Method for detecting target gene using preset probe

Abstract

Disclosed is a method for detecting a target gene using a preset probe. According to one aspect of the present embodiment, provided is a method for detecting a target gene, the method being characterized by comprising: a mixing process of mixing a test subject including a target gene, a magnetic bead solution, and a lysis solution; a first removal process of washing the solution, mixed in the mixing process, with a preset first buffer solution, and then removing a supernatant; an addition process of adding a preset probe into the mixed solution; a second removal process of washing, with the preset first buffer solution, the solution that has undergone the addition process, and then removing a supernatant; a third removal process of washing, with a preset second buffer solution, and then removing a supernatant; and a detection process of eluting and detecting the target gene.

Description

Method for detecting target genes using preset probes

[0001] This embodiment relates to a method for detecting a target gene relatively simply and quickly using a preset probe.

[0002]

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This patent application is the result of research conducted with the support of the Small and Medium Business Technology Information Promotion Agency (SME Information Promotion Agency) funded by the Korean government (Ministry of SMEs and Startups) in 2024 (Project ID: 2420015149, Subproject ID: RS-2024-00508795, Project Name: Startup Growth Technology Development Project (TIPS), Project Name: Development of a Molecular Diagnostic Platform for Rapid On-Site Diagnosis Based on an Ultra-sensitive Single-Photon Detector).

[0005]

[0006] In addition, if this patent application claims priority under 35 USC § 119(a) to Korean Patent Application No. 10-2024-0093619, filed in Korea on July 16, 2024, the entire contents of which are incorporated by reference herein. Furthermore, if this patent application claims priority in countries other than the United States for the same reasons, the entire contents of which are incorporated by reference herein.

[0007] The content described in this section merely provides background information for the present embodiment and does not constitute prior art.

[0008] In the past, the following methods were used to test whether a target gene was contained in a test subject such as a cell or phage.

[0009] The tester extracts genes from the test subject and purifies the extracted genes to obtain pure genes (pure DNA or pure RNA) free of foreign substances.

[0010] Afterwards, the inspector injects a probe that complementarily binds to the target gene into the obtained pure genome, allowing the two to combine, and then amplifies the result through processes such as PCR. Previously, inspectors tested the presence of the target gene by subjecting the amplified genes to processes such as electrophoresis.

[0011] However, conventional detection methods have been problematic in that only a relatively small amount of binding occurs between the probe and the pure gene, which has undergone extraction and purification. Consequently, conventional detection methods require an amplification process to detect the target gene.

[0012] However, since the conventional detection method necessarily requires an amplification process, it has the inconvenience of taking a relatively long time, and even if the concentration of the test subject or the target gene within the test subject is (relatively) quite high, there is the inconvenience of having to go through the amplification process.

[0013] One embodiment of the present invention aims to provide a method for detecting a target gene relatively simply and quickly using a preset probe.

[0014] According to one aspect of the present embodiment, a method for detecting a target gene is provided, characterized in that it includes a mixing process of mixing a test subject including a target gene, a magnetic bead solution, and a dissolution solution, a first removal process of washing the mixed solution in the mixing process with a preset first buffer, and then removing the supernatant, an addition process of adding a preset probe to the mixed solution, a second removal process of washing the solution that has undergone the addition process with the preset first buffer, and then removing the supernatant, a third removal process of washing with the preset second buffer, and then removing the supernatant, and a detection process of eluting and detecting the target gene.

[0015] According to one aspect of the present embodiment, the mixing process is characterized in that the test subject, the magnetic bead solution, and the dissolution solution are loaded into a container and then mixed.

[0016] According to one aspect of the present embodiment, the mixing process is characterized in that the reaction is carried out by leaving the container at room temperature for several minutes.

[0017] According to one aspect of the present embodiment, the preset probe is characterized in that it includes a backbone structure implemented as a PNA.

[0018] According to one aspect of the present embodiment, the backbone structure is characterized by having a base sequence complementary to the target gene.

[0019] According to one aspect of the present embodiment, the preset probe is characterized in that it is neutral.

[0020] According to one aspect of the present embodiment, the preset probe is characterized in that it is formed by combining a fluorescent material with a backbone structure.

[0021] According to one aspect of the present embodiment, the fluorescent material is characterized in that it is implemented as an inorganic fluorescent material, an organic fluorescent material, or a quantum dot.

[0022] According to one aspect of the present embodiment, the preset probe is characterized in that it is added at a preset concentration.

