

[0019] FIG. 4 shows a schematic cross-sectional view of a reaction tube having a reaction space section and a reaction reservoir section of the present invention.

[0020] FIG. 5 shows a schematic cross-sectional view of a reaction tube having a reaction channel portion of the present invention, wherein the reaction channel inclined portion and the reaction channel branching portion have a certain angle.

[0021] FIG. 6(a) shows a schematic cross-sectional view of a reaction tube filled with a sample of the present invention, and FIG. 6(b) shows a schematic cross-sectional view of a reaction tube filled with a sample of the present invention and mineral oil.

[0022] FIG. 7(a) shows a photograph of reaction tubes having a reaction space section without a reaction reservoir section, with 29.5 µl, 20 µl, 29.7 µl, and 30.8 µl injected; FIG. 7(b) shows a photograph of multiple reaction tubes in which 320 µl of elution buffer is rotated at 2,000 rpm inside a PCR device, 25 µl is filled into the reaction space section, and the excess volume is filled into the reaction reservoir section; FIG. 7(c) shows a photograph of multiple reaction tubes in which mineral oil is injected and rotated at 2,000 rpm to inject mineral oil into the reaction reservoir section.

[0023] Figure 7(b) shows a photograph of a reaction tube filled with 20 µl of the sample of the present invention, Figure 7(c) shows a photograph of a reaction tube filled with 40 µl of the sample of the present invention, and Figure 7(c) shows a photograph of a reaction tube filled with 40 µl of the sample of the present invention and 40 µl of mineral oil.

[0024] FIG. 8 shows a flowchart of a sample quantification control method using a PCR device equipped with a reaction tube of the present invention.

[0025] In the following, a reaction tube for sample quantitative control according to an exemplary embodiment, a PCR device equipped with a reaction tube, and a method for sample quantitative control of the PCR device will be described in detail with reference to the attached drawings.

[0026] FIG. 1(a) shows a cross-sectional schematic diagram of a PCR device equipped with a plurality of reaction tubes (100), FIG. 1(a) shows a cross-sectional schematic diagram of a rotating PCR device equipped with a plurality of reaction tubes (100). As shown in FIG. 1, the initial position of the sample (60) is located in the center of the interface (220) within the separation chamber (200). When the PCR device begins to rotate, the sample (60) moves outward through four separation channels (210) and is injected into four reaction tubes (100) connected to the separation channels (210).

[0027] FIG. 2(a) shows a schematic cross-sectional view of a reaction tube (100), and FIG. 2(b) shows a schematic cross-sectional view of a reaction tube (100) filled with a sample (60). As shown in FIG. 2, the reaction tube (100) includes a reaction body portion (10), a reaction channel portion (50) located inside the reaction body portion (10) for moving the sample (60), and a reaction space portion (20) connected to the reaction channel portion (50) for storing a portion of the sample (60). Here, the sample (60) flows into the reaction tube (100) by the rotation of the PCR device and is stored in the reaction space portion (20).

[0028] FIG. 3(a) shows a photograph of a reaction tube (100) with 20 µl of sample filled in the reaction tube (100), FIG. 3(b) shows a photograph of a reaction tube (100) with 25 µl of sample filled in the reaction tube (100), FIG. 3(c) shows a photograph of a reaction tube (100) with 32 µl of sample filled in the reaction tube (100), and FIG. 3(d) shows a photograph of a reaction tube (100) with 38 µl of sample filled in the reaction tube (100). As shown in FIG. 3, the volume of sample (60) distributed to each channel is difficult to adjust consistently due to the non-uniformity of the fluid. Differences in the volume of sample (60) induced to each channel inevitably occur due to differences in minute surface tension or the characteristics of the fluid moving randomly during injection. In the prechamber responsible for the primary distribution of the interface (220), a sample volume of approximately 20 µl to 40 µl is injected with a difference, and this sample volume is injected as is, causing a volume difference between each tube.

[0029] FIG. 4 shows a schematic cross-sectional view of a reaction tube (100) having a reaction space section (20) and a reaction reservoir section (30) of the present invention. As shown in FIG. 4, a reaction tube (100) for sample quantitative control according to one embodiment of the present invention includes: a reaction body section (10); a reaction channel section (50) located inside the reaction body section (10) for sample movement; a reaction space section (20) connected to the reaction channel section (50) for storing a portion of the sample (60); and a reaction reservoir section (30) connected to the reaction channel for storing the remaining portion of the sample (60).

