KR20230058722A - Nucleic acid reaction chamber, nucleic acid reaction method using the same, and sample processing cartridge including the same - Google Patents

Abstract

The present invention relates to a nucleic acid reaction chamber, comprising a flow path including a first flow path and a second flow path, and a body including a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path, , The first flow passage and the second flow passage respectively provide a nucleic acid reaction chamber in communication with the outside. According to the present invention, despite the small volume of the sample solution and the viscosity of the reaction mixture, the sample solution and the reaction mixture can be uniformly mixed and the reliability of the detection result can be improved.

Description

Nucleic acid reaction chamber, nucleic acid reaction method using the same, and sample processing cartridge including the same

The present invention relates to a nucleic acid reaction chamber, a nucleic acid reaction method using the same, and a sample processing cartridge including the same.

As interest in modern people's health increases and life expectancy increases, the importance of nucleic acid-based in vitro molecular diagnosis, such as accurate analysis of pathogens and gene analysis of patients, is increasing, and the demand is increasing.

Nucleic acid-based molecular diagnosis is performed by extracting nucleic acids from a specimen sample and then confirming the presence or absence of a target nucleic acid in the extracted nucleic acids. A sample treatment process of extracting nucleic acids from a sample includes sequentially mixing the sample and various reagents and removing residues other than nucleic acids. Since such a sample handling process requires precise handling of a small amount of solution, most of the samples were performed manually by the experimenter or by liquid handling equipment capable of precise control. Existing liquid handling equipment has problems in that the cost of the device itself is high and that specialized personnel are required. In addition, since it is a system that simultaneously processes a large amount of samples, it takes a long time from sample collection to test result confirmation when the number of samples generated per hour is small, so it is not suitable for use in local hospitals or clinics with a small number of samples to be processed.

The POC system for nucleic acid detection handles extraction and nucleic acid detection from samples in one-step process with one cartridge. The POC system has advantages in local medical settings because it is designed to allow the sample handling process to proceed immediately upon collection of the sample. Such a POC-based extraction process and nucleic acid detection process are performed by sequentially moving samples through a plurality of sample processing chambers and nucleic acid reaction chambers. The movement of the sample or its processed material between the chambers includes a method of forming a flow path between the chambers and controlling it with a valve, and a method of moving the solution between the chambers by a lyotropic means. A cartridge used in a POC system for detecting nucleic acids includes an extraction chamber for performing a sample processing process for extracting nucleic acids as well as a nucleic acid reaction chamber for performing an amplification reaction of the extracted nucleic acids and optical measurement for detecting target nucleic acids.

A nucleic acid amplification reaction known as the polymerase chain reaction (PCR) involves a repeated cycle of denaturation of double-stranded DNA, annealing of an oligonucleotide primer to a DNA template, and extension of the primer by DNA polymerase. (Mullis et al., US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).

The sample solution containing the extracted nucleic acid is mixed with a reaction mixture containing dNTPs to perform an amplification reaction, and the temperature of the mixed solution is changed according to a predetermined sequence to perform an amplification reaction. DNA denaturation is performed at about 95°C, and primer annealing and extension are performed at 55°C to 75°C, which is lower than 95°C. At this time, the temperature and reaction time for each step of denaturation, annealing, and extension should be set differently depending on the sequence of oligos such as primers and probes used for each sample, nucleic acid to be analyzed, and analysis. In this method, the sample solution and reaction mixture are viscous, including various substances such as dNTPs, enzymes, and buffers. If the sample solution and the reaction mixture are not uniformly mixed, it is difficult to detect the target nucleic acid by the amplification reaction. In addition, since the amplification reaction is performed with a small amount of solution, even a slight evaporation of the mixed solution causes a fatal error in the detection result.

Therefore, it is necessary to develop a nucleic acid reaction chamber and a nucleic acid reaction method capable of uniformly mixing the sample solution and the reaction mixture and preventing evaporation of the mixed solution during the amplification reaction.

Against the above background, the present inventors have tried to develop a nucleic acid reaction chamber and a nucleic acid reaction method capable of uniformly mixing a sample solution containing extracted nucleic acid and a reaction mixture for performing a nucleic acid amplification reaction. In addition, efforts have been made to develop a nucleic acid reaction chamber and a nucleic acid reaction method capable of preventing evaporation of a mixed solution during a nucleic acid amplification reaction. As a result, the present inventors include a body portion including a flow path including a first flow path and a second flow path and a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path, and the first flow path A nucleic acid reaction chamber in communication with the outside of the passage and the second passage, respectively, a cartridge for sample analysis including the same, and a nucleic acid reaction method using the same have been developed.

Accordingly, an object of the present invention includes a body portion including a flow path including a first flow path and a second flow path and a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path, The first flow path and the second flow path each provide a nucleic acid reaction chamber communicating with the outside.

Another object of the present invention is to provide a cartridge for sample analysis including the nucleic acid reaction chamber.

In addition, an object of the present invention is to provide a nucleic acid reaction method using the nucleic acid reaction chamber.

Other objects and advantages of the present invention will become more apparent with the following embodiments, claims and drawings.

In order to achieve the above object, the present invention includes a body portion including a flow path including a first flow path and a second flow path and a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path. And, the first flow path and the second flow path each provide a nucleic acid reaction chamber in communication with the outside.

In addition, the present invention provides a cartridge for sample processing including the nucleic acid reaction chamber.

In addition, the present invention is a nucleic acid reaction method using the nucleic acid reaction chamber, including an injection step of injecting a sample solution through the flow path, mixing the reaction mixture present in the nucleic acid reaction chamber and the sample solution to form a mixed solution. It provides a nucleic acid reaction method comprising the steps of forming a liquid layer of a water-immiscible substance on the upper side of the mixed solution, and performing a nucleic acid reaction by adjusting the temperature of the sample area.

According to the present invention, despite the small volume of the sample solution and the viscosity of the reaction mixture, the sample solution and the reaction mixture can be uniformly mixed and the reliability of the detection result can be improved.