[0023] According to one aspect of the present embodiment, the target gene detection method is characterized in that it further includes a drying process for drying the magnetic beads that have undergone the third removal process or the remaining components combined therewith.

[0024] As described above, according to one aspect of the present embodiment, there is an advantage in that a target gene can be detected relatively easily and quickly using a preset probe.

[0025] Figure 1 is a flowchart illustrating a target gene detection method according to one embodiment of the present invention.

[0026] Figures 2 and 3 are drawings illustrating the configuration and structure of a preset probe according to one embodiment of the present invention.

[0027] Figures 4 to 9 are diagrams illustrating a target gene detection process according to one embodiment of the present invention.

[0028] Figures 10 and 11 are drawings comparing the detection results of a conventional detection method and a detection method according to an embodiment of the present invention.

[0029] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated and described in detail in the drawings. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. Throughout the description of each drawing, similar reference numerals have been used to designate similar components.

[0030] Terms such as first, second, A, and B may be used to describe various components, but these components should not be limited by these terms. These terms are used solely to distinguish one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. The term "and / or" includes a combination of multiple related items described herein or any of multiple related items described herein.

[0031] When a component is referred to as being "connected" or "connected" to another component, it should be understood that it may be directly connected or connected to that other component, but that there may be other components intervening. Conversely, when a component is referred to as being "directly connected" or "connected" to another component, it should be understood that there are no other components intervening.

[0032] The terminology used in this application is solely for the purpose of describing specific embodiments and is not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. It should be understood that terms such as "comprise" or "have" in this application do not preclude the presence or possibility of addition of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification.

[0033] 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 the present invention belongs.

[0034] Terms defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless expressly defined in this application.

[0035] In addition, each configuration, process, procedure or method included in each embodiment of the present invention may be shared within a scope that is not technically inconsistent with each other.

[0036] FIG. 1 is a flowchart illustrating a target gene detection method according to one embodiment of the present invention, FIGS. 2 and 3 are drawings illustrating the configuration and structure of a preset probe according to one embodiment of the present invention, and FIGS. 4 to 9 are drawings illustrating a target gene detection process according to one embodiment of the present invention.

[0037] A method for detecting a target gene according to one embodiment of the present invention detects whether a target gene is present in a test subject. The method according to one embodiment of the present invention can minimize loss of the target gene by minimizing unnecessary purification processes. Accordingly, the method according to one embodiment of the present invention can detect the target gene using a device such as a spectrophotometer without an amplification process such as PCR. Furthermore, when the concentration of the test subject or target gene is relatively high (above a preset reference value), the presence or absence of the target gene can be detected with the naked eye.

[0038] Additionally, a method according to one embodiment of the present invention utilizes a preset probe to detect a target gene. By utilizing a preset probe rather than a probe implemented with DNA or RNA, as is typically the case, target genes can be detected more easily than with conventional detection methods.

[0039] A target gene detection method according to one embodiment of the present invention includes the following process and can be performed by various devices such as a target gene detection device.

[0040] The test subject containing the target gene, the magnetic bead solution, and the solution are mixed (S110).

[0041] A magnetic bead solution and a lysate (Lysis) are each loaded into a container such as a tube, each containing a test subject containing an appropriate amount of a target gene. In the method according to one embodiment of the present invention, the test subject may be included in an amount of several to several tens of μl, and the magnetic solution and the lysate are each included in an amount of several tens to several hundred ml. The lysate dissolves the test subject, and separates / extracts other components, including the target gene, within the test subject. When the amount of the test subject included increases, the detection of the target gene can become easier (detectable with the naked eye, etc.).

[0042] The container in which each ingredient is loaded is mounted on equipment such as a vortex mixer or tube invertor, and the equipment mixes the ingredients loaded in the container.

[0043] The components loaded into the container are mixed and then allowed to react at room temperature for a period of time, more specifically, about 2 minutes.

[0044] This process is illustrated in Fig. 4. As illustrated in Fig. 4(a), if the target gene does not exist in the test subject, the magnetic beads (410) and the solution are mixed with the test subject. On the other hand, as illustrated in Fig. 4(b), if the target gene exists in the test subject, the test subject is dissolved by the magnetic beads (410), the solution, and the solution, and the separated / extracted target gene (420) are mixed.

[0045] Referring again to Figure 1, after washing with the preset first buffer, the supernatant is removed (S120).

[0046] When the individual components are mixed, a preset first buffer solution (WS1) is loaded into the container into which the individual components are loaded, in an amount of tens to hundreds of ml, more specifically, an amount equal to or within a preset error range of the magnetic solution and dissolution solution. Thereafter, the aforementioned equipment additionally mixes them.