[0030] It is preferable that the reaction space section (20) be located below the reaction reservoir section (30). After a portion of the sample (60) is filled into the reaction space section (20), the remaining portion of the sample (60) is filled into the reaction reservoir section (30). For example, if a rotational force of 1,000 rpm to 3,000 rpm is maintained, a centrifugal force of 30 G to 60 G is generated, and the sample (60) present in the separation channel (210) is injected into the reaction channel section (50). The above reaction channel section (50) comprises a reaction channel connection section (54) into which a sample (60) is injected from the outside to the inside of the reaction body section (10), a reaction channel introduction section (51) connected to the reaction channel connection section (54) and which moves the sample (60) with a cross-sectional area smaller than that of the reaction channel connection section (54), a reaction channel inclination section (52) formed at an angle with respect to the direction of the reaction introduction section and connected at one end to the reaction channel introduction section (51) and connected at the other end to the reaction space section (20), and a reaction channel branching section (53) which branches off at one end from the reaction channel inclination section (52) and connected at the other end to the reaction reservoir section (30), and a part of the sample (60) is to the reaction space section (20) through the reaction channel connection section (54), the reaction channel introduction section (51), and the reaction channel inclination section (52). The sample (60) is injected, and the remaining portion of the sample (60) is injected into the reaction reservoir (30) through the reaction channel connection (54), the reaction channel introduction (51), the reaction channel gradient (52), and the reaction channel branching (53). For example, when centrifugal force is continuously applied, the sample (60) solution is separated into the reaction space (20) and the reaction reservoir (30). Here, the reaction space (20) is preferably injected with 20 µl to 30 µl, and more preferably with 25 µl.

[0031] FIG. 5 shows a schematic cross-sectional view of a reaction tube (100) having a reaction channel section of the present invention, wherein the reaction channel inclination section (52) and the reaction channel branching section (53) have a certain angle. As shown in FIG. 5, the reaction channel inclination section (52) includes a first reaction channel inclination section (52) formed to allow a sample (60) to move from the top to the bottom in an inclined direction relative to the ground, a second reaction channel inclination section (52) connected to the first reaction channel inclination section (52) and formed to allow the sample (60) to move from the bottom to the top in an inclined direction relative to the ground, and a third reaction channel inclination section (52) connected to the second reaction channel inclination section (52) and formed to allow the sample (60) to move from the top to the bottom in an inclined direction relative to the ground. When the first reaction channel gradient (52) to the third reaction channel gradient (52) are used, the sample (60) has the effect of not flowing back after PCR rotation.

[0032] It is preferable that the angle (θ) formed by the reaction channel slope portion (52) and the reaction channel branch portion (53) be between 30 and 150 degrees. If the angle is less than 30 degrees, it becomes difficult for the remaining portion of the sample (60) to be injected into the reaction reservoir portion (30) even when the PCR device is rotated, and if the angle exceeds 150 degrees, the sample (60) is injected into the reaction reservoir portion (30) before it is filled into the reaction space portion (20), which is not good.

[0033] FIG. 6(a) shows a schematic cross-sectional view of a reaction tube (100) filled with a sample of the present invention, and FIG. 6(b) shows a schematic cross-sectional view of a reaction tube (100) filled with a sample of the present invention and mineral oil. As shown in FIG. 6, after filling the reaction tube (100) with the sample (60), mineral oil (63) is injected into the reaction tube (100). When using the reaction tube (100) equipped with the reaction space section (20) and the reaction reservoir section (30) of the present invention, if the sample (60) is injected in excess into the reaction tube (100), the excess sample (60) is mixed with the reagent rather than the amount of sample (60) entering the reaction tube (100), causing a problem of diluting the concentration of the reagent. Due to this diluted concentration, the reagent that is adjusted to the volume cannot perform a proper PCR reaction in a diluted state, and the PCR efficiency is inhibited.

[0034] Meanwhile, even if the sample (60) is injected in excess into the reaction tube (100), the sample (60) is filled into the reaction space (20), and the remaining sample (60) is injected into the reaction reservoir (30). The sample (60) in the reaction space (20) and the sample (60) in the reaction reservoir (30) are separated using mineral oil (63), so that a certain amount of the sample (60) reacts with the reagent in the reaction space (20). Therefore, even if there is a difference in the amount of sample (60) injected, the same amount of sample (60) and reagent can always react.

[0035] Therefore, by adding mineral oil (63) and applying centrifugal force in the same way, the injection and separation are not completed, so the remaining sample (60) is completely separated and moved, and the rear space of the sample (60) is filled with mineral oil (63), thereby preventing vaporization that may occur during the PCR process.