In addition, according to the present invention, it is possible to prevent evaporation from occurring in the mixed solution of the sample solution and the reaction mixture due to high temperature during the nucleic acid amplification reaction, thereby improving the accuracy of the detection result.

In addition, according to the present invention, the mixed solution can be easily heated or cooled during the nucleic acid amplification reaction, and the target nucleic acid can be smoothly detected using the fluorescence of the optical module.

1 is a perspective view of a nucleic acid reaction chamber according to an embodiment of the present invention.
2 is a front view of a nucleic acid reaction chamber according to an embodiment of the present invention.
3 is a perspective view of a nucleic acid reaction chamber according to an embodiment of the present invention.
4 is a front view of a nucleic acid reaction chamber according to a nucleic acid reaction step according to an embodiment of the present invention.
5 is a view of a portion of a nucleic acid reaction chamber according to an embodiment of the present invention.
6 is a side view of a cartridge for sample analysis according to an embodiment of the present invention.
7 is a flowchart of a nucleic acid reaction method according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

In adding reference numerals to components of each drawing, it should be noted that the same components have the same numerals as much as possible even if they are displayed on different drawings. In addition, in describing the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description will be omitted.

Also, terms such as first, second, A, B, (a), and (b) may be used in describing the components of the present invention. These terms are only used to distinguish the component from other components, and the nature, order, or order of the corresponding component is not limited by the term. When an element is described as being “connected,” “coupled to,” or “connected” to another element, that element is directly connected or connectable to the other element, but there is another element between the elements. It will be understood that elements may be “connected”, “coupled” or “connected”.

According to one aspect of the present invention, the present invention includes a body portion including a flow path including a first flow path and a second flow path and a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path And, the first flow path and the second flow path each provide a nucleic acid reaction chamber in communication with the outside.

1 is a front view of a nucleic acid reaction chamber according to an embodiment of the present invention.

The term sample herein may include biological samples (eg, cells, tissues, and fluids from biological sources) and non-biological samples (eg, food, water, and soil). The biological sample may be virus, bacteria, tissue, cell, blood (eg whole blood, plasma and serum), lymph, bone marrow fluid, saliva, sputum, swab, aspiration, milk, urine , feces, ocular fluid, semen, brain extract, spinal fluid, joint fluid, thymus fluid, bronchial lavage fluid, ascites fluid, and amniotic fluid. A sample may also include natural and synthetic nucleic acid molecules isolated from biological sources. According to one embodiment of the present invention, the sample may include an additional substance such as water, deionized water, saline solution, pH buffer, acidic solution, or basic solution.

Sample processing refers to a series of processes in which a substance to be analyzed is obtained in a state in which a detection reaction is possible by primarily separating an analyte from the sample. The sample processing may be used in the sense of further including a process of detecting a target analyte from a material capable of a detection reaction. The analyte may be, for example, a nucleic acid. According to one embodiment of the present invention, the sample processing may be a nucleic acid extraction process.

A nucleic acid reaction refers to a series of physical and chemical reactions that generate signals depending on the presence or amount of nucleic acids of a specific sequence in a sample. The nucleic acid reaction may be a reaction including binding of a nucleic acid of a specific sequence in a sample with another nucleic acid or material, or replication, cleavage, or degradation of a nucleic acid of a specific sequence in the sample. The nucleic acid reaction may be a reaction involving a nucleic acid amplification reaction. The nucleic acid amplification reaction may include amplification of a target nucleic acid. The nucleic acid amplification reaction may be a reaction that specifically amplifies a target nucleic acid.

The nucleic acid reaction may be a signal-generating reaction, which is a reaction capable of generating a signal depending on the presence/absence or amount of a target nucleic acid in a sample. This signal-generating reaction may be a genetic analysis process such as PCR, real-time PCR, or microarray.

Various methods are known for generating an optical signal indicative of the presence of a target nucleic acid using a nucleic acid reaction. Representative examples include:

TaqMan ^™ probe method (U.S. Patent No. 5,210,015), molecular beacon method (Tyagi et al., Nature Biotechnology v.14 MARCH 1996), Scorpion method (Whitcombe et al., Nature Biotechnology 17:804-807 (1999)), line Sunrise or Amplifluor method (Nazarenko et al., 2516-2521 Nucleic Acids Research, 25(12):2516-2521 (1997), and U.S. Patent No. 6,117,635), Lux method (U.S. Patent No. 7,537,886) , CPT (Duck P, et al. Biotechniques, 9:142-148 (1990)), LNA method (U.S. Patent No. 6,977,295), Plexor method (Sherrill CB, et al., Journal of the American Chemical Society, 126 :4550-4556 (2004)), Hybeacons ^TM (DJ French, et al., Molecular and Cellular Probes (2001) 13, 363-374 and US Pat. No. 7,348,141), a dual-labeled self-quenching probe (Dual- labeled, self-quenched probe; U.S. Patent No. 5,876,930), hybridization probe (Bernard PS, et al., Clin Chem 2000, 46, 147-148), PTO cleavage and extension (PTOCE) method (WO 2012/096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO2013/115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (PCT/KR2013/012312) and CER method (WO 2011/ 037306).

A target nucleic acid can be amplified in a variety of ways. For example, methods for amplifying a target nucleic acid molecule include the polymerase chain reaction (PCR) and ligase chain reaction (LCR) (U.S. Patent Nos. 4,683,195 and 4,683,202). PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691- 6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996) ) Vuorinen, et al., J. Clin. Microbiol. 2 (1991)), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999); Hatch et al., Genet. Anal. 15(2):35 -40 (1999)) and Q-Beta Replicase (Lizardi et al., BiolTechnology 6:1197 (1988)).

In particular, a nucleic acid amplification reaction can be performed while accompanying a change in temperature. For example, in order to amplify DNA (deoxyribonucleic acid) having a specific nucleotide sequence, the nucleic acid amplification device includes a denaturing step and an annealing step. step), and an extension (or amplification) step.