[0047] After mixing, the device removes the supernatant using a magnet or the like. After mixing, the device brings the magnet near the container, particularly, the bottom of the container. As described above, since magnetic beads (410) exist in the solution loaded and mixed in the container, when the magnet approaches, the components (target genes and magnetic beads) in the solution approach the magnet, and other foreign substances do not. After bringing the magnet near, the device removes the supernatant. After removing the supernatant, the device separates the magnet brought near the container. Since the magnetic solution is included, the supernatant can be easily removed using the magnet.

[0048] The preset probe is added to the mixed solution and cultured (S130).

[0049] The device adds a preset probe to a mixed solution from which the supernatant has been removed. The preset probe has the configuration and structure shown in FIGS. 2 and 3.

[0050] Referring to Fig. 2, unlike the conventional method, the backbone structure (220) of the preset probe (230) is implemented with PNA (Peptide Nucleic Acid). A fluorescent material (210) is bound to the oligo within the aforementioned backbone structure (220) of the preset probe (230).

[0051] The fluorescent material (210) may be implemented as an inorganic fluorescent material or an organic fluorescent material as in the past, or may be implemented as a quantum dot.

[0052] The backbone structure (220) has a base sequence complementary to the target gene to be detected. However, referring to FIG. 3, while the conventional probe implemented with DNA or RNA has a negatively charged backbone structure, the backbone structure (220) is implemented with PNA and thus becomes neutral. Since the backbone structure (220) is implemented with PNA and has a neutrality, it can be electrically distinguished from the target gene implemented with DNA or RNA. For example, in a process for distinguishing a target gene from other components using a magnet, if the probe is implemented with DNA or RNA as in the conventional method, there is an inconvenience in that not only the target gene (and the probe bound to it) but also the conventional probe has polarity, so it has to be approached with a magnet. Accordingly, in the conventional method, the presence or absence of the target gene could not be recognized until all processes (including amplification, etc.) were completed and detection was performed. On the other hand, when using a probe (230) implemented with PNA, as in the detection method according to one embodiment of the present invention, since only the target gene and the probe bound to it approach the magnet, the tester can determine whether the target gene exists even before the final detection process if the concentration of the target gene is sufficient. If the target gene exists, the target gene and the probe (230) bound to it approach the magnet together. On the other hand, if the target gene does not exist, no component approaches the magnet. In this way, the preset probe (230) is implemented with PNA, is neutral, and complementarily binds to the target gene.

[0053] The preset probe (230) is added in the form of a solution in the mixed solution. However, the solution of the preset probe (230) may contain only the preset probe (230), but may further contain a buffer or an additive. The buffer or additive may contain some or all of SDS, LDS, Sarkocyl, Tween20, Tween80, Triton X100, and Nonidet P-40. The buffer or additive improves the luminescence efficiency of the fluorescent material (210) and allows the backbone structure (220) to unfold more smoothly, thereby improving the binding affinity or binding efficiency with the target gene. The buffer or additive may be added to the solution of the preset probe (230) at a preset concentration. Here, the preset concentration may be 0.001 to 1% based on the solution of the preset probe (230).

[0054] As such, the following advantages can be achieved by directly adding the preset probe to the washed mixed solution. Since the probe is bound to the target gene, separation between the target gene and the supernatant can be performed more quickly and accurately. In addition, if the probe is injected in the final stage (when the target gene has been completely purified), an additional purification process must be performed. In particular, as described above, if the probe is DNA or RNA rather than PNA, an additional purification process must be performed. On the other hand, in the detection method according to one embodiment of the present invention, there is no need for a separate additional process since the preset probe (230) is directly added to the washed mixed solution. Even if the same concentration or amount of the target gene is used in the detection process, a relatively large concentration or amount of the target gene may ultimately remain in the detection method according to one embodiment of the present invention. Accordingly, according to the detection method according to one embodiment of the present invention, detection can be performed using a conventional optical measuring device or, in some cases, can be determined by the naked eye without going through an amplification process as in the prior art.

[0055] The process of adding a preset probe in this way is illustrated in Fig. 5. As shown in Fig. 5(a) or Fig. 5(b), the preset probe is added and generates light. As shown in Fig. 5(a), when the target gene does not exist in the test subject, the preset probe emits light in the solution, and as shown in Fig. 5(b), when the target gene exists in the test subject, the preset probe emits light when bound to the target gene. The preset probe can be added at a concentration of about 100 nM / 20 μl.