[0036] FIG. 7(a) shows a photograph of reaction tubes having a reaction space section without a reaction reservoir section, with 29.5 µl, 20 µl, 29.7 µl, and 30.8 µl injected; FIG. 7(b) shows a photograph of multiple reaction tubes in which 320 µl of elution buffer is rotated at 2,000 rpm inside a PCR device, 25 µl is filled into the reaction space section, and the excess volume is filled into the reaction reservoir section; FIG. 7(c) shows a photograph of multiple reaction tubes in which mineral oil is injected and rotated at 2,000 rpm to inject mineral oil into the reaction reservoir section. As shown in FIG. 7, the sample (60) primarily fills the reaction space section (20), and the remaining amount of sample (60) remains in the reaction channel section (50) and the reaction reservoir section (30). However, when mineral oil (63) is added and injected by centrifugal force, the reaction space section (20) and the reaction reservoir section (30) are completely separated. After injecting the sample (60) and mineral oil (63), the device is rotated at 500 rpm for a certain period of time to prevent the mineral oil (63) from evaporating. Therefore, it has the advantage of being able to measure using an accurate sample (60).

[0037] FIG. 8 shows a flowchart of a sample quantitative control method using a PCR device equipped with a reaction tube of the present invention. As shown in FIG. 8, the sample quantitative control method using a reaction tube for sample quantitative control comprises: a first step (S10) in which a sample is injected into a separation chamber and the separation chamber is rotated so that the sample passes through a separation channel and is injected into a reaction chamber; a second step (S20) in which a portion of the sample passes through a reaction channel and is injected into a reaction tube to completely fill the reaction tube, and then the remaining portion of the sample is injected into a reaction reservoir; and a third step (S30) in which mineral oil passes through the reaction channel and is injected into the reaction reservoir.

[0038] Step (S10), in which a sample is injected into the separation chamber of the first step and the separation chamber is rotated so that the sample passes through the separation channel and is injected into the reaction chamber, can be rotated at 1,000 rpm to 3,000 rpm for 10 seconds to 50 seconds to inject the sample into the reaction chamber.

[0039] Step (S20), in which a portion of the sample from the second step is injected into the reaction tube section after passing through the reaction channel section and completely filling the reaction tube section, and the remaining portion of the sample is injected into the reaction reservoir section, wherein a portion of the sample is injected into the reaction space section through the reaction channel connection section, the reaction channel introduction section, and the reaction channel gradient section, and the remaining portion of the sample is injected into the reaction reservoir section through the reaction channel connection section, the reaction channel introduction section, the reaction channel gradient section, and the reaction channel branching section.

[0040] The step (S30) in which the mineral oil of the third step is injected into the reaction reservoir by passing through the reaction channel section can be injected into the reaction chamber by rotating at 1,000 rpm to 3,000 rpm for 10 to 50 seconds. Here, the mineral oil is not injected into the reaction space section, but can be injected into the reaction reservoir section.

Claims

1. Reaction body portion; A reaction channel portion located inside the above-mentioned reaction body portion for sample movement; A reaction space portion connected to the reaction channel portion above, in which a portion of the sample is stored; and A reaction tube for sample quantitative control comprising: a reaction reservoir portion connected to the reaction channel above, in which the remainder of the sample is stored.

2. In Claim 1 The above reaction space section is a reaction tube for sample quantity control located at the bottom of the above reaction reservoir section.

3. In Claim 1 The reaction channel section comprises a reaction channel connection section into which a sample is injected from the outside to the inside of a reaction body section, a reaction channel introduction section connected to the reaction channel connection section and moving the sample with a cross-sectional area smaller than the cross-sectional area of ​​the reaction channel connection section, a reaction channel inclined section formed at an angle with respect to the sample flow direction of the reaction introduction section, one end of which is connected to the reaction channel introduction section and the other end of which is connected to a reaction space section, and a reaction channel branching section, one end of which branches off from the reaction channel inclined section and the other end of which is connected to a reaction reservoir section, for a reaction tube for sample quantitative control.

4. In Claim 3 A reaction tube for sample quantitative control, comprising: a first reaction channel inclined portion formed to allow a sample to move inclinedly from top to bottom relative to the ground; a second reaction channel inclined portion connected to the first reaction channel inclined portion and formed to allow the sample to move inclinedly from bottom to top relative to the ground; and a third reaction channel inclined portion connected to the second reaction channel inclined portion and formed to allow the sample to move inclinedly from top to bottom.

5. In Claim 3 A reaction tube for sample quantitative control having an angle formed by the reaction channel inclined portion and the reaction channel branching portion of 30 to 150 degrees.

6. A PCR device equipped with a reaction tube for sample quantification control of Claim 1.

7. A sample quantitative control method using a reaction tube for sample quantitative control according to Claim 1, and A first step in which a sample is injected into a separation chamber and the separation chamber is rotated so that the sample passes through a separation channel and is injected into a reaction chamber; A second step in which a portion of the above sample passes through a reaction channel and is injected into a reaction tube to completely fill the reaction tube, and then the remaining portion of the above sample is injected into a reaction reservoir; and A method for controlling sample quantification of a PCR device comprising: a third step in which mineral oil is injected into the reaction reservoir portion after passing through the reaction channel portion.

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