Referring to FIG. 1 , a nucleic acid reaction chamber 100 according to an embodiment of the present invention includes a body portion 110. The body portion 110 includes a flow path and a sample area 150 . The flow path includes a first flow path 130 and a second flow path 140 . In other words, the body part 110 includes a first flow path 130, a second flow path 140, and a sample area 150, and a nucleic acid reaction of the sample solution is performed in the sample area 150.

First, referring to FIG. 6, an embodiment of the cartridge 600 for sample analysis according to the present invention will be described. The nucleic acid reaction chamber 100 may constitute a part of the cartridge 600 for sample analysis. The port unit 120 of the nucleic acid reaction chamber 100 may be coupled to the cartridge 600 for sample analysis. The cartridge 600 for sample analysis includes a plurality of chambers, and some of the plurality of chambers may be the nucleic acid reaction chamber 100 . A plurality of chambers may be disposed in the longitudinal direction of the cartridge 600 . The plurality of chambers are spaces in which substances necessary for a process of extracting a substance to be detected from a sample are stored and physical and chemical processes for extraction are performed. The substance to be detected may be, for example, a nucleic acid.

The plurality of chambers may include a sample chamber, a holding chamber, and the like. The sample chamber is a chamber for accommodating the collected sample, and the holding chamber is a chamber for storing materials necessary for the sample treatment reaction, such as magnetic particles, shattering solution, washing solution, and elution solution.

The number of chambers included in the cartridge 600 of the present invention may be configured differently depending on the sample processing method. The number of chambers is not particularly limited, but may be, for example, 5, 6, 7, or 8 or more, and may be 20, 15, 14, 13, or 12 or less. In addition, the number of nucleic acid reaction chambers 100 included in the cartridge 600 of the present invention is not particularly limited, but may be, for example, 1 or 2 or more, and may be 5, 10 or 20 or less. .

The cartridge 600 of the present invention is operated by a sample processing device (not shown) and a desired reaction may proceed. The cartridge 600 is accommodated in the sample processing device and may interact with various parts included in the sample processing device, such as a movement module and a heat transfer module.

The solution may be transferred between the chambers by the pipette module and the transfer module of the sample processing device. After the cartridge 600 is accommodated in the sample processing device, the pipette module may transfer the solution while moving between the plurality of chambers by the moving module.

The sample solution containing the extracted nucleic acid is injected into the nucleic acid reaction chamber 100 by the pipette module. That is, the sample solution may be injected into the nucleic acid reaction chamber 100 through the first opening 121 of the first flow path 130 and/or the second opening 122 of the second flow path 140.

Next, look at the nucleic acid reaction chamber 100 of the present invention with reference to FIGS. 1 to 4 and the nucleic acid reaction method 700 of the present invention with reference to FIG. 7 .

The nucleic acid reaction method 700 of the present invention is a nucleic acid reaction method using the nucleic acid reaction chamber 100, and includes an injection step (S710), a mixing step (S720), a covering step (S730) and a nucleic acid reaction step (S740). .

The injection step (S710) is a step of injecting a sample solution through the first flow path 130 and/or the second flow path 140 (see FIG. 4 (A) and (B)), and the injected sample solution is It is located in area 150. The mixing step (S720) is a step of mixing the reaction mixture (R) for nucleic acid reaction and the sample solution to form a mixed solution (see FIG. 4 (B) to (E)), the reaction mixture is a nucleic acid reaction chamber (100 ) can exist in The covering (masking, coating, anti-vaporizing) step is a step of forming a liquid layer of a water-immiscible (hydrophobia, anti-vaporizing) material on the upper side of the mixed solution (Fig. 4 (E) and (F) reference), it is a step for preventing evaporation of the mixed solution in the nucleic acid reaction step. The nucleic acid reaction step (S740) is a step of performing a nucleic acid reaction by adjusting the temperature of the sample area 150. For example, a thermal module of the sample processing device heats or cools the nucleic acid reaction chamber 100 .

According to one embodiment of the nucleic acid reaction chamber 100 of the present invention, the body portion 110 includes a first flow path 130, a second flow path 140, and a sample area 150 as well as a receiving portion 160 and compartments. A unit 112 and the like may be further provided.

According to one embodiment of the present invention, the body portion 110 is configured in a thin plate shape, and the first flow path 130, the second flow path 140, and the sample area 150 may be formed by intaglio on one surface. . The first flow path 130, the second flow path 140, and the sample area 150 may be covered by a barrier layer 310 attached to one surface (see FIG. 3).

The first flow path 130 and the second flow path 140 connect the first opening 121 and the first sample area 151 and connect the second opening 122 and the second sample area 152, respectively. am. The body portion 110 is provided with at least one accommodating portion 160 formed to communicate with the flow path. In other words, the body portion 110 is provided with at least one accommodating portion 160 configured to communicate with at least one of the first flow path 130 and the second flow path 140 . According to one embodiment of the present invention, the body portion 110 is provided with at least one accommodating part 160 communicating with the first flow path 130 and the second flow path 140, and the first flow path 130 has The first accommodating part 161 communicates with and the second accommodating part 162 communicates with the second flow path 140 . The first flow path 130 includes a first auxiliary sample area 131 and the second flow path 140 includes a second auxiliary sample area 141, which is the first sample area 151 and the second auxiliary sample area 141 is located above the second sample area 152.

The sample region 150 is individually connected to the lower portions of the first flow path 130 and the second flow path 140 . That is, the lower portion of the first flow path 130 and the lower portion of the second flow path 140 are connected to the sample region 150, and the lower portion of the first flow path 130 is connected to the first sample region 151. The lower part of the second flow path 140 is connected to the second sample region 152 . The body portion 110 may include a partition wall 111 provided between the first passage 130 and the second passage 140, and the first passage 130 and the second passage 140 may include a partition wall ( 111) can be defined. In addition, the first flow path 130 and the second flow path 140 communicate with the outside so that the sample solution can be injected, and the sample solution is injected into the first flow path 130 and/or the second flow path in the injection step (S710). (140). The sample solution is located in the sample area 150. The first flow path 130 and the second flow path 140 may be inclined upward or vertically from one end to the other so that the injected sample solution may be located in the sample area 150.