[0056] Referring again to Figure 1, the device reacts by adding a preset probe to a mixed solution from which the supernatant has been removed, and then allowing it to stand for several minutes, more specifically, about two minutes, at room temperature.

[0057] After washing with the preset first buffer again, the supernatant is removed (S140). After adding the preset probe, the device repeats the washing and supernatant removal process with the preset first buffer again.

[0058] After washing with a preset second buffer, the supernatant is removed (S150). The device washes with a preset first buffer, and then with a preset second buffer. The preset second buffer (WS2) is loaded in an amount of tens to hundreds of ml, more specifically, in an amount greater than the magnetic solution and the dissolution solution by a preset error range (e.g., within tens of ml), and is additionally mixed by the aforementioned equipment.

[0059] After mixing has taken place, the device uses a magnet to remove the supernatant as described above.

[0060] The washing and supernatant removal process using the preset second buffer may be performed only once, but more preferably, it may be performed twice or more times.

[0061] Additionally, in some cases, in order to completely remove the supernatant during the process of removing the supernatant (in the process using the preset first buffer and / or the process using the preset second buffer), centrifugation may be performed for an additional time of about 10 seconds.

[0062] The washing and supernatant removal process can be carried out as shown in Figures 6 and 7.

[0063] As shown in Fig. 6(a), when the target gene does not exist in the test subject, when the magnet is brought close, the magnetic bead is positioned close to the magnet, whereas the remaining components are not.

[0064] Accordingly, when the washing and supernatant removal process is performed as shown in Fig. 7(a), all remaining components are removed except for the magnetic beads (410) located close to the magnet and receiving a magnetic field from the magnet.

[0065] As shown in Fig. 6(b), when a target gene exists in the test subject, when the magnet is brought into proximity, the magnetic bead (410), the target gene (420), and the preset probe (230) bound to the target gene are positioned together in proximity to the magnet as the magnetic bead (410) moves.

[0066] Accordingly, when the washing and supernatant removal process is performed as shown in Fig. 7(b), the magnetic bead (410) located close to the magnet and receiving a magnetic field from the magnet, the target gene (420), and the preset probe (230) bound to the target gene remain, and the remaining foreign material components are removed.

[0067] Referring back to Figure 1, after washing with a preset first buffer and a preset second buffer and removing the supernatant, the beads are dried (S160). The device dries the magnetic beads (410) or the remaining components (420, 230) combined therewith in the air for approximately 1 minute.

[0068] The target gene is eluted and detected (S170). The device injects the elution buffer into the solution, which has undergone washing, supernatant removal, and drying, and then mixes it. After injecting and mixing the elution buffer, the device reacts by allowing it to stand at room temperature for several minutes, or more specifically, for approximately one minute. Through this process, only the target gene is eluted.

[0069] The device then detects the target gene. As the magnet is brought close and the supernatant is recovered, the eluted target gene is separated from the magnetic beads. The elution and detection process of the target gene is illustrated in Figures 8 and 9.

[0070] As shown in Fig. 8(a), when the target gene does not exist in the test subject, when the eluate is injected and the magnet is brought close, only the magnetic beads are positioned close to the magnet, and no component is eluted separately.

[0071] Accordingly, as shown in Fig. 9(a), even if supernatant removal is performed, a separate target gene does not exist.

[0072] On the other hand, as illustrated in Figure 8(b), if a target gene exists within the test subject, the target gene is eluted into the solution after washing, etc. On the other hand, due to the approach of the magnet, the magnetic bead is positioned close to the magnet.

[0073] Accordingly, as shown in Fig. 9(b), when supernatant removal is performed, the device can detect only the target gene.

[0074] Through this process, only the target gene can be extracted at a high concentration. This can be confirmed in Figures 10 and 11.

[0075] Figures 10 and 11 are drawings comparing the detection results of a conventional detection method and a detection method according to an embodiment of the present invention.

[0076] Referring to Figure 10(a), the results of detecting a target gene using a conventional detection method in a solution containing 20 μL of the test subject (target gene) are illustrated. Referring to the results, it can be confirmed that the luminescence intensity (by the probe) is in the hundreds.

[0077] On the other hand, referring to Fig. 10(b), the result of detecting a target gene using a detection method according to an embodiment of the present invention in a solution containing 5 μl of a test subject (target gene) is illustrated. Referring to the result, it can be confirmed that the luminescence intensity (by the probe) is in the tens of thousands, and the degree (concentration or number) of the target gene detected numerically is several tens to hundreds of times higher.