According to one embodiment of the present invention, the nucleic acid reaction chamber 100 is provided on the upper side of the body portion 110 and the first opening 121 and the second flow path 140 communicating with the first flow path 130 communicate with each other. The second opening 122 may further include a port portion 120 provided on an upper surface thereof. The body portion 110 and the port portion 120 may be integrally formed. The first flow path 130 and the second flow path 140 may communicate with the outside through the first opening 121 and the second opening 122 provided on the upper surface of the port unit 120 , respectively. Injection of the sample solution may be performed by a pipette module of the sample processing device, and a pipette tip of the pipette module accommodating the sample solution is inserted into the first opening 121 and/or the second opening 122, and the sample solution can be injected. In addition, the sample solution may be mixed with the reaction mixture (R) by injecting or sucking air through the first opening 121 or the second opening 122, and a nozzle for injecting or sucking air is provided through the first opening ( 121) or the second opening 122.

In addition, inside the body portion 110, the first passage 171 connecting the upper portion of the first flow path 130 to the first opening 121 and the second opening 122 connecting the upper portion of the second flow path 140 to the first opening 121 A second passage 172 connecting to may be formed. As described above, the first flow path 130, the second flow path 140, and the sample area 150 may be engraved on one surface of the body portion 110, and the first passage 171 and the second passage 172 Inside the body portion 110 and the port portion 120 , a passage formed on one surface of the body portion 110 and an opening formed on an upper surface of the port portion 120 are connected.

In addition, the port part 120 may include at least one groove 123 recessed along the circumference of the first opening and/or the second opening. That is, on the upper surface of the port part 120, grooves 123 are formed around the first opening 121 and the second opening 122, respectively, or the circumference of the first opening 121 and the second opening ( A groove 123 may be formed in one of the openings 122 . The nucleic acid reaction chamber 100 may be formed of a material having predetermined elasticity (eg, polycarbonate (PC) or polypropylene (PP)). When the pipette tip or nozzle is inserted into the opening of the port part 120, the opening is widened outward by the groove 123, and the pipette tip or nozzle can be closely coupled to the opening by an elastic force.

The sample solution located in the sample area 150 is mixed with the reaction mixture (R) through a mixing step (S720). The reaction mixture (R) means a mixture of substances necessary for a nucleic acid reaction. The reaction mixture (R) may include at least one material from the group consisting of dNTPs, primers, probes, buffers, salts, and DNA polymerases. The mixed solution in which the sample solution and the reaction mixture (R) are mixed is heated or cooled in the nucleic acid reaction step (S740), and the nucleic acid amplification reaction is performed.

According to one embodiment of the present invention, the reaction mixture R may be provided in the sample area 150 . The reaction mixture (R) may be provided in the sample area 150 in a solid state, and is injected in the injection step (S710) and mixed with the sample solution located in the sample area 150. The solid reaction mixture may be lyophilized or freeze-dried to be in the form of pellets or cakes. Freeze-drying of reagents can be performed by methods known in the art, including dehydration by sublimation in a vacuum environment. The solid reaction mixture may include a lyoprotectant such as sugar or polyalcohol for lyophilization. The reaction mixture (R) may be injected through the opening of the first flow path 130 and/or the opening of the second flow path 140, but it is more convenient and quicker to prepare the nucleic acid in advance in the body portion 110. The reaction can be performed, and the amount of the reaction mixture mixed with the sample solution can be more accurately controlled. For example, the first flow path 130, the second flow path 140, and the sample area 150 are formed by being engraved on one surface of the body portion 110 and covered by a barrier layer 310 attached to the engraved surface. In this case, the reaction mixture (R) can be prepared in advance in the inside of the body portion 110 by providing the reaction mixture (R) in the sample area 150 through the engraved surface and attaching the barrier layer 310 . The reaction mixture R may be prepared in the first sample area 151 .

After the sample solution is injected into the sample area 151 where the reaction mixture is located, it is necessary to mix the sample solution in the sample area 151 in order to uniformly mix the reaction mixture with the sample solution. According to one embodiment of the present invention, the mixing of the sample solution and the reaction mixture R may be performed by injecting air or sucking air through the first flow path 130 or the second flow path 140 . That is, according to one embodiment of the present invention, the mixing step (S720) includes an injection step of injecting air through the first flow passage 130 or the second flow passage 140 and a suction step of sucking air. In the mixing step (S720), the injection step and the suction step may be alternately performed at least twice or more each. The injection step and the suction step are performed, and the sample solution located in the sample area 150 is directed from the first flow path 130 toward the second flow path 140 or the second flow path ( 140) moves in the direction toward the first flow path 130, and thus the sample solution and the reaction mixture R are mixed.

According to one embodiment of the present invention, the body portion 110 of the nucleic acid reaction chamber 100 extends the sample area 150 in the longitudinal direction so that the sample solution and the reaction mixture R can be uniformly mixed. It further includes a compartment 112 dividing the first sample area 151 and the second sample area 152, and the bottom of the compartment 112 and the lower surface of the sample area 150 are spaced apart from each other, and the first sample area 151 and the second sample area 152 are connected to each other.

Since the body portion 110 includes the partition portion 112, the sample solution and the reaction mixture R can be uniformly mixed even when the sample solution is small. Despite the viscosity of the reaction mixture (R), the sample solution and the reaction mixture (R) can be uniformly mixed. When the nucleic acid reaction step (S740) is performed on a solution in which the sample solution and the reaction mixture (R) are not uniformly mixed, the nucleic acid amplification reaction is performed smoothly only in an area where the content of some reaction mixture (R) is relatively high. In the region where the content of the remaining reaction mixture (R) is relatively low, no amplification reaction is performed or only a very small number of amplification reactions are performed, and even if the amplification reaction is performed, the target nucleic acid may not be amplified. may cause a decrease in reliability. Since the compartment 112 is provided, non-uniform mixing of the sample solution and the reaction mixture R can be prevented.

The fact that the partition 112 partitions the sample region 150 in the longitudinal direction means that the sample region 150 is partitioned in the longitudinal direction of the first flow path 130 and the second flow path 140 . That is, the sample area 150 is partitioned into a first sample area 151 connected to the first flow path 130 and a second sample area 152 connected to the second flow path 140 by the partition 112 . .

The first sample area 151 and the second sample area 152 are provided to be connected to each other, and the sample solution flows in a direction from the first flow path 130 to the second flow path 140 or from the second flow path 140. It moves in the direction toward the first flow path 130 and is mixed with the reaction mixture R as it moves back and forth between the first sample area 151 and the second sample area 152.

The partition 112 may be formed in a vertical direction, and may be formed from a lower end of the partition wall 111 to a lower side. That is, the barrier rib 111 partitions the first flow path 130 and the second flow path 140, and the partition 112 partitions the first sample area 151 and the second sample area 152. The upper end of the compartment 112 is connected to the partition wall 111 and the lower end is provided to be spaced apart from the lower surface of the sample area 150, and the first sample area 151 and the second sample area 152 are connected.

Also, according to one embodiment of the present invention, the partitioning portion 112 may be formed to partition the first sample area 151 larger than the second sample area 152 . The fact that the first sample area 151 is partitioned larger than the second sample area 152 means that the sample area 150 is not partitioned exactly in half by the partition 112, but one is partitioned larger than the other. means that The fact that the first sample area 151 is larger than the second sample area 152 means that its volume is large. Preferably, the first sample region 151 has an average cross-sectional area larger than that of the second sample region 152 . In other words, the volume ratio of the first sample region 151 and the second sample region 152 is the portion of the first sample region 151 connected to the first flow path 130 and the portion connected to the second sample region 152. The length ratio between the path between the regions and the path between the region of the second sample region 152 connected to the second channel 140 and the region connected to the first sample region 151 may be greater than the length ratio.

As the first sample area 151 is partitioned larger than the second sample area 152, when the sample solution moves back and forth between the first sample area 151 and the second sample area 152, the first sample area ( A faster flow rate is formed in the second sample area 152 than in 151), and the sample solution and the reaction mixture R are mixed quickly and uniformly due to the difference in flow rate. That is, when the sample solution moves from the first sample region 151 to the second sample region 152, a slow flow rate is formed in the first sample region 151, but in the second sample region 152 the first sample region ( When the sample solution moves to 151), the fast flow rate formed in the second sample area 152 generates a strong flow in the first sample area 151, and the sample solution and the reaction mixture R are mixed quickly and uniformly.

According to one embodiment of the present invention, the compartment 112 may be positioned out of the center of the sample area 150 . The compartment 112 may be positioned outside the center of the sample area 150, and one of the first sample area 151 and the second sample area 152 may be partitioned larger than the other. The fact that the compartment 112 is positioned away from the center of the sample area 150 means that the sample area 150 is connected to the first flow path 130 and the second flow path 140. It means that it is located biasedly in a direction toward the flow path 130 or the second flow path 140 . The compartment 112 is positioned adjacent to the inner surface of the sample area 150 and the second sample area 152 may be formed as a narrow passage. Accordingly, the first sample region 151 may be formed as a passage wider than the second sample region 152 as the remaining region.

In addition, since the compartment 112 is positioned away from the center of the sample area 150, target nucleic acid detection using fluorescence of the optical module can be smoothly performed. For example, the sample processing device may include an optical module including a light source and a photodetector to detect target nucleic acids, emit excitation light to a solution that has passed through the nucleic acid reaction step (S740), and detect fluorescence emitted from the nucleic acids. . It is preferable that the partition 112 is positioned away from the center of the sample area 150 to prevent obstruction of the optical path of excitation light and fluorescence.

According to one embodiment of the present invention, the first flow path 130 includes the first auxiliary sample area 131 provided above the first sample area 151, and the second flow path 140 is provided to the second sample area 151. A second auxiliary sample area 141 provided above the area 152 may be included. In the first auxiliary sample region 131 and the second auxiliary sample region 141, the sample solution moving between the first sample region 151 and the second sample region 152 is passed through the first flow path 130 or the second auxiliary sample region 141. 2 to prevent leakage through the opening of the flow path 140.

That is, in the mixing step (S720), the sample solution is supplied to the first sample area 151 and the second sample area 152 by injecting or sucking air into the opening of the first flow path 130 or the second flow path 140. ) to move between When the sample solution in the second sample area 152 moves to the first sample area 151, the amount of the sample solution in the first sample area 151 increases, and a portion of the sample solution flows through the first flow path 130. ) can enter. Conversely, when the sample solution in the first sample area 151 moves to the second sample area 152, the amount of the sample solution in the second sample area 152 increases, and a portion of the sample solution flows into the second flow path. (140). The solution flowing from the first sample area 151 into the first flow path 130 leaks through the opening of the first flow path 130 or the solution flowing from the second sample area 152 into the second flow path 140 leaks through the opening of the first flow path 130. If leaking through the opening of the second flow path 140, the nozzle for injecting air may be contaminated or the sample solution may be lost, causing errors in detection results. The solution introduced from the first sample area 151 into the first flow path 130 is accommodated in the first auxiliary sample area 131 (see (C) of FIG. The solution introduced into the flow path 140 is accommodated in the second auxiliary sample area 141 (see (D) of FIG. 4), and contamination of the nozzle or loss of the sample solution is prevented.