[0078] This can also be confirmed in Fig. 11. According to the conventional detection method, when the purity (impurity) of the probe is low (7%), it can be confirmed that 480 RFU (Relative Fluorescence Unit) is detected in the situation where the target gene does not exist (Negative), and 572 RFU is detected in the situation where the target gene exists (Positive). On the other hand, when the purity (impurity) of the probe is high (0.1%), it can be confirmed that 487 RFU and 699 RFU are detected in each situation (Negative, Positive in that order). It can be confirmed that in the case of the conventional detection method, the difference in the degree of detection in each situation is not large regardless of the purity. Therefore, according to the conventional detection method, an amplification process must be performed to confirm an accurate detection result.

[0079] On the other hand, according to the detection method according to one embodiment of the present invention, it can be confirmed that 422 RFU and 1808 RFU are detected in each situation (in the order of negative and positive) when the purity (impurity) of the probe is low (7%). It can be confirmed that 443 RFU and 1193 RFU are detected in each situation (in the order of negative and positive) when the purity (impurity) of the probe is high (0.1%). That is, according to the detection method according to one embodiment of the present invention, it can be confirmed that the detection result differs by at least 3 times and at most nearly 6 times depending on the presence or absence of the target gene. Accordingly, according to the detection method according to one embodiment of the present invention, detection can be performed without a separate amplification process.

[0080] Although each process is described as being executed sequentially in FIG. 1, this is merely an illustrative description of the technical idea of ​​one embodiment of the present invention. In other words, a person of ordinary skill in the art to which one embodiment of the present invention pertains may modify and apply various modifications and variations, such as changing the order described in each drawing and executing the process, or executing one or more of the processes in parallel, without departing from the essential characteristics of one embodiment of the present invention. Therefore, FIG. 1 is not limited to a chronological order.

[0081] Meanwhile, the processes illustrated in FIG. 1 can be implemented as computer-readable code on a computer-readable recording medium. A computer-readable recording medium includes all types of recording devices that store data that can be read by a computer system. That is, a computer-readable recording medium includes storage media such as magnetic storage media (e.g., ROM, floppy disks, hard disks, etc.) and optical readable media (e.g., CD-ROMs, DVDs, etc.). In addition, a computer-readable recording medium can be distributed across network-connected computer systems, so that the computer-readable code can be stored and executed in a distributed manner.

[0082] The above description is merely an example of the technical idea of ​​the present embodiment, and those skilled in the art to which the present embodiment pertains may make various modifications and variations without departing from the essential characteristics of the present embodiment. Therefore, the present embodiments are not intended to limit the technical idea of ​​the present embodiment, but to explain it, and the scope of the technical idea of ​​the present embodiment is not limited by these embodiments. The protection scope of the present embodiment should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of rights of the present embodiment.

Claims

1. In a method for detecting a target gene, A mixing process of mixing a test subject containing a target gene, a magnetic bead solution, and a dissolution solution; A first removal process in which the mixed solution is washed with a preset first buffer solution during the above mixing process, and then the supernatant is removed; An addition process of adding a preset probe into a mixed solution; A second removal process in which the solution that has gone through the above addition process is washed with the first buffer solution set above, and then the supernatant is removed; After washing with the above-described second buffer, a third removal process of removing the supernatant; and Detection process for extracting and detecting target genes A method for detecting a target gene, characterized by including:

2. In paragraph 1, The above mixing process is, A target gene detection method characterized by loading a test subject, a magnetic bead solution, and a dissolving solution into a container and mixing them.

3. In paragraph 2, The above mixing process is, A method for detecting a target gene characterized by allowing the container to stand at room temperature for several minutes and then react.

4. In paragraph 1, The above preset probe is, A method for detecting a target gene, characterized in that it comprises a backbone structure implemented with PNA.

5. In paragraph 4, The above backbone structure is, A method for detecting a target gene, characterized in that it has a base sequence complementary to the target gene.

6. In paragraph 4, The above preset probe is, A method for detecting a target gene characterized by being neutral.

7. In paragraph 4, The above preset probe is, A method for detecting a target gene characterized in that a fluorescent material is formed by combining with a backbone structure.

8. In paragraph 7, The above fluorescent material is, A method for detecting a target gene, characterized in that it is implemented with an inorganic fluorescent substance, an organic fluorescent substance or a quantum dot.

9. In paragraph 1, The above preset probe is, A target gene detection method characterized in that a preset concentration is added.

10. In paragraph 1, A target gene detection method characterized by further including a drying process for drying the magnetic beads or the remaining components combined therewith that have undergone the third removal process.

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