The first sub-sample area 131 and the second sub-sample area 141 are located between a portion of the first flow path 130 connected to the first sample area 151 and the first opening 121 and the second flow path ( 140) is provided between a portion connected to the second sample area 152 and the second opening 122. The first auxiliary sample region 131 may be adjacent to the first sample region 151 and the second auxiliary sample region 141 may be adjacent to the second sample region 152 . The first auxiliary sample area 131 and the second auxiliary sample area 141 are parts of the first flow path 130 and the second flow path 140, respectively, and are part of the first flow path 130 and the second flow path 140. It is formed to have a larger cross-sectional area than the rest and can accommodate the sample solution. For example, the first auxiliary sample area 131 and the second auxiliary sample area 141 may be formed by recessing the partition wall 111 .

According to one embodiment of the present invention, the second auxiliary sample area 141 may have a larger volume than the second sample area 152 . That is, since the first sample area 151 is partitioned larger than the second sample area 152, the volume of the solution moved from the first sample area 151 to the second sample area 152 in the mixing step (S720) is The volume of the solution flowing from the second sample region 152 into the second channel 140 may be greater than the volume of the second sample region 152 . Accordingly, it is preferable that the volume of the second auxiliary sample region 141 is greater than that of the second sample region 152 in order to prevent the solution from leaking through the opening of the second flow path 140 .

Also, according to one embodiment of the present invention, the volume of the second auxiliary sample region 141 may be equal to or greater than that of the first auxiliary sample region 131 . In other words, the volume of the first auxiliary sample region 131 may be equal to or smaller than that of the second auxiliary sample region 141 . Since the first sample area 151 is partitioned larger than the second sample area 152, the amount of sample solution accommodated in the first sample area 151 is greater than the amount of sample solution accommodated in the second sample area 152. . Therefore, when the solution moves between the first sample area 151 and the second sample area 152 in the mixing step (S720), the volume of the solution flowing from the second sample area 152 into the second flow path 140 is greater than the volume of the solution flowing from the first sample region 151 into the first flow path 130, and therefore, the volume of the second auxiliary sample region 141 is equal to or greater than the volume of the first auxiliary sample region 131. Larger is preferred.

After mixing the sample solution and the reaction mixture (R) through the mixing step (S720), a liquid layer of the water-immiscible material (C) is formed on the upper side of the mixed solution through the covering step (S730) (FIG. 4 see (F)). In the nucleic acid reaction step (S740), which is the next step after the covering step (S730), the nucleic acid reaction is performed by adjusting the temperature of the sample area. During the nucleic acid reaction, the mixed solution is heated to a temperature higher than about 95 ° C. Since the evaporation of the mixed solution by the high temperature as described above can cause a fatal error in the detection result, in the covering step (S730), which is a step before the nucleic acid reaction step (S740), the water-immiscible substance (C) By forming a liquid layer of the evaporation of the mixed solution is prevented. This water-immiscible material (C) should not be substantially evaporated even at 110 ° C., should have a lower specific gravity than the mixed solution, and should not affect the nucleic acid reaction. The water-immiscible material (C) may be, for example, paraffin, wax or oil, but is not limited thereto.

The water-immiscible material (C) may be injected into the body portion 110 through the openings of the first flow path 130 and the second flow path 140 after forming a mixed solution of the sample solution and the reaction mixture (R). , Similar to the reaction mixture (R), it is possible to carry out the nucleic acid reaction more conveniently and quickly when the water-immiscible material (C) is prepared in advance inside the body portion 110.

According to one embodiment of the present invention, the body portion 110 is provided with at least one accommodating portion 160 formed to communicate with the flow path. As will be described later in detail, the water-immiscible material (C) may be provided in a solid state in the accommodating portion 160 . The accommodating portion 160 is formed to communicate with the first flow path 130 and the second flow path 140 . One or more accommodating parts 160 are provided, and when one accommodating part 160 is provided, the one accommodating part 160 communicates with both the first flow path 130 and the second flow path 140 . When two or more accommodating parts 160 are provided, one or more accommodating parts communicating with the first passage 130 and one accommodating part communicating with the second passage 140 are respectively provided. The accommodating portion 160 is formed to be recessed inside the body portion 110, and, for example, as shown in the drawing, it is provided on the partition wall 111 and communicates with the flow path by an opening facing the flow path.

As will be described later, the water-immiscible material (C) is provided in a solid state in the accommodation unit 160, the solid water-immiscible material (C) is liquefied and moved to the sample area 150, where the liquefied water-immiscible material (C) It is that the part communicating with the passage of the accommodation part 160 is formed inclined in the direction toward the passage so that all of the accommodation part 160 can be moved from the accommodation part 160 to the sample area 150. desirable. For example, when the first flow path 130 and the second flow path 140 are formed in the vertical direction, the portion where the receiving part 160 communicates with the first flow path 130 and the second flow path 140 is upward. It may be formed inclined downward without a protruding part. Referring to Figure 5, the opening communicating with the passage of the receiving portion 160 is formed between the upper and lower surfaces facing in the vertical direction, and the lower surface is at least flat in the horizontal direction (see reference numeral 512) or lower It may be formed to be inclined (see reference numeral 514).

According to one embodiment of the present invention, the accommodating part 160 includes a first accommodating part 161 communicating with the first passage 130 and a second accommodating part 162 communicating with the second passage 140. can do. The first accommodating part 161 and the second accommodating part 162 may communicate with the first passage 130 and the second passage 140 respectively upstream from the sample area 150 . Here, the upstream refers to a direction from the first flow path 130 and the second flow path 140 toward the sample region 150 . In other words, as shown in the drawing, when the first flow path 130 and the second flow path 140 are formed in the vertical direction, the first accommodating part 161 and the second accommodating part 162 are larger than the sample area 150. It communicates with the first flow path 130 and the second flow path 140 from the upper side, respectively.

The water-immiscible material (C) is provided in the accommodating part 160 . That is, the accommodating portion 160 is formed by intaglio on one surface of the body portion 110, similarly to the first flow path 130, and the water-immiscible material (C) is provided to the accommodating portion 160 through the intaglio surface. do. By attaching the barrier layer 310, the water-immiscible material (C) may be previously positioned in the accommodation part 160. The water-immiscible material (C) provided in the accommodating portion may be prepared in an amount sufficient to cover both the upper side of the mixed solution in the first sample area 151 and the second sample area 152 .

When the water-immiscible material (C) is previously provided in the accommodating unit 160, the water-immiscible material (C) is preferably provided in the accommodating unit 160 in a solid state. That is, the water-immiscible substance (C) in a liquid state may leak from the accommodating unit 160 due to shaking of the nucleic acid reaction chamber 100 or the like. In this case, it flows out through the opening of the flow path, and evaporation occurs because the upper side of the mixed solution cannot be completely covered, or it flows into the sample area 150 and interferes with the mixing of the sample solution and the reaction mixture R in the mixing step S720. Therefore, it is preferable to provide the water-immiscible material (C) in a solid state in the receiving part 160. The shape of the solid water-immiscible substance (C) is not particularly limited, and may be, for example, circular, elliptical, triangular, quadrangular, polygonal or irregular.

The water-immiscible material (C) provided in a solid state in the receiving portion 160 is heated in the covering step (S730) and phase-changed into a liquid state. A thermal module for heating or cooling the mixed solution can heat and liquefy the water-immiscible material (C) in a solid state. The liquefied water-immiscible material (C) is released from the accommodating part 160 and moves to the sample area 150, forming a liquid layer on the upper side of the mixed solution. The water-immiscible substance (C) contained in the first accommodating part 161 forms a liquid layer on the upper side of the mixed solution located in the first sample area 151, and the water-immiscible substance contained in the second accommodating part 162 (C) forms a liquid layer on the upper side of the mixed solution located in the second sample area 152. The water-immiscible material (C) is required to have a low specific gravity so that the liquid phase can be located on the upper side of the mixed solution (for example, the specific gravity of paraffin is 0.9g/cm^3). Since the first accommodating portion 161 and the second accommodating portion 162 communicate with the first flow path 130 and the second flow path 140 upstream of the sample area 150, respectively, the liquefied water-immiscible material (C ) is naturally moved to the sample area 150 and forms a liquid layer without going through a separate device or step.

The heating of the water-immiscible material (C) may be performed separately from the heating of the mixed solution, or may be automatically performed in the process of heating the sample region 150 to perform the denaturation step. That is, the denaturation step is performed at about 95 ° C. When a water-immiscible material (C) with a melting point lower than 95 ° C is used (for example, the melting point of paraffin is 47 to 64 ° C), the thermal module is used as a body part. In the process of heating (110), the solid water-immiscible material (C) is heated together and liquefied, and the liquefied water-immiscible material (C) is moved to the sample area 150 and the mixed solution reaches 95 ° C. It is possible to form a liquid layer on the upper side of the mixed solution before and prevent evaporation.

However, in order to prevent the water-immiscible material (C) provided in the accommodation part 160 in a solid state from escaping to the first flow path 130 or the second flow path 140, the body portion 110 has the accommodation part 160 A structure can be formed to limit the movement of the material located on the . That is, as described above, the part where the receiving part 160 communicates with the flow path is inclined downward so that all of the liquefied water-immiscible material C is moved from the receiving part 160 to the sample area 150. can In this case, a movement limiting structure for preventing the solid water-immiscible material (C) before being liquefied from being separated from the accommodating part 160 needs to be provided in the body part 110. If the solid water-immiscible material (C) is separated from the receiving part 160 before the sample solution is injected, the solid water-immiscible material (C) may block the flow path and prevent the sample solution from being injected into the sample area. .

Referring to FIG. 5, the opening communicating with the passage of the accommodating part 160 is formed between the upper and lower surfaces facing in the vertical direction, and the upper surface protrudes in the direction toward the lower surface and forms a locking jaw. The movement limiting structure may be formed on the body part 110 (refer to reference numeral 511). Alternatively, the locking protrusion may extend in a direction toward the lower surface and form a narrow opening (refer to reference numeral 513). Alternatively, a blocking portion may be provided between the upper and lower surfaces to form the movement limiting structure (refer to reference numeral 515).

After forming the liquid layer of the water-immiscible substance (C) on the upper side of the mixed solution, a nucleic acid reaction step (S740) of performing a nucleic acid reaction by adjusting the temperature of the sample area 150 is performed. Temperature control of the sample area 150 may be performed by, for example, a thermal module of the sample processing device. The thermal module may heat the sample area 150 using a heat conductive film or Peltier element or cool the sample area 150 using a heat sink, heat dissipation fan, or heat sink.

As described above, the channel and the sample region 150 are formed by being engraved on one surface of the body 110, and the barrier layer 310 covering the channel and the sample region 150 is formed on the engraved surface of the body 110. ) may be attached, and temperature control of the sample area 150 may be performed by heat exchange through the barrier layer 310. That is, the barrier layer 310 is formed of a thermally conductive material, and for example, a thermal module may contact the barrier layer 310 to heat or cool the sample region 150 . The barrier layer 310 may be, for example, an aluminum thin film. The barrier layer 310 may be attached only to the engraved surface of the body part 110 as shown in (A) of FIG. 3 or the engraved surface and both side surfaces of the body part 110 as shown in (B) of FIG. can also be attached to As shown in FIG. 6, the both sides face each other when the plurality of nucleic acid reaction chambers 100 are disposed in the longitudinal direction in the sample analysis cartridge 600. When the thermal module controls the temperature of the sample area 150 in the nucleic acid reaction step (S740) by attaching the barrier layer 310 to both sides, heat exchange is performed on both sides as well as the engraved side of the body 110 Heat exchange can be performed more quickly, and heat loss in the heating process can be prevented.

In addition, the body portion 110 may be formed of a transparent or translucent material, which is to more easily detect the target nucleic acid after the nucleic acid is amplified through the nucleic acid reaction step (S740). That is, as described above, the detection of the target nucleic acid can be performed, for example, by the optical module of the sample processing device, and the body portion 110 can detect excitation light emitted by the optical module and emission light emitted from the nucleic acid. ) May be formed of a transparent or translucent material, for example, PC (Polycarbonate) or PP (Polypropene). The optical module may emit excitation light or detect emission light through a side surface opposite to the engraved surface of the body portion 110. Therefore, it is preferable that the barrier layer is not attached to the opposite side surface.

Examples where examples described herein extend to individual elements and concepts described herein, independently of other concepts, ideas or systems, as well as examples including combinations of elements recited elsewhere herein are contemplated. Although examples have been described in detail herein with reference to the accompanying drawings, it will be understood that concepts are not limited to the examples themselves. Accordingly, the scope of the concept is intended to be defined by the following claims and equivalents thereof. It is also contemplated that certain features that are described individually or as part of an example may be combined with other individually described features or portions of other examples even if the other features and examples do not refer to the particular feature. Therefore, the absence of a description of the combination should not preclude the right to such combination.

This application claims priority to Korean Patent Application No. 10-2020-0127008 filed on September 29, 2020, the disclosure of which is incorporated herein by reference.

100: nucleic acid reaction chamber 110: body
111: bulkhead 112: compartment
120: port portion 121: first opening
122: second opening 123: groove
130: first flow path 131: first auxiliary sample area
140: second flow path 141: second auxiliary sample area
150: sample area 151: first sample area
152: second sample area 160: accommodating unit
161: first accommodating part 162: second accommodating part
171 first passage 172 second passage
310: barrier layer 600: cartridge for sample analysis
700: nucleic acid reaction method C: water immiscible substances
R: reaction mixture

Claims (25)

It includes a body portion including a flow path including a first flow path and a second flow path and a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path, the first flow path and the second flow path. Each nucleic acid reaction chamber communicated with the outside. According to claim 1,
The nucleic acid reaction chamber, characterized in that the body portion is provided with at least one receiving portion configured to communicate with the flow path. According to claim 2,
The nucleic acid reaction chamber according to claim 1 , wherein the accommodating part includes a first accommodating part communicating with the first flow path and a second accommodating part communicating with the second flow path. According to claim 3,
The nucleic acid reaction chamber according to claim 1 , wherein the first accommodating part and the second accommodating part communicate with the first flow passage and the second flow passage, respectively, upstream from the sample area. According to claim 2,
The nucleic acid reaction chamber, characterized in that the water-immiscible material is provided in the accommodating part. According to claim 5,
The nucleic acid reaction chamber, characterized in that the water-immiscible substance is provided in the receiving portion in a solid state. According to claim 2,
A nucleic acid reaction chamber, characterized in that a structure for limiting the movement of the material located in the accommodating part is formed in the body part. According to claim 1,
The nucleic acid reaction chamber, characterized in that the reaction mixture for the nucleic acid reaction is provided in the sample area. According to claim 1,
Further comprising a compartment dividing the sample area into a first sample area and a second sample area in the longitudinal direction, wherein a lower end of the compartment and a lower surface of the sample area are spaced apart from each other, and the first sample area and the second sample area are A nucleic acid reaction chamber, characterized in that connected to each other. According to claim 9,
The nucleic acid reaction chamber according to claim 1 , wherein the compartment is configured such that the first sample area is larger than the second sample area. According to claim 9,
The nucleic acid reaction chamber, characterized in that the compartment is located out of the center of the sample area. According to claim 9,
The first flow path includes a first auxiliary sample area provided above the first sample area, and the second flow path includes a second auxiliary sample area provided above the second sample area. A nucleic acid reaction chamber. According to claim 12,
The nucleic acid reaction chamber of claim 1, wherein the second auxiliary sample area has a larger volume than the second sample area. According to claim 12,
The nucleic acid reaction chamber according to claim 1 , wherein the second auxiliary sample area has a volume equal to or greater than that of the first auxiliary sample area. According to claim 1,
The nucleic acid reaction chamber of claim 1, further comprising a port portion provided on an upper side of the body portion and having a first opening communicating with the first flow path and a second opening communicating with the second flow path. According to claim 15,
A nucleic acid reaction chamber, characterized in that a first passage connecting the upper part of the first flow path to the first opening and a second passage connecting the upper part of the second flow path to the second opening are formed inside the body part. According to claim 15,
The nucleic acid reaction chamber further comprises at least one recess formed along the circumference of the opening. According to claim 1,
The nucleic acid reaction chamber of claim 1 , wherein the passage and the sample region are formed by intaglios on one surface of the body, and a barrier layer covering the passage and the sample region is attached to the engraved surface of the body. According to claim 18,
The nucleic acid reaction chamber, characterized in that the barrier layer is composed of a thermally conductive material. According to claim 1,
The nucleic acid reaction chamber, characterized in that the body portion is made of a transparent or translucent material. A cartridge for sample analysis comprising the nucleic acid reaction chamber according to claim 1 . It includes a body portion including a flow path including a first flow path and a second flow path and a sample area, wherein the sample area is individually connected to lower portions of the first flow path and the second flow path, the first flow path and the second flow path. Is a nucleic acid reaction method using a nucleic acid reaction chamber communicated with the outside, respectively,
an injection step of injecting a sample solution through the passage;
a mixing step of mixing the reaction mixture present in the nucleic acid reaction chamber and the sample solution to form a mixed solution;
A covering step of forming a liquid layer of a water-immiscible material on the upper side of the mixed solution; and
a nucleic acid reaction step of performing a nucleic acid reaction by controlling the temperature of the sample area;
Nucleic acid reaction method comprising a. 23. The method of claim 22,
The covering step is
The nucleic acid reaction method characterized in that the liquid phase is formed by heating a water-immiscible material provided in a solid state in at least one receiving part configured to communicate with the flow path inside the body part. 23. The method of claim 22,
In the mixing step,
A nucleic acid reaction method comprising an injection step of injecting air through the first passage or the second passage and a suction step of sucking the air. 25. The method of claim 24,
In the mixing step,
The nucleic acid reaction method, characterized in that the injection step and the suction step are alternately performed at least twice or more each.

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